Monograph Series No. 4 - Equine Inmunology

Proceedings of a Workshop on
EQUINE IMMUNOLOGY IN 2001

24th28th January 2001
Santa Fe, New Mexico
Editors: D. P. Lunn and J. F. Wade
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Havemeyer Foundation
Monograph Series No. 4
Proceedings of a Workshop on
EQUINE IMMUNOLOGY IN 2001
H
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Havemeyer Foundation
Monograph Series No. 4
2001 by R & WPublications (Newmarket) Limited
Suites 3 & 4, 8 Kings Court, Willie Snaith Road, Newmarket, Suffolk CB8 7SG, UK
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First published 2001
ISSN 1472-3158
Published by R &WPublications (Newmarket) Limited
Printed in Great Britain by Quality Print Services (Anglia) Limited
iii
Havemeyer Foundation Monograph Series No. 4
CONTENTS
FOREWORD ....................................................................................................................................Page vi
SESSION I: IMMUNITY & INFECTION
Immune control of equine infectious anaemia virus
T. C. McGuire, S. R. Leib, R. H. Mealey, D. G. Fraser, S. L. Ridgely and D. J. Prieur ....................Page 3
Overview of T cell cytokine research in the horse
D. W. Horohov ....................................................................................................................................Page 7
Rhodococcus equi, a paradigm for equine immunity to intracellular bacteria
S. Gigure ........................................................................................................................................Page 11
Equine herpesvirus-1: immunity in lytic and latent infections
J. Slater ............................................................................................................................................Page 14
Strongyles large and small: immunity
T. R. Klei ..........................................................................................................................................Page 17
Mucosal immunity: an opportunity to prime without prejudice
D. Hannant........................................................................................................................................Page 19
Mucosal antigen delivery in horses
J. F. Timoney and A. S. Sheoran ......................................................................................................Page 21
Regulation of mucosal immune responses
G. Sobboll, D. W. Horohov, C. W. Olsen and D. P. Lunn ................................................................Page 23
The induction of equine herpesvirus-specific antibodies in the upper respiratory tract of the horse
C. C. Breathnach, M. R. Yeargan, A. S. Sheoran and G. P. Allen....................................................Page 26
Immunogenetics defined
D. F. Antczak ....................................................................................................................................Page 29
Organisation of the equine immunoglobulin constant region genes
B. Wagner ..........................................................................................................................................Page 30
Equine MAC regulation of resistance and immune bias
E. Marti ............................................................................................................................................Page 33
A syndrome of anaemia, immunodeficiency and peripheral ganglionopathy in Fell pony foals
M. A. Holmes, S. F. E. Scoles, A. Holliman and P. D. F. May .......................................................Page 35
iv
Immunology in 2001
SESSION II: NEW TECHNOLOGIES IN EQUINE IMMUNITY
Monoclonal antibodies: whats next?
D. P. Lunn ......................................................................................................................................Page 41
Detection of interferon- producing equine T lymphocytes by flow cytometry:
pulmonary responses to Rhodococcus equi challenge
S. Hines, D. Stone, M. Hines, L. Norton and T. C. McGuire ..........................................................Page 42
Il-4 induced CD23 (FCERII) upregulation in equine peripheral blood mononuclear cells and
alveolar macrophages
J. L. Watson, K. A. Jackson and J. L. Stott .....................................................................................Page 44
Oponisation by complement C3 and IgG as measured by a flow cytometric immunoassay
G. Grndahl ...................................................................................................................................Page 46
Flow cytometric characterisation of neutrophil phagocytosis and oxidative burst activity
S. L. Raidal .......................................................................................................................................Page 49
RT-PCR detection of equine cytokines
D. W. Horohov ................................................................................................................................Page 54
Equine cytokines and associated reagents
L. Nicolson, L. McMonagle, S. Taylor, C. Hopkins, L. Sanders, H. van Kuilekom, N. Scholts,
D. Argyle, D. Onions and V. Schijns ...............................................................................................Page 57
Quantitation of equine cytokine mRNA expression by RT-PCR
S. Gigu`ere ........................................................................................................................................Page 59
Type I interferon and interleukin-6 in nasal secretions and serum from ponies infected
with equine influenza A2 (H3N8)
E. Wattrang, D. M. Jessett, P. Yates, L. Fuxler and D. Hannant ...................................................Page 62
Genomic exploration of orthopaedic infection using cDNA microarrays
E. M. Santschi, A. Rink and C. W. Beattie ......................................................................................Page 64
The power of limiting dilution analysis
J. H. Kydd, E. Wattrang, G. P. Allen, T. ONeill and D. Hannant ................................................Page 65
Flow cytometric techniques in clinical investigation
B. R. Rush, M. J. B. F. Flaminio, E. G. Davis and M. J. Wilkerson.................................................Page 67
Simultaneous analysis of phagocytosis and oxidative burst activity of equine phagocytes by
flow cytometry
M. J. B. F. Flaminio, B. R. Rush, E. G. Davis, W. Shuman and M. Wilkerson ................................Page 69
SESSION III: INFLAMMATION
Chronic airway disease in the horse
N. E. Robinson ...............................................................................................................................Page 73
Variation of non-infectious airway disease phenotype with age
L. Viel .............................................................................................................................................Page 77
Biology of airway neutrophils
B. McGorum and T. Brazil ............................................................................................................Page 79
Cytokine immunoregulatory elements in RAO
J. -P. Lavoie and D. W. Horohov .....................................................................................................Page 82
IgG subtypes and clues to the immunopathology of recurrent airway obstruction (heaves)
D. M. Ainsworth, J. A. Appleton, D. F. Antczak. M. A. Santiago and G. A. Aviza ........................Page 84
v
Effects of dust and endotoxin exposures on lung function and airway cytology of horses
L. L. Coutil, M. A. Hunt and F. S. Rosenthal ..............................................................................Page 86
Septicaemia and endotoxaemia in horses
J. N. Moore .....................................................................................................................................Page 87
Pathophysiology of endothelin-1 in equine acute laminitis
A. S. Holm, S. C. Eades, C. S. Venugopal, J. L. Oliver and R. M. Moore .......................................Page 89
Immunomodulation of events in sepsis: role of P-selectin/P-selectin ligand system
B. J. Darien.......................................................................................................................................Page 91
Joint disease in the horse
C. W. McIlwraith...............................................................................................................................Page 94
Chondrocytic response to joint inflammation and corticosteroids
J. MacLeod........................................................................................................................................Page 97
Regulation of inducible nitric oxide expression and activity in equine chondrocytes
J. T. Tung, P. J. Venta and J. P. Caron..............................................................................................Page 99
LIST OF PARTICIPANTS.............................................................................................................Page 102
AUTHOR INDEX...........................................................................................................................Page 103
vi
Immunology in 2001
FOREWORD
T
he Havemeyer Foundation Workshop
on Equine Immunology in 2001 was
convened in order to bring together a
broad range of equine scientists with a
common interest in immunological studies,
both basic and applied. The goal of the
organisers was to ensure that the latest and
greatest were presented, and that innovative
scientists working in different disciplines
would have the opportunity to learn about
new tools and establish new collaborations.
We were delighted by the quality of the
presentations and the meeting in general, the
strength of which is well documented by the
outstanding papers in this monograph. The
standard of research and results presented
illustrates clearly the ongoing progress in the
field of equine immunology, and promises
much for the future. It is our hope and
expectation that the relationships and
collaborations established by this workshop
will foster this progress.
We are extremely grateful to the
Havemeyer Foundation, and to Mr Gene
Pranzo, for providing the major sponsorship
for the meeting. In addition we would like to
thank the Veterinary Immunology
Committee of the International Union of
Immunological Societies, Heska, Bayer and
Intervet for additional support. We are also
extremely grateful to Mrs Jan Wade and
R&W Publications for managing the
organisation of the meeting and for the
preparation of this monograph.
We hope the reader finds much of value in
this publication, and feel that it is a fitting
testament to the breadth and strength of
equine immunological science in this new
millennium.
D. Paul Lunn
David W. Horohov
Doug F. Antczak
vii
HAVEMEYER SCIENTIFIC WORKSHOPS
1981 First International Workshop on Lymphocyte Alloantigens of the Horse
October - New York City, USA
Organiser: Dr D. F. Antczak
1982 Second International Workshop on Lymphocyte Alloantigens of the Horse
October - Cornell University, Ithaca, New York, USA
1983 Third International Workshop on Lymphocyte Alloantigens of the Horse
April - New Bolton Center, University of Pennsylvania, USA
1984 First International Symposium on Equine Embryo Transfer
October - Cornell University, Ithaca, New York, USA
Organisers : Drs D. F. Antczak and W. R. Allen
1985 Fourth International Workshop on Lymphocyte Alloantigens of the Horse
October - University of Kentucky, USA
Organisers: Drs D. F. Antczak and E. Bailey
1986 Workshop on Corynebacterium equi Pneumonia of Foals
July - University of Guelph, Canada
Organiser: Dr J. F. Prescott
1987 Fifth International Workshop on Lymphocyte Alloantigens of the Horse
October - Louisiana State University, USA
Organisers: Drs D. F. Antczak and J. McClure
1989 Second International Symposium on Equine Embryo Transfer
February - Banff, Alberta, Canada
Organisers : Drs D. F. Antczak and W. R. Allen
1990 International Workshop on Equine Sarcoids
April - Interlaken, Switzerland
Organisers: Dr D. F. Antczak and Professor S. Lazary
1992 Workshop on Equine Neonatal Medicine
January - Naples, Florida
Organisers: Drs D. F. Antczak and P. D. Rossdale
Third International Symposium on Equine Embryo Transfer
February - Buenos Aires, Argentina
Organisers : Drs D. F. Antczak, W. R. Allen, J. G. Oriol and R. Pashen
viii
Immunology in 2001
1995 Equine Perinatology
July - Cambridge, England
Organiser: Dr P. D. Rossdale
Second International Equine Leucocyte Antigen Workshop
July - Lake Tahoe, California, USA
Organisers : Drs D. F. Antczak, P. Lunn and M. Holmes
First International Workshop on Equine Gene Mapping
October - Lexington, Kentucky, USA
Erection and Ejaculation in the Human Male and Stallion: A Comparative
Study
October - Mount Joy, Pennsylvania, USA
Organiser: Dr S. M. McDonnell
Bone Remodelling Workshop
October - Corcord, Massachusetts, USA
Organiser: Dr H. Seeherman
1997 Second International Workshop on Equine Gene Mapping
October - San Diego, California, USA
Maternal Recognition of Pregnancy in the Mare
January - Dominican Republic
Organisers: Drs W. R. Allen and T. A. E. Stout
Uterine Clearance
March - Gainesville, Florida, USA
Organiser: Dr M. M. LeBlanc
Trophoblast Differentiation
September - Edinburgh, Scotland
Organisers: Drs D. F. Antczak and F. Stewart
1998 Third International Genome Workshop
January - San Diego, California, USA
Third International Workshop on Perinatology: Genesis and Post Natal
Consequences of Abnormal Intrauterine Developments: Comparative
Aspects
February - Sydney, Australia
Organiser: Dr P. D. Rossdale
Horse Genomics and the Genetic Factors Affecting Race Horse Performance
March - Banbury Center, Cold Spring Harbor, New York, USA
Organisers: Drs D. F. Antczak, E. Bailey and J. Witkowski
ix
Allergic Diseases of the Horse
April - Lipica, Slovenia
Organisers: Drs D. F. Antczak, S. Lazary and E. Marti
Equine Placentitis Workshop
October - Lexington, Kentucky, USA
Organisers: Drs D. F. Antczak, W. R. Allen and W. Zent
Septicemia II Workshop
November - Boston, Massachusetts, USA
Organiser: Dr M. R. Paradis
1999 Equine Genome Project
Third International Equine Genome Workshop
June - Uppsala, Sweden
Organisers: Drs D. F. Antczak, E. Bailey and K. Sandberg
Fourth International Meeting of OIE and WHO Experts on Control of
Equine Influenza
August - Miami, Florida, USA
Organiser: Dr J. Mumford
European Equine Gamete Workshop
September - Lopuszna, Poland
Organisers: Drs W. R. Allen and M. Tischner
Fetomaternal Control of Pregnancy
November - Barbados, West Indies
Organisers: Drs T. Stout and W. R. Allen
2000 Equine Genome Project
Uterine Infections in Mares and Women: A Comparative Study
March - Naples, Florida, USA
Organiser: Dr M. M. LeBlanc
5th International Symposium on Equine Embryo Transfer
Saari, Finland
Organiser: Dr T. Katila
2001 USDA Internationl Plant & Animal Genome Conference
San Diego, California
Equine Immunology in 2001
Sanata Fe, New Mexico
Organiser: Dr D. P. Lunn
x
Immunology in 2001
The following are monographs available to date at a cost of 9.95 each.
Series No 1
PROCEEDINGS OF THE FIRST MEETING OF THE EUROPEAN EQUINE GAMETE GROUP (EEGG)
Editors: W. R. Allen and J. F. Wade
5th8th September 1999
Lopuszna, Poland
Series No 2
PROCEEDINGS OF A WORKSHOP ON FETOMATERNAL CONTROL OF PREGNANCY
Editors: T. A. E. Stout and J. F. Wade
14th16th November 1999
Barbados, West Indies
Series No 3
PROCEEDINGS OF THE 5TH INTERNATIONAL SYMPOSIUM ON EQUINE EMBRYO TRANSFER
Editors: T. Katila and J. F. Wade
6th9th July 2000
Saari, Finland
Series No 4
PROCEEDINGS OF A WORKSHOP ON EQUINE IMMUNOLOGY IN 2001
If you wish to order copies, please contact R & WPublications Ltd, Suites 3 & 4, 8 Kings Court, Willie
Snaith Road, Newmarket, Suffolk CB8 7SG, UK, Tel: +44 1638 667600, Fax: +44 1638 667229, e-mail:
rw.publications@btinternet.com.
HAVEMEYER MONOGRAPH SERIES
1
SESSION I :
Immunity & infection
Chairman: David Horohov
2
Immunology in 2001
3
IMMUNE CONTROL OF EQUINE INFECTIOUS
ANAEMIA VIRUS
T. C. McGuire, S. R. Leib, R. H. Mealey, D. G. Fraser, S. L. Ridgely and
D. J. Prieur
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman,
Washington 99163-7040, USA
Horses with equine infectious anaemia virus
(EIAV) are infected for life, but control of virus
replication occurs following the viraemic episodes
that usually happen during the first year of
infection. Later, during the carrier state when there
are no clinical signs, the virus is clearly more
tightly controlled and virus replication is difficult
to detect. An adaptive immune response is required
for the control of initial viraemia. This was
demonstrated by the failure of Arabian foals with
severe combined immunodeficiency (SCID) and
lacking functional T and B lymphocytes (McGuire
et al. 1974) to control viraemia after infection with
EIAV (Perryman et al. 1988). Non-SCID foals
controlled viraemia after similar infection. In
addition, adoptive transfer of lymphocytes from an
EIAV-infected horse to a SCID foal matched at the
A locus of MHC class I resulted in control of
viraemia following challenge (Mealey et al. 2001).
At least 2 immunological mechanisms for EIAV
control have been identified, neutralising antibody
and cytotoxic T lymphocytes (CTL). Neutralising
antibody is effective when it is present to
homologous virus. However, in infected horses,
continual antigenic variation of recognised
epitopes limits its effectiveness. In addition, the
late appearance of neutralising antibody after
infection, or after the appearance of variants,
further constrains the effectiveness of neutralising
antibody in EIAV control (Montelaro et al. 1993).
Another possible protective mechanism involving
antibody is antibody dependent cellular
cytotoxicity (ADCC). However, ADCC against
EIAV-infected target cells could not be
demonstrated with sera from infected horses,
whereas ADCC against equine herpesvirus-1
(EHV-1)-infected target cells could be
demonstrated with sera from horses vaccinated
against EHV-1 (Tschetter et al. 1997). Thus, the
absence of antibodies which cause ADCC, and the
late appearance of neutralising antibody as well as
variation of neutralising antibody sensitive
epitopes, dictates additional focus on CTL.
Our research hypothesis is that class I-
restricted, EIAV-specific CD8+ CTL control EIAV
infection. Effector CTL that do not require in vitro
stimulation and kill EIAV-infected target cells are
present in peripheral blood mononuclear cells
(PBMC) within a few days of initial viraemia and
for approximately 3 months after infection
(McGuire et al. 1994). This observation is
consistent with these cells being responsible for
control of the initial viraemia. Later in infection, in
vitro stimulation with antigen is required to
demonstrate MHC class I-restricted, EIAV-specific
CD8+ memory CTL (McGuire et al. 1997;
Hammond et al. 1997). In addition, EIAV-specific,
MHC class II-restricted CD4+ memory CTL have
been demonstrated in EIAV-infected horses
(Hammond et al. 1997). The importance of these
class II-restricted CTL is not known. Many of our
studies use equine kidney (EK) cells which express
MHC class I molecules, but not class II molecules
(McGuire et al. 1997). Therefore, the only CTL
activity that can be measured in this system is that
mediated by CD8+ T lymphocytes. In order to have
autologous and MHC class I-mismatched EK cells
for targets in CTL assays, kidney biopsies are taken
before EIAV infection and the cells are expanded
and frozen for later use. Other investigators have
effectively used stimulated horse T lymphocytes as
target cells in CTL assays (Allen et al. 1995;
ONeill et al. 1999).
To determine if memory CTL were present in
EIAV carriers and in numbers that could account
for virus control, the CTLm frequency in PBMC
4
from 5 inapparent carriers infected for 22 to 50
months was determined by limiting dilution
analysis (McGuire et al. 1997). Limiting dilution
analysis has also been used to determine the
frequency of CTLm to EHV-1 in horses (ONeill et
al. 1999). PBMC from EIAV carrier horses were
diluted, stimulated and tested on EK cell targets
infected with EIAV and recombinant vaccinia
viruses expressing EIAV Env or Gag/Pr proteins.
All 5 carriers had CTLm to EIAV-infected targets;
3 had CTLm to targets expressing either Env or
Gag/Pr proteins, one had CTLm to Env only and
one had CTLm to Gag/Pr only. The CTLm
frequency range was 60468 per million PBMC to
EIAV-infected targets, 4286 to Env, and 25190
to Gag/Pr expressing targets (McGuire et al.
1997). In summary, the mean frequency of
memory CTL to EIAV-infected target cells in
PBMC from carrier horses is 293/million (1/3,413)
when measured by limiting dilution analysis,
which underestimates frequency. This number of
memory CTL is consistent with these cells being
responsible for EIAV control in the carrier state.
Because proteins and epitopes recognised by
predominant memory CTL from inapparent
carriers may be useful for inducing protective
immune responses in nave horses, experiments
were undertaken using these memory CTL to
identify the EIAV proteins and epitopes for further
evaluation in immunisation experiments. To
identify the EIAV proteins with epitopes
recognised by class I-restricted CD8+ CTL from
the greatest number of carrier horses, retroviral
vectors expressing the different EIAV proteins
were made (Lonning et al. 1999a). These retroviral
vectors were used to transduce EK cells for targets
in CTL assays (McGuire et al. 2000). The proteins
recognised by memory CTL from the most carrier
horses was Gag p15 (by CTL from 100% of 7
infected horses), p26 (86%), SU and the middle
third of Pol protein (each by 43%), TM (29%), and
S2 (14%). These results identify the proteins
recognised by the CTL present during times of
virus control and justify experiments to determine
their role.
As most carrier horses had memory CTL that
recognised Gag matrix (p15) and capsid (p26)
proteins, with no killing of target cells expressing
p11 and p9, overlapping synthetic peptides of
1625 amino acids were used to identify p15 and
p26 epitopes (Zhang et al. 1998). Peptides were
identified containing at least 12 Gag CTL epitopes
recognised by virus-stimulated PBMC from 6
long-term EIAV-infected horses with varying class
I haplotypes. Each of the 6 horses had CTL
recognising at least one Gag epitope, and CTL
from one horse recognised at least 8 different Gag
epitopes. None of the identified peptides were
recognised by CTL from all 6 horses. Two
nonamer peptide epitopes were defined from Gag
p26; one (18a) was restricted by class I equine
leucocyte alloantigen (ELA) -A5.1 and the other
(28b-1) by ELA-A9 molecules (Zhang et al.
1998). A 10 nM concentration of peptide 18a was
required to sensitise EK target cells for CTL
killing whereas 28b-1 required 1 nM. The results
demonstrated that diverse CTL responses against
Gag epitopes were generated in EIAV-infected
carrier horses and indicated that ELA-A class I
molecules were responsible for the diversity of
CTL epitopes recognised. Further work
demonstrated that neither epitope 18a or 28b-1
were conserved among EIAV strains indicating
that antigenic variation of these epitopes could
occur (Zhang et al. 1999).
To determine the in vivo role of CD8+ CTL,
we are inducing CTL in horses expressing class I
ELA-A1 molecules by immunisation with
peptides containing mapped A1-restricted CTL
epitopes. When CTL that will kill EIAV-infected
target cells are detected in numbers equal to or
higher than the number of CTLm detected in
carrier horses, then the immunised horses will be
challenged with EIAV and the protective response
mediated by the CTL evaluated. This approach is
being taken to lessen the variables associated with
inducing CTL responses in outbred horses to
protein antigens. To this end, a class I ELA-A1-
restricted EIAV peptide has been defined by initial
mapping with retroviral vector-transduced target
cells expressing all different EIAV proteins
including portions of the Env proteins (McGuire et
al. 2000). CTL assays with these targets identified
an epitope in Env construct 1 (Fig 1). The protein
expressed by Env 1 was expressed in 3 parts by 3
new vectors (a,b,c) (Fig 2) and Env T1c was found
to contain the A1-restricted epitope (Fig 3). Six
overlapping synthetic peptides covering the
protein of Env T1c were constructed and used for
further CTL epitope mapping and peptide 1 was
found to contain the epitope (Fig 3). Further
mapping to define the minimal CTL epitope is
being done. The class I molecules from an A1
horse have been cloned and expressed in order to
identify the gene expressing the A1 molecule. In
addition, EIAV peptides recognised by CD4+ Th1
Immunology in 2001
5
lymphocytes of carrier horses with varying MHC
class II haplotypes have been identified to evaluate
their potential to enhance CTL responses (15,16).
Finally, if induction of CTL in horses is required
for protection against EIAV or any other disease,
immunization strategies must induce CTL to
epitopes in various proteins, in horses with
different MHC class I haplotypes.
REFERENCES
Allen, G., Yeargan, M., Costa, L.R. and Cross, R. (1995)
Major histocompatibility complex class I-restricted
cytotoxic T-lymphocyte responses in horses infected
with equine herpes virus 1. J. Virol. 69, 606-612.
Hammond, S. A., Cook, S. J., Lichtenstein, D. L., Issel,
C. J. and Montelaro, R. C. (1997) Maturation of the
cellular and humoural immune responses to
persistent infection in horses by equine infectious
anaemia virus is a complex and lengthy process. J.
Virol. 71, 3840-3852.
Lonning, S.M., Zhang, W., Leib, S.R. and McGuire, T.C.
(1999a) Detection and induction of equine infectious
anaemia virus-specific cytotoxic T lymphocyte
responses using recombinant retroviral vectors.
J.Virol. 73, 2762-2769.
McGuire, T.C., Poppie, M.J. and Banks, K.L. (1974)
Combined (B- and T- lymphocyte immuno-
deficiency: A fatal genetic disease of Arabian foals.
J. Am. vet. med. Ass. 164, 70-76.
Fig 1: CTL assay with stimulated
PBMC from an EIAV-infected
horse and target cells expressing
the various EIAV proteins as
previously described (McGuire
et al. 2000). Significant lysis
occurred with target cells
expressing p15 and E1 (Env T1).
N
S
3
P
1
S
2
P
3
E
3
S
1
P
2
E
2
E
1
p
9
p
1
1
p
2
6
b
p
2
6
a
p
1
5
60
50
40
30
20
10
0
%

s
p
e
c
i
f
i
c

l
y
s
i
s
Proteins expressed by target cells
Fig 2: The major regions of the
EIAV genome are represented.
Env T1, T2, and T3 are portions
of the env gene expressed by
retroviral vector-transduced
target cells. a, b, and c are
portions of Env T1 expressed by
vectors.
a
b
c
Env T1
Env T2
Env T3
LTR LTR gag pol
env
Fig 3: CTL assay with stimulated
PBMC from an EIAV-infected
horse and target cells expressing
the EIAV Env T1 protein portions
a, b, and c depicted in Fig 2 and
synthetic peptides 1-6 spanning
E1c (Env T1c). N is normal cells
and Blk is a blank column to
separate the vector and peptide
target data. Significant CTL lysis
of targets expressing Env T1c and
peptide 1 occurred.
45
40
35
30
25
20
15
10
5
0
%

l
y
s
i
s
N E1a E1b E1c Blk p1 p2 p3 p4 p5 p6
Protein or peptide
6
McGuire, T.C., Tumas, D.B., Byrne, K.M., Hines, M.T.,
Leib, S.R., Brassfield, A.L., O'Rourke, K.I. and
Perryman, L.E. (1994) Major histocompatibility
complex-restricted CD8+ cytotoxic T lymphocytes
from horses with equine infectious anaemia virus
recognise env and gag/PR proteins. J. Virol. 68,
1459-1467.
McGuire, T.C., Zhang, W., Hines, M.T., Henny, P.J. and
Byrne, K.M. (1997) Frequency of memory cytotoxic
T lymphocytes to equine infectious anaemia virus
proteins in blood from carrier horses. Virology 238,
85-93.
McGuire, T.C., Leib, S.R., Lonning, S.M., Zhang, W.,
Byrne, K.M. and Mealey, R.M. (2000) Equine
infectious anaemia virus proteins with epitopes most
frequently recognised by cytotoxic T lymphocytes
from infected horses. J. gen. Virol. 81, 2735-2739.
Mealey, R.H., Fraser, D.G., Oaks, J.L., Cantor, G.H. and
McGuire, T.C. (2001) Equine infectious anaemia
virus is controlled following immune reconstitution
in an Arabian foal with severe combined immuno-
deficiency. J. clin. Immunol. In press.
Montelaro, R. C., Ball J. M. and Rushlow K. E. (1993)
Equine retroviruses. In: The Retroviridae. Ed: J. A.
Levy, Plenum Press, New York. p. 257-360.
ONeill, T., Kydd, J.H., Allen, G.P., Wattrang, E.,
Mumford, J.A. and Hannant, D. (1999)
Determination of equid herpesvirus 1-specific,
CD8+, cytotoxic T lymphocyte precursor
frequencies in ponies. Vet. Immunol. Immunopathol.
70, 43-54.
Perryman, L.E., O'Rourke, K.I. and McGuire, T.C.
(1988) Immune responses are required to terminate
viraemia in equine infectious anaemia lentivirus
infection. J. Virol. 62, 3073-3076.
Tschetter, J.R., Byrne, K.M., Perryman, L.E. and
McGuire, T.C. (1997) Control of equine infectious
anaemia virus is not dependent on ADCC mediating
antibodies. Virology 230, 275-280.
Zhang, W., Lonning, S.M. and McGuire, T.C. (1998)
Gag protein epitopes recognised by ELA-A-
restricted cytotoxic T lymphocytes from horses with
long-term equine infectious anaemia virus infection.
J. Virol. 72, 9612-9620.
Zhang, W., Auyong, D.B., Oaks, J.L. and McGuire, T.C.
(1999) Natural variation of equine infectious
anaemia virus Gag-protein cytotoxic T lymphocyte
epitopes. Virology 261, 242-252.
Immunology in 2001
OVERVIEW OF T CELL CYTOKINE RESEARCH IN THE
HORSE
D. W. Horohov
Department of Pathobiological Sciences, Louisiana State University, School of Veterinary Medicine,
Baton Rouge, Louisiana 70803, USA
Cytokines are small hormone-like proteins that
influence the functions of various cells. A vast
number of cytokines have been described that
influence lymphocyte function. Work in murine
models has made it clear that the induction of
protective resistance or the exacerbation of disease
is often dependent upon the pattern of cytokine
genes expressed during an immune response
(Mosmann and Coffman 1989; Urban et al. 1996;
Lohoff et al. 1998). Although research into the
role of cytokines in equine immunity and disease
is in its infancy, considerable advances have been
made. The horse presents a number of scenarios
that lend themselves to the study of cytokine
interactions in either disease progression or
prevention. The emphasis of the earliest work in
these areas was on the characterisation of the
cytokine response itself, first through the use of
conventional bioassays (Stott and Osburn 1988;
Morris et al. 1990; May et al. 1990; Morris et al.
1992) and later, with the growing availability of
recombinant cytokines and monoclonal antibodies,
through immunoassays (MacKay and Socher
1992; Franchini et al. 1998) and molecular
analyses (Gigu` ere and Prescott 1999; Swiderski et
al. 1999c; Leutenegger et al. 1999). Although
relatively few groups have been involved in these
efforts, the tools of modern biotechnology have
helped to accelerate the rate of advancement.
Thus, the cloning, sequencing and expression of a
number of equine cytokines have been
accomplished (Table 1). The availability of these
reagents will help lead to a better understanding of
the equine immune system and, perhaps, of the
immunological basis of a number of important
equine diseases.
Recent work has focused on the patterns of
cytokine expression in various disease processes in
the horse and the role that specific cytokines may
play in either protection or pathology. These
studies are modelled in part on the early
observations made in the mouse that T
lymphocytes could be divided into different
subpopulations based on their pattern of cytokine
expression and these patterns of cytokine
expression could be associated with either
protective or pathological responses to various
parasitic agents (Urban et al. 1996; Mosmann and
Coffman 1989; Lohoff et al. 1998). Thus, so-
called Th1 cells produced those cytokines, notably
interferon (IFN)- and interleukin (IL)-2, which
generated protective immune responses to
intracellular parasites (Lohoff et al. 1998). By
contrast, Th2 cells produced those cytokines, IL-4
and IL-5, which played a central role in immunity
to intestinal helminths and other multicellular
parasites (Urban et al. 1996). In addition to these
protective responses, it was also shown that both
subsets could be associated with
immunopathological responses. Many forms of
autoimmune disease appear to be associated with
the induction of a Th1 response to self-antigens
TABLE 1: Published equine cytokine sequences
Cytokine Genebank accession
IL-1 D42146, U92481
IL-2 L06009, X69393
IL-4 L06010
IL-5 U91947
IL-6 U64794, AF005227
IL-8 AF062377
IL-10 U38200
IL-12 Y11129, Y11130
IL-18 Y11131
IFN- M14540
IFN- M14546
IFN- D28520, U04050
TFN- M64087
7
(OGarra et al. 1997). Likewise, allergies are
associated with the induction of a Th2 response
(Umetsu and DeKruyff 1997). Indeed atopic
individuals have a propensity to mount Th2
responses to most foreign antigens (Del Prete
1992). While similar patterns of cytokine
expression have been observed in other species,
their existence in the horse was unknown.
Nevertheless there are several clinical situations in
which it appears likely that Th1 or Th2 cells could
play a central role in either the protective or
pathological response in horses.
Horses affected with equine recurrent uveitis
(ERU) experience painful bouts of ocular
inflammation that can ultimately lead to blindness
in the affected eye (Schwink 1992). Although the
underlying cause of this condition is unknown,
data support the hypothesis that this disease is
immunological in nature (Romeike et al. 1998).
Analysis of mRNA collected from the eyes of
ERU horses demonstrated the presence of
messenger (m)RNA for the Th1 cytokines IL-2
and IFN- (Gilger et al. 1999). Furthermore,
treatment of affected horses with corticosteroid
implants resulted in both a reduction in cytokine
mRNA levels and an improvement of the clinical
condition (Gilger et al. 2000). Direct cause has
yet to be established but these results are
consistent with the notion that ERU is the result of
an ongoing Th1 immune response in the affected
eye.
A Th1 response was also associated with
protective immune responses to equine influenza
virus infection in ponies. This response, primarily
associated with the respiratory lymph nodes of
infected ponies, contrasted with the response in
ponies given a commercial vaccine (Fig 1). Indeed
the latter response was characterised by the lack of
IFN- production and elevated levels of the Th2
cytokine IL-4, presumably as a result of the use of
alum in the commercial product (Grun and Maurer
1989). Given that horses infected with equine
influenza virus exhibit longer term protection than
vaccinates (Hannant et al. 1988), these results are
consistent with murine models demonstrating that
Th1 cells play a central role in protection against
influenza viruses while Th2 cells are non-
protective (Graham et al. 1994).
Immunity to the intestinal parasite Strongylus
vulgaris is associated with production of the Th2
cytokines IL-4 and IL-5 in both peripheral blood
mononuclear cells and colonic lymph nodes
(Swiderski et al. 1999a,b). This is consistent with
the in vivo observation that protection is associated
with the induction of an anamnestic eosinophilia
(Dennis et al. 1993) and that IL-5 plays a key role
in regulating this response (Dent et al. 1990). By
contrast, intramuscular vaccination of ponies with
soluble adult worm antigens in a Ribi adjuvant
resulted in both disease exacerbation following
challenge (Monahan et al. 1995) and the
concomitant induction of a Th1 cytokine response
(Fig 2). By contrast, oral immunisation with
irradiated larvae is associated with a protective
Th2 immune response (Fig 2).
Lastly, lymphocytes obtained from
bronchoalveolar lavage samples from horses
affected with summer pasture-associated
obstructive pulmonary disease (SPAOPD), a
recurrent airway obstructive disease similar to
chronic obstructive pulmonary disease (COPD)
(Seahorn et al. 1996), produced significantly
elevated levels of IL-4 mRNA. Interestingly
mRNA for IL-5 was not detected, suggesting that
this is a modified Th2 response (Beadle et al.
2001). Consistent with this interpretation is the
Fig 1: Cytokine mRNA levels in
lymphocyte cultures from
influenza virus-vaccinated and
infected ponies. Peripheral blood
mononuclear cells (PBMC) and
respiratory lymph node cells
(RLN) were collected from 4
vaccinated and 4 infected ponies
one month post vaccination/
infection. The cells were
stimulated in vitro with equine
influenza virus antigen (A/KY/82)
for 48 h and total mRNA collected
for cytokine mRNA quantitation
(Swiderski et al. 1999c).
Immunology in 2001
8
600
500
400
300
200
100
0
IL-2
IL-4
IFN-
m
R
N
A

c
o
p
y

u
n
i
t
s
PBMC PBMC RLN RLN
Vaccinated Infected
absence of an eosinophilic infiltrate in the lungs of
the affected ponies (Seahorn et al. 1996).
In summary, current data support the
proposition that horses can mount both Th1 and
Th2 immune responses and these can be associated
with either protective or pathological responses
(Table 2).
REFERENCES
Beadle, R.E., Horohov, D.W. and Gaunt, S.D. (2001)
Interleukin-4 and interferon-gamma gene expression
in summer pasture-associated obstructive pulmonary
disease affected horses. Equine vet. J. In press.
Curran, J., Argyle, D., Cox, P., Onions, D. and Nicolson,
L. (1994) Nucleotide sequence of the equine
interferon cDNA. DNA Seq. 4, 405-407.
Del Prete, G. (1992) Human Th1 and Th2 lymphocytes:
their role in the pathophysiology of atopy. Allergy
47, 450-455.
Dennis, V.A., Klei, T.R. and Chapman, M.R. (1993)
Generation and partial characterisation of an
eosinophil chemotactic cytokine produced by
sensitised equine mononuclear cells stimulated with
Strongylus vulgaris antigen. Vet. Immunol
Immunopathol. 37, 135-149.
Dent, L.A., Strath, M., Mellor, A.L. and Sanderson, C.J.
(1990) Eosinophilia in transgenic mice expressing
interleukin 5. J. exp. Med. 172, 1425-1431.
Franchini, M., Gilli, U., Akens, M.K., Fellenberg, R.V.
and Bracher, V. (1998) The role of neutrophil
chemotactic cytokines in the pathogenesis of equine
chronic obstructive pulmonary disease (COPD). Vet.
Immunol. Immunopathol. 66, 53.
Gigue`re, S. and Prescott, J.F. (1999) Quantitation of
equine cytokine mRNA expression by reverse
transcription-competitive polymerase chain reaction.
Vet. Immunol Immunopathol 67, 1-15.
Gilger, B., Malok, E., Cutter, K., Stewart, T., Horohov,
D. and Allen, J. (1999) Characterisation of T-
lymphocytes in the anterior uvea of eyes with
chronic equine recurrent uveitis. Vet. Immunol.
Immunopathol. In Press.
Gilger, B.C., Malok, E., Stewart, T., Horohov, D.,
Ashton, P., Smith, T., Jaffe, G.J. and Allen, J.B.
(2000) Effect of an intravitreal cyclosporine implant
on experimental uveitis in horses. Vet. Immunol.
Graham, M.B., Braciale, V.L. and Braciale, T.J. (1994)
Influenza virus-specific CD4+ T helper type 2 T
lymphocytes do not promote recovery from
experimental virus infection. J. exp. Med. 180, 1273-
1282.
Grun, J.L. and Maurer, P.H. (1989) Different T helper cell
subsets elicited in mice utilising 2 different adjuvant
vehicles: the role of endogenous interleukin 1 in
proliferative responses. Cell Immunol. 121, 134-145.
Hannant, D., Mumford, J.A. and Jessett, D.M. (1988)
Duration of circulating antibody and immunity
following infection with equine influenza virus. Vet.
Rec. 122, 125-128.
Kato, H., Ohashi, T., Nakamura, N., Nishimura, Y.,
Watari, T., Goitsuka, R., Tsujimoto, H. and
Hasegawa, J. (1995) Molecular cloning of equine
interleukin-1 - and - cDNAs. Vet. Immunol.
Leutenegger, C.M., von Rechenberg, B., Huder, J.B.,
Zlinsky, K., Mislin, C., Akens, M.K., Auer, J. and
Lutz, H. (1999) Quantitative real-time PCR for
equine cytokine mRNA in non-decalcified bone
tissue embedded in methyl methacrylate. Calcif.
Tissue Int. 65, 378-383.
Lohoff, M., Gessner, A., Bogdan, C. and Rollinghoff, M.
(1998) The Th1/Th2 paradigm and experimental
murine leishmaniasis. Int. Arch. Allergy Immunol.
115, 191-202.
TABLE 2: Equine Th1 vs Th2 Paradigm
Protection Pathology
Th1 Equine influenza Equine recurrent
virus uveitis
Th2 Strongyles vulgaris SPAOPD
9
6,000,000
5,000,000
4,000,000
3,000,000
2,000,000
1,000,000
0
Infect Irrad Ribi None
IL-4
IL-5
IFN-
C
y
t
o
k
i
n
e

m
R
N
A

c
o
p
y

u
n
i
t
s
Fig 2: Cytokine mRNA expression
in PBMC from parasite-
challenged and control ponies.
Four parasite-free ponies were
immunised with either irradiated
L3 (Irrad) or somatic adult
antigens of S. vulgaris (Ribi)
(Monahan et al. 1995). Vaccinated
and naive ponies (Infect) were
then challenged with 500 S.
vulgaris larvae. Colonic lymph
node samples were collected from
the ponies at necropsy (16 days
post challenge) for mRNA analysis
(Swiderski et al. 1999a).
Unchallenged ponies (None) were
included as negative controls.
MacKay, R.J. and Socher, S.H. (1992) Anti-equine
tumour necrosis factor (TNF) activity of antisera
raised against human TNF- and peptide segments
of human TNF-. Am. J. vet. Res. 53, 921.
May, S.A., Hooke, R.E. and Lees, P. (1990) The
characterisation of equine interleukin-1. Vet.
Immunol. Immunopathol. 24, 169.
Monahan, C., Taylor, H., Chapman, M. and Klei, T.
(1995) Experimental immunisation of ponies with
Strongylus vulgaris radiation-attenuated larvae or
crude soluble somatic extracts from larvae or adult
stages. J. Parasitol. 80, 911.
Morris, D.D., Crowe, N., Moore, J.N. and Moldawer, L.
(1992) Endotoxin-induced production of interleukin
6 by equine peritoneal macrophages in vitro. Am. J.
vet. Res. 53, 1298.
Morris, E.A., McDonald, B.S., Webb, A.C. and
Rosenwasser, L.J. (1990) Identification of
interleukin-1 in equine osteoarthritic joint effusions.
Am. J. vet. Res. 51, 59.
Mosmann, T.R. and Coffman, R.L. (1989) TH1 and TH2
cells: different patterns of lymphokine secretion lead
to different functional properties. Ann. rev. Immunol.
7, 145.
Nicolson, L., Penha-Goncalves, M., Keanie, J., Logan,
N., Argyle, D. and Onions, D. (1999) Cloning and
sequencing of horse interleukin-12 and interleukin-
18 cDNAs. Immunogenetics 50, 94.
OGarra, A., Steinman, L. and Gijbels, K. (1997) CD4+
T-cell subsets in autoimmunity. Curr. Opin.
Immunol. 9, 872.
Romeike, A., Brugmann, M. and Drommer, W. (1998)
Immunohistochemical studies in equine recurrent
uveitis (ERU) Vet. Pathol. 35, 515.
Seahorn, T.L., Groves, M.G., Harrington, K.S. and
Beadle, R.E. (1996) Chronic obstructive pulmonary
disease in horses in Louisiana. J. Am. vet. med.
Assoc. 208, 248.
Schwink, K.L. (1992) Equine uveitis. Vet. clin. North.
Am. equine Pract. 8, 557.
Stott, M.L. and Osburn, B.I. (1988) Establishment of
equine T-lymphocyte cultures dependent on
recombinant human interleukin-2. Am. J. vet. Res
49, 553.
Swiderski, C.E., Klei, T.R., Folsom, R.W., Pourciau,
S.S., Chapman, A., Chapman, M.R., Moore, R.M.,
McClure, J.R., Taylor, H.W. and Horohov, D.W.
(1999a) Vaccination against Strongylus vulgaris in
ponies: comparison of the humoural and cytokine
responses of vaccinates and nonvaccinates. Adv. vet.
Med. 41, 389.
Swiderski, C.E., Klei, T., Pourciau, S., Chapman, M.R.,
Moore, R., McClure, J.R. and Horohov, D.W.
(1999b) T cell cytokine responses to Strongylus
vulgaris in infected and vaccinated ponies. Proc. 8th
int. Conf. equine inf. Dis. Eds: U. Wernery, J.F.
Wade, J.A. Mumford and O.-R. Kaaden. R&W
Publications (Newmarket) Ltd, pp 206-210.
Swiderski, C.E., Klei, T.R. and Horohov, D.W. (1999c)
Quantitative measurement of equine cytokine
mRNA expression by polymerase chain reaction
using target-specific standard curves. J. immunol.
Methods 222, 155.
Umetsu, D.T. and DeKruyff, R.H. (1997) Th1 and Th2
CD4+ cells in the pathogenesis of allergic diseases.
Proc. Soc. exp. Biol. Med. 215, 11.
Urban, J.F. Jr., Fayer, R., Sullivan, C., Goldhill, J., Shea-
Donohue, T., Madden, K., Morris, S. C., Katona, I.,
Gause, W., Ruff, M., Mansfield, L.S. and Finkelman,
F.D. (1996) Local TH1 and TH2 responses to
parasitic infection in the intestine: regulation by IFN-
and IL-4. Vet. Immunol. Immunopathol. 54, 337.
Immunology in 2001
10
RHODOCOCCUS EQUI: A PARADIGM FOR EQUINE
IMMUNITY TO INTRACELLULAR BACTERIA
S. Gigure
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida,
Gainesville, Florida 32610, USA
Rhodococcus equi, a Gram-positive facultative
intracellular pathogen, is one of the most
important causes of pneumonia in foals aged 15
months. Other less common clinical
manifestations of R. equi infections in foals
include ulcerative enterocolitis, colonic or
mesenteric lymphadenopathy, immune-mediated
synovitis and uveitis, osteomyelitis and septic
arthritis. R. equi is widespread in the environment
of stud farms. Unlike most environmental R. equi,
isolates from pneumonic foals typically contain an
8090 kb plasmid encoding a family of 7 closely
related virulence-associated proteins designated
VapA and VapC through VapH (Takai et al. 2000).
Plasmid-cured derivatives of virulent R. equi
strains lose their ability to replicate and survive in
macrophages, and fail to induce pneumonia in
foals, confirming the absolute necessity of the
large plasmid for the virulence of R. equi (Gigure
et al. 1999a). Cell wall mycolic acid-containing
glycolipids may also contribute to virulence of R.
equi. Strains with a longer carbon chain mycolic
are more virulent as determined by lethality and
granuloma formation in mice than those with
shorter chains (Gotoh et al. 1991). Other
unexplored candidates as virulence factors include
capsular polysaccharides as well as cholesterol
oxidase, choline phosphohydrolase and
phospholipase C exoenzymes (equi factors).
However, both capsule and exoenzymes are
produced by virulent as well as by avirulent
strains, suggesting that their contribution to
virulence, if any, is insignificant in comparison to
plasmid-mediated functions.
The ability of R. equi to persist in, and
eventually destroy, alveolar macrophages is the
basis of its pathogenicity. Intracellular persistence
correlates with the absence of phagosome-
lysosome fusion (Hietala and Ardans 1987a; Zink
and Yager 1987). Phagocytosis of R. equi by
equine macrophages is not associated with a
functional respiratory burst (Brumbaugh et al.
1990) and, at least in people, the L-arginine-NO
pathway is not required for intracellular killing of
this organism (Vullo et al. 1998). Optimal binding
of R. equi to mouse macrophages in vitro requires
complement and is mediated by Mac-1, a
leucocyte complement receptor type 3 (CR3,
CD11b/CD18) (Hondalus et al. 1993). Entry of
several microorganisms into macrophages after
adherence to complement receptors has been
shown to allow them to avoid the toxic
consequences of the oxidative burst (Bamler and
Heffron 1995). Opsonisation of R. equi with
specific antibody is associated with increased
phagosome-lysosome fusion and significantly
enhances killing of R. equi by equine macrophages
suggesting that the mechanism of cellular entry
can mediate the fate of the bacteria (Hietala and
Ardans 1987a). As opposed to macrophages,
neutrophils from foals and adult horses are fully
bactericidal and killing of R. equi is considerably
enhanced by specific opsonising antibody (Hietala
and Ardans 1987b; Martens et al. 1988; Takai et
al. 1985; Yager et al. 1987).
Immunity to R. equi pneumonia in foals
probably depends on both the antibody and cell-
mediated components of the immune system but
its exact basis remains to be determined. The age
of development of R. equi pneumonia coincides
with, and may in part be related to, the decline of
maternally-derived antibodies (Hietala et al.
1985). However, the strongest evidence for a role
of antibody in protection against R. equi is the
protective effect of passively transferred anti-R.
equi hyperimmune equine plasma (Madigan et al.
11
1991). The mechanisms by which hyperimmune
plasma confers protection are not completely
understood. The list of possible effector molecules
includes antibody and non-specific factors such as
fibronectin, complement components, collectins,
cytokines and acute phase proteins. Recent studies
have focused more specifically on the role of
antibody against plasmid-encoded virulence-
associated proteins (Vap). First, a monoclonal
antibody to VapA and serum from horses
immunised with partially purified VapA have
opsonising activity (Prescott et al. 1997).
Moreover, purified immunoglobulins obtained
from horses vaccinated with partially purified
VapA protected mice against intraperitoneal
challenge with virulent R. equi compared with
mice administered immunoglobulins from non-
immunised horses (Fernandez et al. 1997). More
recently, iv administration of purified
immunoglobulins obtained from horses
immunised with recombinant VapA and VapC to
foals was found to reduce the severity of
pneumonia following heavy experimental
challenge with R. equi (Hooper-McGrevy et al.
2001). In the same study, the degree of protection
conferred by purified anti-VapA and -VapC
immunoglobulins was similar to that provided by
hyperimmune plasma.
Because of the facultative intracellular nature
of R. equi, cell-mediated immune mechanisms are
thought to be of major importance in resistance.
Almost all knowledge of cell-mediated immunity
to R. equi infections comes from infection of mice.
Deficiencies in the complement component C5,
phagocytic cells and NK cells do not impair the
pulmonary clearance of virulent R. equi (Yager et
al. 1991). In contrast, functional T lymphocytes
are absolutely required for the clearance of
virulent (plasmid and VapA positive) R. equi in
mice (Kanaly et al. 1996; Madrame et al. 1997;
Ross et al. 1996). However, athymic nude mice
(lacking functional T lymphocytes) clear plasmid-
cured derivatives from their lungs within one week
of infection suggesting that, as opposed to virulent
organisms, clearance of avirulent plasmid-negative
strains in mice does not require functional
lymphocytes and depends mainly on innate
defence mechanisms (Madrame et al. 1997). The 2
major mechanisms by which T lymphocytes
mediate clearance of intracellular pathogens are
secretion of cytokines and direct cytotoxicity.
Although both CD4
+
(helper) and CD8
+
(cytotoxic) T cells contribute to host defence
against R. equi in mice, CD4
+
T lymphocytes play
the major role and are absolutely required for
complete pulmonary clearance (Ross et al. 1996;
Kanaly et al. 1993; Nordmann et al. 1992). The
mouse CD4
+
Th cells can be divided in 2 subsets
based on the cytokines they produce. The Th1
subset produces mainly IFN- and IL-2 and is
mainly responsible for macrophage activation and
cell-mediated immunity. The Th2 subset produces
mainly IL-4, IL-5 and IL-10 which mainly
promote humeral immunity. Studies in mice have
shown that a Th1 response is sufficient to effect
pulmonary clearance of R. equi whereas a Th2
response is detrimental (Kanaly et al. 1995, 1996).
How these findings in mice relate to the foal
remains to be determined. Analogy to human
immunodeficiency virus-related R. equi
pneumonia suggests either that foals are
immunocompromised in some way or that
infection with virulent R. equi alters immune
response in foals. The cytokine response of foals
infected with virulent and avirulent R. equi has
recently been investigated. Foals infected
intrabronchially with a virulence plasmid-
containing strain of R. equi showed marked
reduction in IFN- mRNA expression by bronchial
lymph node CD4
+
T lymphocytes compared to
CD4
+
T cells similarly isolated from foals infected
with an avirulent plasmid-cured derivative of the
same strain (Gigure et al. 1999b). In addition, IL-
10, a cytokine known to downregulate a Th1
response in other species, was only expressed in
the lungs of foals infected with the virulent strain
(Gigure et al. 1999b). These findings suggest that
virulent R. equi have an immunomodulating effect
important in the pathogenesis of infection.
REFERENCES
Bamler, A.J. and Heffron, F. (1995) Microbial
resistance to macrophage effector functions:
Strategies for evading microbicidal mechanisms and
scavenging nutrients within mononuclear
phagocytes. In: Virulence Mechanisms of Bacterial
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K.A. Brogden, F.C. Minion and M.J. Wannemuehler.
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Brumbaugh, G.W., Davis, L.E., Thurmon, J.C. and
Savage, D.C. (1990) Influence of Rhodococcus equi
on the respiratory burst of resident alveolar
macrophages from adult horses. Am. J. vet. Res. 51,
766-771.
Fernandez, A.S., Prescott, J.F. and Nicholson, V.M.
(1997) Protective effect against Rhodococcus equi
infection in mice of IgG purified from horses
Immunology in 2001
12
vaccinated with virulence associated protein
(VapA)-enriched antigens. Vet. Microbiol. 56, 187-
192.
Gigure, S., Hondalus, M.K., Yager, J.A., Darrah, P.,
Mosser, D.M. and Prescott, J.F. (1999a) Role of the
85-kilobase plasmid and plasmid-encoded
virulence-associated protein A in intracellular
survival and virulence of Rhodococcus equi. Infect.
Immun. 67, 3548-3557.
Gigure, S., Wilkie, B.N. and Prescott, J.F. (1999b)
Modulation of cytokine response of pneumonic foals
by virulent Rhodococcus equi. Infect. Immunol. 67,
5041-5047.
Gotoh, K., Mitsuyama, M., Imaizumi, S., Kawamura, I.
and Yano, I. (1991) Mycolic acid-containing
glycolipid as a possible virulence factor of
Rhodococcus equi for mice. Microbiol. Immunol. 35,
175-185.
Hietala, S.K., Ardans, A.A. and Sansome, A. (1985)
Detection of Corynebacterium equi-specific
antibody in horses by enzyme-linked
immunosorbent assay. Am. J. vet. Res. 46, 13-15.
Hietala, S.K. and Ardans, A.A. (1987a) Interaction of
Rhodococcus equi with phagocytic cells from
Rhodococcus equi-exposed and non-exposed foals.
Vet. Microbiol. 14, 279-284.
Hietala, S.K. and Ardans, A.A. (1987b) Neutrophil
phagocytic and serum opsonic response of the foal to
Corynebacterium equi. Vet. Immunol.
Hondalus, M.K., Diamond, M.S., Rosenthal, LA.,
Springer, T.A. and Mosser, D.M. (1993) The
intracellular bacterium Rhodococcus equi requires
Mac-1 to bind to mammalian cells. Infect. Immunol.
61, 2919-2929.
Hooper-McGrevy, K., Gigure, S., Wilkie, B.N. and
Prescott, J.F. (2001) Antibody to virulence-
associated proteins A and C in protection against
Rhodococcus equi pneumonia in foals. Am. J. vet.
Res. 68, 1307-1313.
Kanaly, S.T., Hines, S.A. and Palmer, G.H. (1993) Failure
of pulmonary clearance of Rhodococcus equi
infection in CD4
+
T-lymphocyte-deficient transgenic
mice. Infect. Immunol. 61, 4929-4932.
Kanaly, S.T., Hines, S.A. and Palmer, G.H. (1995)
Cytokine modulation alters pulmonary clearance of
Rhodococcus equi and development of
granulomatous pneumonia. Infect. Immunol. 63,
3037-3041.
Kanaly, S.T., Hines, S.A. and Palmer, G.H. (1996)
Transfer of a CD4
+
Th1 cell line to nude mice
effects clearance of Rhodococcus equi from the
lung. Infect. Immunol. 64,1126-1132.
Madarame, H., Takai, S., Matsumoto, C., Minamiyama,
K., Sasaki, Y., Tsubaki, S., Hasegawa, Y. and
Nakane, A. (1997) Virulent and avirulent
Rhodococcus equi infection in T-cell deficient
athymic nude mice: pathologic, bacteriologic and
immunologic responses. FEMS Immunol. Med.
Microbiol. 17, 251-262.
Madigan, J.E., Hietala, S. and Muller, N. (1991)
Protection against naturally acquired Rhodococcus
equi pneumonia in foals by administration of
hyperimmune plasma. J. Reprod. Fert. Suppl 44,
571-578.
Martens, R.J., Martens, J.G. and Renshaw, H.W. (1988)
Rhodococcus (Corynebacterium) equi: bactericidal
capacity of neutrophils from neonatal and adult
horses. Am. J. vet. Res. 49, 295-299.
Nordmann, P., Ronco, E. and Nanciel, C. (1992) Role of
T-lymphocyte subsets in Rhodococcus equi
infection. Infect. Immun. 60, 2748-2752.
Prescott, J.F., Nicholson, V.M., Patterson, M.C., Zandona
Meleiro M.C., Caterino de Araujo, Yager, J.A. and
Holmes, M.A. (1997) Use of Rhodococcus equi
virulence-associated protein for immunisation of
foals against R. equi pneumonia. Am. J. vet. Res. 58,
356-359.
Ross, T.L., Balson, G.A., Miners, J.S., Smith, G.D.,
Shewen, P.E., Prescott, J.F. and Yager, J.A. (1996)
Role of CD4
+
, CD8
+
and double negative T-cells in
the protection of SCID/beige mice against
respiratory challenge with Rhodococcus equi. Can.
J. vet. Res. 60, 186-192.
Takai, S., Morozumi, Y., Higaskiyama, S. and Tsubaki,
S. (1985) Nitroblue tetrazolium reduction by
neutrophils of newborn foals, adult horses, and a foal
infected with Rhodococcus (Corynebacterium) equi.
Jpn. J. vet. Sci. 48, 405-408.
Takai, S., Hines, S.A., Sekizaki, T., Nicholson, V.M.,
Alperin, D.A., Osaki, M., Takamatsu, D., Nakamura,
M., Suzuki, K., Ogino, N., Kakuda, T., Dan, H. and
Prescott, J.F. (2000) DNA sequence and comparison
of virulence plasmids from Rhodococcus equi
ATCC33701 and 103. Infect. Immun. 68, 6840-6847.
Vullo, V., Mastroianni, C.M., Lichtner, M., Mengoni, F.,
D'Agostino, C., Forcina, G., Corpolongo, A. and
Delia, S. (1998) Rhodococcus equi infection of
monocytes/macrophages from human immuno-
deficiency (HIV)-infected patients and healthy
individuals: evaluation of intracellular killing and
nitric oxide production. FEMS Immunol. med.
Microbiol. 21, 11-17.
Yager, J.A., Duder, C.K., Prescott, J.F. and Zink, M.C.
(1987) The interaction of Rhodococcus equi and foal
neutrophils in vitro. Vet. Microbiol. 14, 287-294.
Yager, J.A., Prescott, C.A., Kramar, D.P., Hannah, H.,
Balson, G.A. and Croy, B.A. (1991) The effect of
experiemental infection with Rhodococcus equi on
immunodeficient mice. Vet. Microbiol. 28, 363-376.
Zink, M.C. and Yager, J.A. (1987) Electron microscopic
investigation of intracellular events after ingestion of
Rhodococcus equi by foal alveolar macrophages.
Vet. Microbiol. 14, 295-305.
13
EQUINE HERPESVIRUS-1: IMMUNITY IN LYTIC
AND LATENT INFECTIONS
J. Slater
Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge
CB3 0ES, UK
SUMMARY
Understanding of the pathogenesis of lytic and, to a
lesser extent, latent EHV-1 infections has improved
dramatically. Understanding of the immune
response is sufficient to identify that circulating
antibody responses alone do not provide a correlate
of protective immunity. Preliminary evidence
suggests that CTL responses may provide a
correlate and CTL responses in the lamina propria
and circulation thus require further study. Mucosal
immunity is a second area of EHV-1 immunity that
requires further investigation.
INTRODUCTION
Equine herpesvirus-1 (EHV-1) establishes invasive
infections which result in viraemic dissemination
of virus from the respiratory tract to organs
throughout the body, notably the uterus and central
nervous system (CNS). The virus has a complex
lifecycle which involves infection of multiple cell
types including epithelial cells, endothelial cells,
mononuclear leucocytes and neurones. In addition
there are 2 distinct cycles of infection: lytic
(primary or acute) and latent infection. During
lytic infection there is virus replication in infected
cells with extensive virus gene transcription and
expression of virus antigens on the surface of the
infected cell. Lytic infection results in destruction
of the infected cell, subsequent release of progeny
virions from the cell, infection of adjacent cells
and virus shedding from the horse. In contrast,
during the latent infection cycle, there is no virus
replication; gene transcription is restricted to a
single region of the genome and there is no
translation of this mRNA. Virus antigens are thus
not expressed on the cell surface, the latently-
infected cell is not destroyed and progeny virions
are not released. During latency, the virus thus
resides within latently infected cells in a near-
quiescent state. Periodically, however, latent virus
undergoes reactivation which may result
ultimately in the establishment, once more, of lytic
infection in respiratory epithelium, shedding of
infectious virus from the respiratory tract and
viraemia. The lifecycle of EHV-1 thus presents a
major challenge to the immune system because
both prevention and termination of infection
require an integrated, multi-component immune
response to control lytic and latent infection. In
recent years, great advances have been made in the
understanding of the immune response to EHV-1
and the role of the immune response in
pathogenesis. Nonetheless, several key virological
and immunological aspects of EHV-1
pathogenesis require further study. The aims of
this presentation are to 1) review the current state
of knowledge of the immune response to EHV-1 in
lytic and latent infections and 2) highlight areas of
the immune response that require further study and
to consider the tools and techniques currently
available to study these areas.
REVIEW OF THE IMMUNE RESPONSE TO
EHV-1
Lytic infection
During lytic infection virus gene transcription, and
therefore virus antigen expression by infected
cells, is tightly regulated into 3 sequential phases:
immediate-early, early and late with each phase
up-regulating transcription of later phase genes as
well as down-regulating earlier phase genes. The
first virus gene to be transcribed and the initiator
of the lytic infection cycle is the single immediate-
early gene, ORF 64. Certain virus antigens have
Immunology in 2001
14
already been identified as key immunological
targets eg ORFs 64 and 5 are potent stimulators of
cytotoxic T lymphocyte (CTL) responses and virus
glycoproteins gB, gC and gD are targets for
neutralising antibody.
The immunological events following initial
exposure to EHV-1 can be summarised as follows:
1) At the mucosal surface humoural and cell
mediated responses are likely to occur. The
mucosal humoural immune response has not been
fully characterised although it is likely that
neutralising Ig appears in nasal secretions. CD8
+
T lymphocytes are released into the
bronchoalveolar space 23 weeks post infection,
although the cytotoxic activity of these cells has not
been investigated; 2) In the lamina propria, EHV-1
infects trafficking mononuclear cells which
subsequently appear in drainage lymph nodes and
the circulation resulting in viraemia. Although the
identity of infected mononuclear cells in the lamina
propria has not been identified, the viraemic
population of cells are predominantly CD8
+
T
lymphocytes. In the circulation, and presumably in
the lamina propria as well, infected lymphocytes
express virus antigens only transiently and, from
around 3 weeks post infection, virus exists in
circulating lymphocytes in an antigenically silent,
latent form. Endothelial cells are also infected in
the lamina propria and these probably act as
another route by which trafficking lymphocytes are
infected. The humoural and cellular immune
responses in the lamina propria have not, as yet,
been fully elucidated although this is one area of
current EHV-1 research. Of particular importance,
the virus antigens that drive CTL responses in the
lamina propria have not been elucidated. This
information is likely to provide the key to
protective immunity. Related to this, the interaction
between dendritic cells and EHV-1 has not yet been
studied in detail; 3) In the circulation, high titres of
circulating antibody develop, directed mainly
against virus glycoproteins gB, gC and gD. There
is a short-lived CF antibody response and a more
durable neutralising antibody response. However,
neutralising antibody titres do not correlate with
protection and do not shorten the duration of
viraemia, although high titres of circulating
antibody may correlate with reduced duration of
virus shedding from the nasopharynx. Circulating
CD8
+
class 1 restricted CTL and CTL precursor
(memory) cells also appear in the circulation.
CTLp cells persist in the circulation for months
after infection and low frequencies of CTL
memory cells may be associated with increased
susceptibility to infection; 4) In other organs,
notably the uterus and CNS, virus is transferred
from trafficking lymphocytes to endothelial cells
inducing thrombosis, ischaemia, possibly virus
translocation into the foetus and, rarely, neurones
of the CNS. The immune responses during this
phase of infection have, for reasons of practical
difficulty, not been studied in detail. it is known
that endothelial cells in the uterus express virus
antigens, making these candidate targets for
clearance by CTL. Whether virus transfer from
lymphocytes to endothelial cells occurs by
extracellular release of virions or by fusion of
infected cells is unknown. Thus, the role of
antibody and CTL responses in controlling this
phase of infection is not understood. It is possibile
that CTL responses targeting infected endothelial
cells could result in immunopathological damage
to the uterus and CNS and this is the basis for the
debate about the clinical use of corticosteroids in
the management of paresis.
Latent infection
The immune control of latency and reactivation is
less well understood. It is likely that the lytic and
latent infection cycles are separate and the latency
is established in the early stage of infection in
parallel to the lytic infection cycle. In other words,
the outcome of infection of a cell is either lytic
infection and death, or latent infection and
survival. It is not known what role, if any, the
immune response plays in this process.
It is well established that latent infections are
established in mononuclear cells, mainly CD8
+
T
lymphocytes, in both drainage lymph nodes and in
the circulation; and that latency in these cells
persists for lengthy periods, probably for the life of
the horse. Latent infections are also established in
trigeminal ganglionic neurones. Latently infected
cells do not express virus antigens and are not
susceptible to immune surveillance. Thus,
systemic virus neutralising antibody (VNA) titres
and presumably the frequency of CTL memory
cells gradually declines during latency.
The major role of the immune system probably
lies in controlling virological events during
reactivation, although this area has not received
detailed study for EHV-1 infections. It is known
that reactivation events are marked by increases in
circulating VNA titres, together with shedding of
infectious virus from the respiratory tract and
15
viraemia. Immune responses probably act not by
determining whether virus reactivates from
lymphocytes or trigeminal ganglionic neurones but
by limiting the extent of replication of virus once
it has reactivated. Thus, humoural immune
responses act at the respiratory epithelial surface
mucosa by neutralising free virions via VNA and
cellular immune responses eliminate infected cells
via CTL responses. In the circulation, reactivating
lymphocytes may transiently express virus
antigens, rendering them susceptible to clearance
by CTL.
The mechanism of transfer of reactivating
virus from circulating latently-infected
lymphocytes to the respiratory epithelium and to
uterine and CNS endothelium is not understood.
The opportunity for the immune response to
intervene in this process is thus also not
understood.
AREAS THAT REQUIRE FURTHER STUDY
There is general consensus that circulating
antibody responses (CF and VNA) are not the sole
determinants of immunity to EHV-1. There is no
correlation between either vaccine-induced or
natural infection-induced circulating antibody
levels and protection from infection or reinfection;
and viraemia occurs in the face of neutralising
antibody. CTL response is therefore the area of
immune response that urgently requires further
study.
There are 4 key questions
1. Which virus antigens are responsible for
driving CTL responses in the lamina propria
and in the circulation (including the
interactions between EHV-1 and dendritic
cells)?
2. What is the role of CTL in the termination and
prevention of EHV-1 induced viraemia?
3. Can the measurement of CTL provide a
quantitative measure of protection or
susceptibility to infection?
4. Do CTL responses provide an immunological
means of preventing transfer of virus from
lymphocytes to endothelial and epithelial
cells?
Two further important immunological aspects
are worthy of study
1. Mucosal antibody responses to infection and
correlation between these and protection.
2. The role of mucosal and systemic antibody
and CTL responses in controlling latency and
reactivation.
Two key techniques have been developed that
provide an opportunity for more accurate dissection
of the cellular immune responses to EHV-1: 1)
estimation of CTLp frequency by limiting dilution
analysis; and 2) semi-quantitative and quantitative
measurement of equine cytokine mRNA. However,
although these techniques are being applied both to
peripheral blood samples and to tissues collected
post mortem, it remains to be seen whether
endoscopic biopsies will provide adequate tissue
samples for the study of lamina propria and other
tissue CTL responses in the live horse.
ACKNOWLEDGEMENTS
The ideas presented in this presentation are the
result of collaborative projects with Drs. Julia
Kydd and Ken Smith of the Animal Health Trust,
Newmarket, UK and Dr George Allen of the
University of Kentucky, USA. Grant support for
the work discussed here was provided by the
Equine Virology Research Foundation, the
Horserace Betting Levy Board, The Wellcome
Trust, The BBSRC and the Grayson Jockey Club
Research Foundation.
Immunology in 2001
16
STRONGYLES LARGE AND SMALL: IMMUNITY
T. R. Klei
Department of Pathobiological Sciences, School of Veterinary Medicine and Department of
Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State University,
It is generally accepted that horses acquire an
immunity to nematode parasite infections. Some of
these responses such as those to Strongyloides
westeri and Parascaris equorum occur rapidly in
foals, are complete and long lasting. Most others are
not. Detailed studies on acquired resistance to
nematode infections of horses is, however, limited
and pertains almost exclusively to strongyle
infections. Based on their biology it is not surprising
that the acquisition and character of immunity to
infections of the large strongyles, specifically
Strongylus vulgaris and the small strongyles or
cyathostomes differ markedly. What is known about
both will be summarised briefly (Klei 1992, 2000).
Although the large strongyles of equids are the
most pathogenic nematodes found in the horse they
are controlled readily by the use of the macrocyclic
lactone anthelmintics, ivermectin and moxidectin.
On closed, well managed, horse farms these
parasites are no longer considered to pose a
significant health problem. Nonetheless, infections
of parasite free ponies with known numbers of S.
vulgaris infective larvae remains a useful model to
study equine immune responses to nematode
parasites and the most detailed information on
equine immunity to nematodes has come from this
model system (Klei 1992, 2000). The concomitant
immunity to S. vulgaris where both adult and larval
stages of the parasite live unaffected within the
lumen of the caecum and mesenteric vasculature
while incoming infective third stage larvae (L3) are
killed, is acquired early after initial exposure to L3.
In this system a strong acquired immunity can be
induced by vaccination with 2 doses of orally
administered irradiated L3. This vaccination
provides >90% protection against challenge
infection, prevents arterial lesions and protects
completely from clinical signs associated with the
early arterial migrations of these parasites. This
protection has been associated in secondary
infections with an anamnestic eosinophilia which
does not occur in primary infections until the
parasites are established. In vitro culture of L3 with
immune serum, or Ig from immune serum,
mediates the adherence of cells to the surface of the
L3. This adherence immobilises and kills the larvae
if the cell mixtures contain eosinophils from
eosinophilic horses. Neutrophils or macrophages
do not mediate this reaction. Eosinphils from
eosinophilic ponies infected with S. vulgaris have
been demonstrated to possess increased numbers of
Fc and complement receptors and be hypodense,
all of which are indicators of an activated state.
Only these activated eosinophils, and not normal
eosinophils, are active in the in vitro ADCC of L3.
The antibodies which mediate this response are
species- and stage-specific. Although these data
suggest a role for antibody in the protective
immunity seen, passive transfer of hyperimmune
serum does not protect from challenge infection.
However, this passive transfer does reduce the
severity of intravascular lesions while promoting a
marked and significant perivascular inflammation
which is predominately eosinophils. An
anamnestic eosinophilia was also seen in the ponies
which received hyperimmune serum but not
controls. These observations indicate that a cellular
immune response was central to this protective
immunity. Early studies demonstrated that
lymphocytes from immune ponies cultured with
soluble somatic antigen of S. vulagaris produced a
factor(s) which was chemotactic for eosinophils in
vitro. The stimulation of factor production was
species specific in that similar antigens produced
from S. edentatus did not induce chemotatic factor
induction in immune lymphocyte cultures.
Interestingly, field studies using the irradiated L3
vaccine also showed a species specificity when
17
vaccinated ponies were challenged with mixed
species of Strongylus. The character of this T-cell
response has been partially defined in this model by
measuring the in situ and in vitro expression of
cytokine genes using quantitative RT-PCR
measurements of cytokine mRNA in peripheral
blood mononuclear cells (PBMC) and colonic
lymphocytes. These protective immune responses
coincide with the induction of a marked increase in
gene expression of IL-5 but not IL-4 or IFN in
PBMC and colonic lymph nodes (Swiderski et al.
1999). Clear differences have yet to be seen in the
compartmentalisation of this response to the large
intestine. Earlier studies have shown that
vaccination with somatic antigens of S. vulgaris in
RIBI adjuvant induced a response to challenge
infection which was more severe and characterised
by an antibody response in the absence of an
anamnestic eosinophilia, and a dominance of IFN
mRNA. Attempts have been made to alter the
effectiveness of the irradiated L3 vaccination by
superimposing the RIBI vaccination, and its
associated Th-1 IFN response, upon the
irradiated L3 vaccination. Reductions were seen in
eosinophilia and IL-5 mRNA by pre-treatment with
the RIBI immunisation which induced elevated
levels of IFN mRNA. However, these effects
were insufficient to alter the protective immune
responses to challenge infection. Data from these
later experiments are still being collected and
analysed. Nonetheless, alternative approaches will
be necessary to define the requirements for
eosinophils and associated Th2 responses in the
protective immunity seen in this system.
Unlike the large strongyles the cyathostomes
continue to be found in horses on well managed
farms and have developed resistance to all classes
of anthelmintic except the macrocyclic lactones.
Concern is increasing over the potential
development of macrocyclic lactone resistant
populations of cyathostomes. Data on field
observations suggest that horses acquire immunity
to cyathostome infections with age (Klei and
Chapman 1999). When examining this resistance
using strongyle faecal egg counts, it is clear and
not surprising that horses respond to strongyle
nematode infections like other hosts. The
frequencies of EPGs are evenly dispersed within
the yearling population but over dispersed in the
mature horse population. In this situation very few
mature animals are infected with large numbers of
parasites. It is likely this difference in acquired
resistance is genetically regulated. Examination of
adult worm recoveries from ponies selected
because they were infected, ie they were examined
post mortem as controls in anthelminthic efficacy
trials, indicates that although this selected older
population had worm numbers similar to those of
younger individuals, the EPG were significantly
lower than in the young animals. This suggests that
some immune response affects the fecundity of the
adult worms without affecting its survival. In
general, the protective immune response against
cyathostomes is slow to develop and incomplete.
Evidence from experimental infections is more
convincing and indicates that resistance is
acquired with exposure and is directed against all
stages of the parasite life cycle. Some immunity
against developing larval stages in the mucosa,
however, appears to require less exposure and is
acquired early. Protection against invasion or
establishment of the early L3 is only seen in older
chronically infected animals. Infection with L3
induces some non-specific events which cause the
expulsion of existing lumen dwelling adults. This
self cure like phenomenon is not species-
specific. Antibody responses to somatic extracts of
parasites, not surprisingly, do not correlate with
protective immunity. Serendipitous observations
have suggested that an increase in IL-4 expression
is associated with the spontaneous expulsion of
adult parasites. However, this has yet to be
demonstrated in controlled experiments. Further
examination of this experimental model is required
to define the immune response to these parasites.
REFERENCES
Klei, T.R. (2000) Equine immunity to parasites. Vet. Clin.
North Am. Equine Pract. 16, 69-78.
Klei, T.R. (1992) Recent observations on the
epidemiology, pathogenesis and immunology of
equine helminth infections. In: Proceedings of the
Sixth International Conference on Equine Infectious
Diseases. Eds: W. Plowright, P.D. Rossdale & J.F.
Wade. R.&W Publications (Newmarket) Ltd UK. pp
129-137.
Klei, T.R. and Chapman, M.R. (1999) Immunity in
equine cyathostome infections. Vet. Parasitol. 86,
191-202.
Swiderski, C.E., Klei, T.R., Folsom, R.W., Pourciau, S.S.,
Chapman, A., Chapman, M.R., Moore, R.M.,
McClure, J.R., Taylor, H.W. and Horohov, D.W.
(1999) Vaccination against Strongylus vulgaris in
ponies: Comparison of the humoral and cytokine
responses of vaccinates and nonvaccinates. In:
Advances in Veterinary Medicine Veterinary
Vaccines and Diagnostics. Ed: R.D. Schultz, Vol. 41,
Academic Press, New York, pp 389-404.
Immunology in 2001
18
MUCOSAL IMMUNITY: AN OPPORTUNITY
TO PRIME WITHOUT PREJUDICE
D. Hannant
Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK
By far the majority of immunological effort is
focused on both innate and adaptive immune
responses that occur at sites of infection within the
respiratory, gastrointestinal and other mucosal
surfaces (McGhee and Kiyono 1993). These
mucosal reactions can occur independently of
systemic immunity or they may act in concert.
Thus, pathogen-specific antibody detected at
mucosal surfaces can result from local secretion by
recruited antibody secreting cells (ASCs) or
transudation of antibody from plasma. Typically,
after initial stimulation of lymphocytes at the site
of infection (the induction site), lymphocyte
activation at local lymph nodes occurs. These
activated cells (including ASCs) enter the blood
system and return to the mucosal sites where they
secrete antibody (the effector site). Because there
is linkage of immune responses at different
mucosal sites, antibody secretion may be detected
at both the site of initial infection and at other
mucosal sites. For example, stimulation of the gut
immune system may result in production and
detection of specific antibody in the trachea
(Mestecky et al. 1994).
Pathogen-specific and other antibodies,
particularly of the IgA isotype, are found
commonly in secretions of the upper respiratory
tract and gut of normal healthy and diseased
animals. The predominance of these antibodies
results from a combination of events including the
high rate of IgA isotype switching in ASCs, their
selective localisation and proliferation at mucosal
effector sites (Husband et al. 1999). Because of the
relatively short half-life of cells, the continuous
supply of antibodies at these sites is achieved by
their constant replacement. The regulation of this
process is not only by mucosal CD4+ lymphocytes
in the sub-mucosae but, significantly, by mucosal
epithelial cells. These produce many cytokines
including those that are critical for maturation of
cells and IgA secretion such as TGF- and IL-6, as
well as IL-2 and IL-10 (Ehrhardt et al. 1992; Salvi
and Holgate 1999).
The histological and functional features of the
equine mucosal immune system were first
described by Mair et al. (1987a) and these showed
many similarities with other species. As with other
species (Neutra and Kraehenbuhl 1992), the
equine microfold (M) cell is a critical component
in the cascade from antigen deposition at mucosal
surfaces to transepithelial transport and
development of mucosal immunity. Antigens are
transported into mucosal lymphoid tissues by M
cells, which are only present in the follicle-
associated epithelium overlying organised
lymphoid tissue in the nasal and oral cavities as
well as the intestine and bronchi.
Several studies have demonstrated
immunoglobulins in the respiratory secretions of
normal horses (Mair et al. 1987b) and virus-
specific IgA and IgG antibodies in the upper
respiratory tract of horses after influenza infection
(Hannant et al. 1989). Further support for the
function of a mucosal immune system in the horse
has come from the demonstration of traffic of
virus-specific ASCs in the blood (en route from
induction site to effector sites) after virus infection
or intranasal vaccination (Hannant et al. 1996).
The detection of virus-specific antibody in nasal
wash samples 23 days after the transient
appearance of ASCs in the blood, confirmed there
was a locally-recirculating mucosal immune
system in the horse (Hannant et al. 1999).
Exploitation of this knowledge in horses has
occurred by experimental studies using inactivated
virus vaccines given intranasally (with cholera
toxin B chain, CTB, or other adjuvants) or
mucosal application of virus gene products and/or
19
DNA (Lunn et al. 1999). CTB is an efficient
mucosal adjuvant because not only does it
stimulate IgA class-switching, but it upregulates
TGF- activity at mucosal surfaces, which is
essential for IgA-secreting B cell maturation and
expansion (Kim et al. 1998).
This work has opened many possibilities for
the rational design of improved vaccines for
equine respiratory pathogens. One potential
application derives from the observations that in
other species, the mucosal immune system of
neonates may be primed under cover of maternal
antibody. This may offer a route to overcome the
well-known interference of systemic
immunological priming in foals.
There is great potential now for mucosal
vaccination of horses with inactivated antigens or
DNA of pathogens in combination with exogenous
cytokines. More than one vaccine manufacturer is
investigating intranasal presentation of vaccine
antigens in horses as a route to boost mucosal
immunity. It is to be hoped that this approach will
be exploited further to take the best advantage
from stimulation of this important immune
mechanism.
REFERENCES
Ehrhardt, R.O., Stober, W. and Harriman, G.R. (1992)
Effect of transforming growth factor (TGF)- 1 on
IgA isotype expression. TGF- 1 induces a small
increase in sIgA+ B cells regardless of the method of
B cell activation. J. Immunol. 148, 3830-3836.
Hannant, D., Jessett, D.M., ONeill, T. and Mumford,
J.A. (1989) Antibody isotype responses in the serum
and respiratory tract to primary and secondary
infections with equine influenza virus (H3N8). Vet.
Microbiol. 19, 293-303.
Hannant, D., Easman, R.L., Evers, E. and Mumford, J.A.
(1996) Mucosal vaccination with inactivated
influenza virus stimulates neutralising antibody in
the nasopharynx of horses. Vet. Immunol.
Immunopathol. 54, 205.
Hannant, D., Easman, R.L. and Mumford, J.A. (1999)
Equine mucosal immune system: intranasal
vaccination with inactivated equine influenza virus
protects from infection. Proc. 8th int. Conf. equine
inf. Dis. Eds: U. Wernery, J.F. Wade, J.A. Mumford
and O.-R. Kaaden. R&W Publications (Newmarket)
Ltd, pp 50-56.
Husband, A.J., Bao, S. and Beagley, K.W. (1999)
Analysis of the mucosal microenvironment: factors
determining successful responses to mucosal
vaccines. Vet. Immunol. Immunopathol. 72, 135-142.
Kim, P.H., Eckmann, L., Lee, W.J., Han, W. and
Kagnoff, M.F. (1998) Cholera toxin and cholera
toxin B subunit induce IgA switching through the
action of TGF- 1. J. Immunol. 160, 1198-1203.
Lunn, D.P., Soboll, G., Schram, B.R., Quass, J.,
McGregor, M.W., Mackiln, M.D., McCabe, D.E.,
Swain, W.F. and Olsen C.W. (1999) Antibody
responses to DNA vaccination of horses using the
influenza virus hemagglutinin gene. Vaccine 17,
2245-2258.
McGhee, J.R. and Kiyono, H. (1993) New perspectives
in vaccine development: mucosal immunity to
infections. Infect. Agents and Disease 2, 55-73.
Mair, T.S., Batten, E.H., Stokes, C.R. and Bourne, F.J.
(1987a) The histological features of the immune
system of the equine respiratory tract. J. comp.
Pathol. 97, 575-586.
Mair, T.S., Batten, E.H., Stokes, C.R. and Bourne, F.J.
(1987b) Quantification of immunoglobulins in
respiratory tract secretions of the horse. Vet.
Immunol. Immunopathol. 14, 187-203.
Mestecky, J., Abraham, R. and Ogra, P.L. (1994)
Common mucosal immune system and strategies for
the development of vaccines effective at the mucosal
surfaces. In: Handbook of Mucosal Immunology
Eds: P.L. Ogra, J. Mestecky, M.E. Lamm, W.
Strober, J.R. McGhee and J. Bienenstock. Academic
Press, San Diego, California, USA, 357-372.
Neutra, M.R. and Kraehenbuhl, J.-P. (1992)
Transepithelial transport and mucosal defence I: the
role of M cells. Trends cell Biol. 2, 134-138.
Salvi, S. and Holgate, S.T. (1999) Could airway
epithelium play an important role in mucosal
immunoglobulin A production? Clin. exp. Allergy
29, 1597-1605.
Immunology in 2001
20
MUCOSAL ANTIGEN DELIVERY IN HORSES
J. F. Timoney and A. S. Sheoran
Gluck Equine Research Centre, University of Kentucky, Lexington, Kentucky 40546-0099, USA
Inductive sites in the nasopharynx are an attractive
target for new generation vaccines because of their
accessibility and absence of chemical barriers such
as acidity and hydrolytic enzymes which might
degrade immunogens administered per os.
However, an intranasal vaccine must be presented
in a manner that will circumvent mucociliary
clearance in the nasopharynx and resist the
mechanical scouring of the lingual and palatine
tonsillar surfaces that accompanies swallowing.
The vaccine must not be affected by naturally
occurring polyreactive IgA, must not induce
tolerance and should induce a memory B and T
lymphocyte response. Intranasal delivery systems
evaluated in the horse include microparticle
encapsulation, a mucoadhesive carrier, a cholera
toxin chimera and an avirulent salmonella.
MICROPARTICLE ENCAPSULATION
Microencapsulation involves coating of antigen
with a biodegradable polymer such as poly DL-
lactide-co-glycolide so that, after antigen release
in associated lymphoid tissue, mucosal and
systemic antibodies are elicited. However, ponies
immunised with 250 g of an immunogenic
peptide (SeMF3) of the SeM protein of S. equi on
Days 0, 7 and 42 made no detectable serum
antibody. SeM specific mucosal IgA responses
were detected in 2 vaccinates on Day 21 and in all
3 on Day 49. The absence of a systemic response
may have been due to failure of release of antigen
from the mucosal compartment.
MUCOADHESIVE COMPOUNDS
Bioadhesive polymers such as sodium alginate
have been used for mucosal application of killed
influenza vaccine to elicit specific mucosal and
systemic antibody responses. Non-polymeric
(food grade) sucrose acetate isobutyrate (SAIB) is
a highly viscous excipient which becomes much
less viscous in appropriate solvents including
ethanol in which it can be aerosolised readily.
After deposition on the nasopharyngeal mucosa,
the solvent evaporates and the SAIB and dissolved
antigens form a sticky film which degrades very
slowly. Adult Thoroughbred mares were
inoculated intranasally with 200 mg SeMF3 in 2
ml 75:25 SAIB-ethanol solution on Day 0 and
again 28 days later. Control mares were given 200
mg SeMF3 alone. Of the mares vaccinated with
SeMF3-SAIB 91% had made SeM specific serum
and mucosal IgA by Day 42. Control mares made
only short-lived mucosal and no serum responses.
Interestingly, serum and mucosal antibodies of the
same mares showed different patterns of reactivity
with linear epitopes of SeM consistent with the
conclusion that SAIB was effective in delivery of
SeMF3 to both the mucosal and systemic immune
compartments.
CHOLERA TOXIN-SEMF3 CHIMERAS
Cholera toxin (CT) is a potent mucosal
immunogen and adjuvant. The pentameric B
(CTB) subunit binds to GM1 gangliosides on
epithelial cells and M cells thereby enhancing its
own uptake by mucosal associated lymphoid
tissue. Ponies inoculated intranasally with a CT-
SeMF3 chimera containing the entire ctB gene, the
nontoxic A2 region and SeMF3 made strong CTB
and SeMF3 specific serum IgGb responses after
the first immunisation but no subsequent serum
antibody responses. In contrast, specific mucosal
IgA responses were boostable but varied in time of
onset for each pony and did not attain the
amplitude observed for convalescent responses.
21
The failure to stimulate anamnestic serum
antibody responses is unexplained and is a serious
limitation of the CT-chimera approach.
AVIRULENT SALMONELLA TYPHIMURIUM
MGN 707
Avirulent mutants of S. typhimurium are attractive
mucosal vaccine delivery systems because of their
safety and ability to stimulate both the mucosal
and systemic immune compartments. MGN 707, a
cya crp mutant of an equine isolate of S.
typhimurium, genetically engineered for
temperature regulated expression of SeMF3, was
inoculated intranasally into ponies on Days 0, 35,
56 and 161. Strong SeM specific serum responses
occurred in all ponies and delayed but strong
mucosal IgA responses were noted in 4 of 6
ponies. Induction of mucosal antibody responses
required induction of SeMF3 expression prior to
administration. Serum and mucosal responses to
salmonella antigens and to SeMF3 were boostable
suggesting that pre-existing antibody does not
cause immune exclusion.
Immunology in 2001
22
REGULATION OF MUCOSAL IMMUNE RESPONSES
G. Soboll, D. W. Horohov*, C. W. Olsen and D. P. Lunn
School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin; * Louisiana State
University, Baton Rouge, Louisiana, USA
Equine influenza is endemic in Europe and the
USA and remains one of the most common
infectious diseases in the horse. However, current
vaccination programmes are largely ineffective
(Mumford 1992; Morley et al. 1999) Nelson et al.
(1998) showed that inactivated vaccines offered no
protection 3 months after vaccination and induced
non-protective IgGT antibody isotype responses.
In contrast, natural infection is associated with
serum IgGa and IgGb in addition to mucosal IgA,
and protects from re-infection for up to one year.
Lunn et al. (1999) showed that DNA vaccination
with the equine influenza hemagglutinin gene
protects horses from homologous challenge
infection and induces appropriate IgG isotype
responses. However, this approach failed to induce
a mucosal IgA response.
An interesting observation made in an initial
DNA vaccination experiment (Lunn et al. 1999)
was that, in the absence of a mucosal IgA
response, the production of mucosal IgGb appears
to be critical for protection. Although appropriate
IgG responses are often sufficient for protection
against influenza, induction of a mucosal IgA
response remains important and may improve
protection in terms of heterotypic immunity and
better control of viral shedding. An attempt was
made to elicit mucosal IgA production by co-
administration of IL-6 DNA and hemagglutinin
DNA (HA DNA). This experiment failed to switch
the immune response at the mucosal level but did
indeed switch the immune response at the level of
serum IgG responses, as indicated by up-
regulation of serum IgG(T) and down-regulation
of serum IgGb responses (Soboll et al. 1999).
For induction of mucosal IgA production it is
critical to stimulate the mucosal associated
lymphoid tissue (MALT) directly (Underdown and
Mestecky 1994; McGhee and Kiyone 1992).
Following a decision to change vaccination
strategy, animals were primed with an intranasally
administered vaccine. Because intranasal vaccines
by themselves do not typically generate IgA
(Holmgren et al. 1993, 1994; Tamura et al. 1994),
cholera toxin was used as an adjuvant. Cholera
toxin has been shown highly efficacious in
inducing strong mucosal IgA responses, as well as
boosting the immune system in general (Holmgren
et al. 1993; Tamura et al. 1990, 1991, 1994;
Hirabayashi et al. 1990, 1992; Hornquist and
Lycke 1993).
The primary goal of this experiment was to
determine the effect of cholera toxin and
hemagglutinin DNA co-administration on
antibody isotype responses. As a secondary
objective, we examined the duration of the
immune response to DNA vaccination in terms of
protection from challenge infection, antibody
responses and the presence of influenza specific
memory cells at 3 months post vaccination.
Two intranasal DNA vaccinations were
followed by 2 gene gun vaccinations. For the
intranasal vaccine the DNA was administered
through a narrow gauge catheter positioned in the
nasopharynx. The gene gun vaccinations were
delivered at skin and mucosal sites using the
Powderject XR gene gun. Three groups of 4
influenza negative ponies were established; CT-
HA DNA vaccinates, HA DNA vaccinates and
controls. For the intranasal vaccines, the CT-HA
DNA vaccination group received hemagglutinin
DNA mixed with cholera toxin and the HA DNA
group received hemagglutinin DNA alone. Both
groups were then given 2 further gene gun
vaccinations. The control pony group initially
comprised 2 sentinel ponies, used to confirm that
influenza virus was not accidentally introduced
into the pony groups. Fourteen days prior to
23
challenge infection the number of control ponies
was increased to 4. All ponies were challenged by
nebulisation of a high dose of homologous virus 3
months after the last vaccination. Antibody isotype
responses in serum and nasal secretions were
analysed by ELISA. Nasal swabs for virus
isolation were collected for 12 days after challenge
infection and the egg infectious dose 50%, or
EID50, was determined. Immediately prior to
challenge infection, PBMCs were prepared from 2
ponies and used to generate dendritic cells.
Influenza virus-specific proliferation assays were
then conducted using autologous dendritic cells or
PBMCs as stimulator cells, which were either
infected with influenza virus or transfected with
HA DNA using the gene gun.
Following challenge infection, clinical disease
was significantly reduced in both vaccination
groups when compared to the control group but
not significantly different between the vaccination
groups. Virus shedding was seen in all groups,
nevertheless there was a reduction in the amount
and level of virus shed in the vaccination groups,
when compared to the control animals. Again no
significant differences could be found between the
2 vaccination groups. The IgG isotype responses
resembled closely what had been reported in our
previous studies. DNA vaccination induced
significant IgGa&b antibody titres following
vaccination as determined by paired T-tests.
However, no significant differences between the 2
vaccination groups in terms of serum IgG
responses could be found. In contrast to previous
reports a low titred but significant IgA response
could be detected in the CT-HA DNA group post
vaccination. By the time of challenge infection
antibody titres waned, but post challenge infection
the CT-HA DNA vaccinates again produced a
significantly higher IgA response. An additional
experiment was performed in 2 vaccinated ponies
prior to challenge infection to measure influenza
virus specific memory responses, and to examine
differences between dendritic cells and PBMCs as
antigen presenting cells. Memory cells were
clearly present as indicated by high stimulation
indices, but only dendritic cells were effective as
stimulator cells when transfected with HA DNA.
Overall, we found that DNA vaccination
protects horses from influenza infection but it
appears to do so in the absence of a mucosal IgA
response. It was implied that mucosal IgGb might
play an important role in mucosal immunity in this
circumstance. In addition, IL-6 HA DNA co-
administration does not switch the immune
response at the mucosal surface but it appears to
influence the systemic immune response. Lastly,
intranasal CT-HA DNA co-administration induces
a mucosal IgA response as well as the appropriate
IgG responses. This additional IgA response is,
however, of low titre and does not result in
improved protection of the animal. In conclusion
these results represent an improvement over
current inactivated vaccines. However, we believe
the responses must be amplified further to produce
longer lasting protection with practical value in the
field.
REFERENCES
Hirabayashi, Y., Kurata, H., Funato, H., Nagamine, T.,
Aizawa, C., Tamura, S., Shimada, K. and Kurata, T.
(1990) Comparison of intranasal inoculation of
influenza HA vaccine combined with cholera toxin
B subunit with oral or parenteral vaccination.
Vaccine 8, 243-248.
Hirabayashi, Y., Tamura, S.I., Shimada, K. and Kurata, T.
(1992) Involvement of antigen-presenting cells in
the enhancement of the in vitro antibody responses
by cholera toxin B subunit. Immunology 75, 493-
498.
Holmgren, J., Lycke, N. and Czerkinsky, C. (1993)
Cholera toxin and cholera B subunit as oral-mucosal
adjuvant and antigen vector systems. Vaccine 11,
1179-1184.
Holmgren, J., Czerkinsky, C., Lycke, N. and
Svennerholm, A.M. (1994) Strategies for the
induction of immune responses at mucosal surfaces
making use of cholera toxin B subunit as
immunogen, carrier and adjuvant. Am. J. trop. med.
Hyg. 50, 42-54.
Hornquist, E. and Lycke, N. (1993) Cholera toxin
adjuvant greatly promotes antigen priming of T
cells. Eur. J. Immunol. 23, 2136-2143.
Lunn, D.P., Soboll, G., Schram, B.R., Quass, J.,
McGregor, M.W., Drape, R., Macklin, M.D.,
McCabe, D.E., Swain,W.F. and Olsen, C.W. (1999)
DNA vaccination of horses using the influenza virus
hemagglutinin gene. Vaccine 17, 2245.
McGhee, J.R. and Kiyono, H. (1992) Mucosal immunity
to vaccines: current concepts for vaccine
development and immune response analysis. Adv.
exp. Med. Biol. 327, 3-12.
Morley, P.S., Townsend, H.G., Bogdan, J.R. and Haines
D.M. (1999) Efficacy of a commercial vaccine for
preventing disease caused by influenza virus
infection in horses. J. Am. vet. med. Ass. 215, 61-66.
Mumford, J.A. (1992) Progress in the control
of equine influenza. Proc. 6th inf. Conf. equine inf.
Dis. Eds: W. Plowright, P.D. Rossdale and J.F.
Wade, R&W Publications (Newmarket) Ltd, pp 207-
217.
Nelson, K.M., Schram, B.R., McGregor, M.W., Olsen,
C.W. and Lunn, D.P. (1998) Local and systemic
Immunology in 2001
24
isotype-specific antibody responses to equine
influenza virus infection versus conventional
vaccination. Vaccine 16, 1306-1313.
Soboll, G., Horohov, D.W., Olsen, C.W., McGregor,
M.W., Drape, R.J., Macklin, M.D., Swain, W.F.,
Larsen, D. and Lunn, D.P. (1999) Mechanisms of
protective immunity against equine influenza virus
infection following DNA vaccination,
conventional vaccination or influenza virus
infection in the horse. J. vet. int. Med. 13, 231.
Tamura, S., Funato, H., Hirabayashi, Y., Kikuta, K.,
Suzuki, Y., Nagamine, T., Aizawa, C., Nakagawa,
M. and Kurata, T. (1990) Functional role of
respiratory tract haemagglutinin-specific IgA
antibodies in protection against influenza. Vaccine 8,
479-485.
Tamura, S., Funato H., Hirabayashi, Y., Suzuki, Y.,
Nagamine, T., Aizawa, C. and T. Kurata. (1991)
Cross-protection against influenza A virus infection
by passively transferred respiratory tract IgA
antibodies to different hemagglutinin molecules.
Eur. J. Immunol. 21, 1337-1344.
Tamura, S., Yamanaka, A., Shimohara, M., Tomita, T.,
Komase, K., Tsuda, Y., Suzuki, Y., Nagamine, T.,
Kawahara, K., Danbara, H. (1994) Synergistic
action of cholera toxin B subunit (and Escherichia
coli heat-labile toxin B subunit) and a trace amount
of cholera whole toxin as an adjuvant for nasal
influenza vaccine. Vaccine 12, 419-426.
Underdown, B.J. and Mestecky, J. (1994) Mucosal
immunoglobulins. In: Handbook of Mucosal
Immunol. Eds: P.L. Ogra, W. Strober, J. Mestecky,
J.R. McGhee, M.E. Lamm and J. Bienenstock,
Academic Press Inc., San Diego, USA. 79-85.
25
THE INDUCTION OF EQUINE HERPESVIRUS-SPECIFIC
ANTIBODIES IN THE UPPER RESPIRATORY
TRACT OF THE HORSE
C. C. Breathnach, M. R. Yeargan, A. S. Sheoran and G. P. Allen
Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington,
Kentucky 40546-0099, USA
INTRODUCTION
Equine herpesvirus-1 (EHV-1) is a leading cause
of viral abortion in mares despite the availability
of commercial vaccines. The pathogenesis of EHV
abortion is a complex series of events (Allen et al.
1999). The virus initially infects and replicates in
the upper respiratory tract epithelium. Progeny
virions can then infect lymphocytes and
monocytes in the underlying subepithelial tissue
and draining lymph nodes, giving rise to a pool of
infected leucocytes which can establish viraemia.
This leucocyte-associated viraemia is critical in
the pathogenesis of EHV-1-induced abortion, as it
allows contact between infectious virus and the
permissive endothelium of the uterine vasculature,
where subsequent vasculitis or transplacental
spread of infectious virus to the fetus can lead to
premature fetal expulsion.
The mucosal immune system offers the
potential for immune exclusion of virus infection at
the respiratory tract epithelium portal of entry
(Wilkie 1982), thereby precluding the establishment
of infection. Little is known about induction of local
EHV-specific antibodies and their role in this first
line of immunological defence against EHV
abortion. This represents a fundamental gap in our
understanding of the effector components of EHV-1
immunity and hinders our ability to stimulate more
effective herpesvirus abortion defence strategies in
the horse by vaccination.
EXPERIMENTAL DESIGN
Twenty mixed-breed weanlings were assigned to 5
equal groups so that they were age-matched. Each
group received 2 doses of EHV antigen, 3 weeks
apart, and all 20 weanlings were challenged
simultaneously. Group 5 was divided into 2 pairs
of animals. One pair was allowed to become
infected with Army 183 by contact exposure on 2
occasions. The other pair remained untreated until
final challenge (Table 1).
Inoculation of weanlings with Army 183 and
with Rhinomune (Pfizer Animal Health Inc.,
Philadelphia, USA) was performed in identical
fashion. Both antigens were delivered by direct
intranasal instillation of 3 ml of a 2.35 x 10
7
PFU/ml cell-free suspension of live virus onto the
nasal turbinates. Pneumabort K (Fort Dodge
Animal Health, Kansas, USA) was administered
im. Rectal temperature and nasal discharge,
together with virus isolation from nasopharyngeal
swabs and heparinised blood samples, were
monitored for up to 14 consecutive days post
inoculation.
TABLE 1: Experimental design
No of Primary Secondary Challenge
weanlings inoculation inoculation
Group 1 4 Army 183 Army 183 Army 183
Group 2 4 Rhinomune Rhinomune Army 183
Group 3 4 Rhinomune Pneumabort K Army 183
Group 4 4 Pneumabort K Pneumabort K Army 183
Group 5a 2 Army 183 (contact) Army 183 (contact) Army 183
Group 5b 2 None None Army 183
Immunology in 2001
26
MEASUREMENT OF NASAL WASH IMMUNO-
GLOBULINS
Total IgA in each nasal wash sample was measured
by ELISA by coating 96-well plates with an
affinity purified sheep-anti-equine IgA polyclonal
antiserum (Bethyl Laboratories Inc., Texas, USA).
Nasal washes were titrated in duplicate columns
on each plate, and immunoglobulin reference
serum standard (Bethyl Laboratories Inc.) was
titrated in the remaining pair of columns, such that
IgA was at a starting concentration of 1 mg/ml.
The reference serum allowed a standard curve for
IgA content to be set up for each plate. Using the
standard curve, OD
490
readings corresponding to
the amount of total IgA in each nasal wash could
be translated into milligram quantities of IgA per
millilitre of nasal wash. This served as an internal
control for normalising virus-specific immuno-
globulin isotype levels.
Individual antibody isotype responses to EHV
were measured in nasal wash samples using
monoclonal antibodies (mAbs) specific for equine
IgGa (CVS 45), IgGb (CVS 39), IgG(T) (CVS 38),
IgA (BVS 2) (Sheoran et al. 1997, 1998) and IgM
(CM7; Custom Monoclonals International,
California, USA). Ten columns of 96-well plates
were coated with whole EHV-1 virus antigen, and
the remaining 2 columns with polyclonal isotype-
specific capture antibody specific for the isotype
being measured for the establishment of a standard
curve as above. Using the standard curve,
microgram quantities of virus specific antibody of
the isotype in question were calculated for each
nasal wash. All results were standardised to 1 mg
total IgA.
RESULTS
Exposure of seronegative weanling foals to
virulent EHV-1 (Army 183) gave rise to the
induction of a local mucosal humoural immune
response. This was demonstrated by the detection
of a wide range of virus-specific antibody
isotypes in the nasal washes (Fig 1). Virus-
specific IgA was elicited by EHV-1 inoculation,
and proved durable in the local environment of the
upper respiratory tract. Appreciable levels of
EHV-specific IgA were still detectable in the
nasal washes on the final sampling day, 13 weeks
post final challenge (Fig 1). Virus-specific IgGa
and IgGb isotypes were found at high
concentrations in nasal washes for brief periods
during the acute phase of disease. IgG(T) was
generally present in tandem with IgGa and IgGb,
but was usually found at lower quantities. This
pattern of appearance of IgG species in the upper
respiratory tract secretions is suggestive of
inflammation-dependent exudation of serum
antibody. It seems plausible that the rapid decline
in nasal wash IgG species was due to both the
restoration of the natural epithelial barrier during
convalescence from EHV-1 infection, thereby
preventing further passage of IgG into the
respiratory lumen, and the short half-life of
monomeric immunoglobulins in the nasal cavity.
All 6 seronegative weanlings innoculated with
Army 183 (Groups 1 & 5a) showed clinical signs of
disease consistent with primary exposure to virulent
EHV-1 antigen, following primary experimental
inoculation. However, they were protected from
upper respiratory tract disease following both
subsequent challenges. EHV-specific IgA and other
Fig 1: Mean EHV-specific
antibody isotype responses of 6
weanlings (Groups 1 and 5a), to
experimental inoculation with
Army 183. Arrowheads denote
times of inoculation with Army
183.
IgGa
IgGb
IgG(T)
IgA
Weeks post primary inoculation
6
5
4
3
2
1
0
g

E
H
V
-
s
p
e
c
i
f
i
c

a
n
t
i
b
o
d
y
/
m
g

t
o
t
a
l

I
g
A
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26
27

antibody isotypes were present in the respiratory
secretions, albeit at low levels. It seems likely that
local antibody against the challenge virus, whether
pre-existing or rapidly recalled, contributed to the
protective immune response.
Intranasal administration of Rhinomune
(Group 2) was not effective in the stimulation of
local EHV-specific antibody detectable by ELISA
(data not shown). The vaccine strain may have
been over-attenuated for this experimental
purpose. EHV-specific antibodies of all isotypes
remained at baseline levels following both
primary and secondary administration of
Rhinomune, and remained as such until challenge
of the animals with Army 183. A similar situation
arose among the animals in Group 3 (Rhinomune
intranasally followed by Pneumabort K im) and
Group 4 (Pneumabort K im twice; data not
shown). Each group of vaccinates demonstrated a
reduction in clinical signs following challenge,
suggesting that vaccination had conferred some
limited protection from clinical disease on those
animals, despite its failure to induce detectable
levels of pre-challenge local EHV-specific
antibody. The post challenge local antibody
response of the individuals in these groups was
clearly dominated by IgA. The amount of virus-
specific IgG isotypes in the nasal washes
remained low, presumably because reduced
clinical signs resulted in less epithelial damage.
Pathogen-specific antibody present at various
mucosal surfaces is reported to protect from
several viruses (Zeitlin et al. 1999) including
herpesviruses (Israel et al. 1992; Whaley et al.
1994). The demonstration of induction of nasal
IgA in the horse following experimental
inoculation with EHV-1 suggests a role for this
antibody as a first line of defence against
colonisation by this important upper respiratory
tract pathogen. Previous studies suggested that
IgA is the most important antibody isotype at the
nasal mucosa for protection of horses from other
notorious upper respiratory tract pathogens
(Sheoran et al. 1997; Nelson et al. 1998; Hannant
et al. 1999).
REFERENCES
Allen, G.P., Kydd, J.H., Slater, J.D. and Smith, K.C.
(1999) Recent advances in understanding the
pathogenesis, epidemiology and immunological
control of equid herpesvirus-1 (EHV-1) abortion.
Proc. 8th int. Conf. equine inf. Dis. Eds: U. Wernery,
J.F. Wade, J.A. Mumford and O.-R. Kaaden. R&W
Publications (Newmarket) Ltd, pp 129-146
Hannant, D., Easeman, R. and Mumford, J.A. (1999)
Equine mucosal immune system: intranasal
vaccination with inactivated equine influenza virus
protects from infection. Proc. 8th int. Conf. equine
inf. Dis. Eds: U. Wernery, J.F. Wade, J.A. Mumford
and O.-R. Kaaden. R&W Publications (Newmarket)
Ltd, pp 50-56.
Israel, B.A., Herber, R., Gao, Y. and Letchworth III, G.J.
(1992) Induction of a mucosal barrier to bovine
herpesvirus 1 replication in cattle. Virology 188,
256-264.
Nelson, K.M., Schram, B.R., McGregor, M.W., Sheoran,
A.S., Olsen, C.W. and Lunn, D.P. (1998) Local and
systemic isotype-specific antibody responses to
equine influenza virus infection versus conventional
Sheoran, A.S., Sponseller, B.T., Holmes, M.A. and
Timoney, J.F. (1997) Serum and mucosal antibody
isotype responses to M-like protein (SeM) of
Streptococcus equi in convalescent and vaccinated
horses. Vet. Immunol. Immunopathol. 59, 239-251.
Sheoran, A.S., Lunn, D.P. and Holmes, M.A. (1998)
Monoclonal antibodies to subclass-specific
antigenic determinants on equine immunoglobulin
gamma chains and their characterisation. Vet.
Whaley, K.J., Zeitlin, L., Barratt, R.A., Hoen, T.E. and
Cone, R.A. (1994) Passive immunisation of the
vagina protects mice against vaginal transmission of
genital herpes infections. J. inf. Dis. 169, 647-649.
Wilkie, B.N. (1982) Respiratory tract immune response
to microbial pathogens. J. Am. vet. med. Ass. 181,
1074-1079.
Zeitlin, L., Cone, R.A. and Whaley, K.J. (1999) Using
monoclonal antibodies to prevent mucosal
transmission of epidemic infectious diseases. Emerg.
Inf. Dis. 5, 54-64.
Immunology in 2001
28
29
IMMUNOGENETICS DEFINED
D. F. Antczak
College of Veterinary Medicine, The James A Baker Institute for Animal Health, Cornell University,
Ithaca, New York 14853, USA
The term 'immunogenetics' has enjoyed many
meanings over the past century, ranging from the
practical use of antibodies to detect polymorphic
gene products to the early, arcane studies of the
genes controlling tissue transplantation. For the
purposes of this discussion a broader definition
will be employed.
Immunogenetics is the study of genes
affecting and controlling immune responses.
Today this term encompasses complementary
investigations of the structure, function, and
polymorphism of these genes. Some of the genetic
regions influencing immune responses are well-
known, although not yet completely understood.
These include the major histocompatibility
complex (MHC), and the immunoglobulin and T
cell receptor regions. Others, such as the genes
encoding the cytokines and their receptors, are
less well-studied. Progress towards sequencing
the entire human genome and the development of
comparative gene maps of many species,
including the horse, allows construction of a
genomic map of the equine immune system. The
rapidly expanding array of molecular tools
developed by the Horse Genome Project will
enable sophisticated studies of the function of
genes of the equine immune system and
applications in production and disease.
ORGANISATION OF THE EQUINE IMMUNOGLOBULIN
CONSTANT REGION GENES
B. Wagner
Immunology Unit, Hannover School of Veterinary Medicine, Germany
Immunoglobulin (Ig) effector functions during
immune response are dependent on the isotypes
which are expressed after B-cells have recognised
their specific antigens. Whether immunoglobulins
induce protection, have no effect on the outcome
of the immune response or mediate autoaggression
or allergy is influenced by the isotype.
Many studies have been performed to
characterise equine Ig isotypes using serological
and biochemical methods (Table 1), These studies
allowed the description of IgM, 5 to 6 IgG
isotypes, IgE and one or 2 IgA subclasses, but
particularly for IgG and IgA the exact number of
isotypes could not be determined. To resolve the
dilemma of how many isotypes exist in the horse,
the molecular basis of equine Ig isotype expression
was investigated.
Ig isotypes are encoded by the constant heavy
chain genes (CH-genes), which are located on the
immunoglobulin heavy chain gene locus (IgH-
locus). The IgH-locus contains the 5 located
variable, diversity and joining genes (VDJ genes),
representing the gene segments encoding the
variable domain and the different CH-genes (Fig.
1). The IgH-loci of mouse and rat are very similar
having 5 located C and C genes, 4 C genes
clustered together and one C and one C gene at
the 3 ends (Shimizu et al. 1982). In contrast, the
human IgH-locus shows a duplication of a
fragment which occurred during evolution. This
leads to the expression of four C genes, one C
gene, but 2 C genes in man (Flanagan and
Rabbitts 1982). Further, the rabbit constant heavy
chain genes are completely different, with only
one C gene, but 13 C genes
To analyse the equine constant heavy chain
genes we used human and murine probes to isolate
equine C, C and C genes from a genomic phage
library. One C and one C gene were isolated and
clustered together on one DNA fragment,
represented by 4 overlapping phage clones
(Wagner et al. 1997). In addition, 6 different C
gene containing clones were obtained by the same
rationale. Subsequently, these equine CH-genes
were used as DNA probes to analyse equine
genomic DNA from PBMC by restriction analysis.
The restriction analysis confirmed the existence of
6 equine C genes, one C and one C gene.
To align the 6 C genes, which did not overlap,
and to determine their relative position to the C-
C cluster, as well as to the equine C gene,
deletion analysis of heterohybridomas expressing
equine IgM and IgG was performed (Wagner et al.
1995).
The deletion analysis was based on class
switch recombination, which occurs during B-cell
development. The isotype expression is known to
be regulated by different cytokines which are
produced by T-helper cells (Fig 2).
Cytokines secreted by T-helper 1 cells, like
IFN induce the expression of isotypes other than
TABLE 1: Equine isotypes characterised by
serology or biochemistry
IgM Hill and Cebra (1965);
Montgomery (1973)
IgGa, IgGb, IgGc Rockey (1967);
Montgomery (1973)
IgG(T), with probably Weir and Porter (1966);
2 subclasses Helms and Allen (1970);
Sheoran and Holmes (1996)
AI/IgG(B) Helms and Allen (1970);
Seide et al. (1987)
IgA Vaerman et al. (1971);
Pahud and Mach (1972)
IgE Suter and Fey (1981);
Halliwell and Hines (1985)
Immunology in 2001
30
T-helper 2 cytokines, like IL4. For example, IL4
induces expression of murine IgG1 and in higher
concentrations to IgE. In contrast, IFN inhibits
both steps, but induces IgG3 and IgG2a expression
(Siebenkotten and Radbruch 1995).
Early in the B-cell development, the variable
genes on the IgH-locus on one chromosome
undergo a VDJ recombination to express a
functional variable heavy chain domain together
with the first 3 located constant gene, the C
gene. This leads to the expression of IgM on the
cell surface, as well as to IgM secretion.
The class switch recombination is initiated
when the B-cell recognises a specific antigen. The
class switch occurs between highly repetitive
intron sequences, known as switch regions, which
are located at 5 of the associated CH-gene. A part
of the S region and the switch region of the newly
expressed CH-gene are re-arranged and the DNA
between both switch regions is looped out and
becomes deleted from the chromosome.
In heterohybridomas expressing a defined
isotype, only the expressed gene and the 3 located
constant genes are present while all genes at 5 of
the expressed one are deleted. Deletion analysis of
equi-murine heterohybridoma DNA, using the
equine DNA probes for C, C and C genes lead
to the alignment of the equine CH-genes (Fig 3),
with the 5 located C gene, 6 C genes clustered
together, one C and one C gene at the 3 end.
The designation of the equine C genes as C1 to
C6 was carried out according to their position
from the 5 to 3 direction. The corresponding
isotypes for 3 of the 6 C genes were determined
according to heterohybridoma expression: C1
encoding IgGa, C3 IgG(T) and C4 IgGb
(Wagner et al. 1998).
In addition, the serological data strongly
indicate that the remaining C genes are also
expressed.
The genetic data offer general information that
can be used for equine Ig isotype diagnosis in
several diseases. In addition, the analysis of
functional aspects like cytokine regulation of
isotype expression or the characterisation of the
equine constant heavy chain gene evolution by
nucleotide or amino acid sequence comparison can
be performed.
We decided to use the DNA for the expression
of rare equine isotypes, such as recombinant
proteins like IgE. The equine IgE was expressed as
a complete chimeric Ig molecule in mammalian
cells (Fig. 4).
The chimeric IgE contained murine light
chains and a murine VH domain, forming 4-
(hydroxy-3-nitro-phenyl) acetyl (NP) specific
Fig 1: IgH-loci of different species.
Fig 2: Immunoglobulin class switching occurs in
activated B-cells and is strictly regulated by cytokines.
Fig 3: Alignment of equine C, C, C and C genes and
corresponding immunoglobulin isotypes.
Fig 4: Expression of recombinant equi-murine NP-
specific IgE.
31
antigen binding sites, and a complete equine IgE
constant region (NP-IgE). This NP-IgE had a
molecular weight of 230.000 daltons, was highly
glycosilated and able to induce an immediate skin
reaction after crosslinking with antigen. Thus, the
recombinant equine IgE is very similar to the
native protein and will be a useful reagent for the
production of monoclonal antibodies specific for
equine IgE.
REFERENCES
Flanagan, J.G. and Rabbitts, T.H., (1982) Arrangement
of human immunoglobulin heavy chain constant
region genes implies evolutionary duplication of a
segment containing , and genes. Nature 300,
709-713.
Shimizu, A., Takahashi, N., Yaoita, Y. and Honjo, T.,
(1982) Organisation of the constant-region gene
family of the mouse immunoglobulin heavy chain.
Cell 28, 499-506.
Siebenkotten, G. and Radbruch, A. (1995) Towards a
molecular understanding of immunoglobulin class
switching. The Immunologist 3, 141-145.
Wagner, B., Radbruch, A., Richards, C. and Leibold, W.
(1995) Monoclonal equine IgM and IgG
immunoglobulins. Vet. Immunol. Immunopathol. 47,
1-12.
Wagner, B., Siebenkotten, G., Leibold, W., Radbruch, A.,
(1997) Organisation of the equine immunoglobulin
constant heavy chain genes. I. C and C genes. Vet.
Wagner, B., Overesch, G., Sheoran, A.S., Holmes,
M.A., Richards, C., Leibold, W., Radbruch, A.,
(1998) Organisation of the equine immunoglobulin
heavy chain constant region genes. III. Alignment
of C, C, C and C genes. Immunobiol. 199,
105-119.
32
Immunology in 2001
EQUINE MHC REGULATION ON RESISTANCE AND
IMMUNE BIAS
E. Marti
Division of Immunogenetics, Institute of Animal Breeding, Bremgartenstrasse 109A, 3012-Berne,
Switzerland
MHC class I and class II genes encode
membranebound molecules whose function is to
bind and present peptides to T cells and thus play
a key role in the initiation of an immune response
and influence susceptibility to diseases. Some
MHC genes and gene products exhibit a
remarkably high level of polymorphism. The
major focus of MHC research in the horse has
been identification of homology between the
equine MHC and the MHC of other species,
characterisation of the MHC genes of the horse
and investigation of associations between MHC
alleles and disease susceptibility or antibody
production.
The equine leucocyte antigen (ELA) class I
alleles are still determined serologically and over
20 different alleles have been described at the so-
called ELA-A locus. MHC class II typing is now
also performed with molecular techniques such as
SSCP and PCR-RFLP (reviewed by Bailey et al.
2000). Disease association studies, however, were
performed before such information was available,
and were carried out with a limited number (5) of
serologically determined MHC class II alleles.
The strongest association which has been
found so far is the association between the equine
MHC class II allele DW13 and susceptibility to
sarcoid tumours (Lazary et al. 1994), a skin
tumour of the horse caused by a virus closely
related or identical to bovine papilloma virus
(Otten et al. 1993). This association has been
described in the USA, Sweden and Switzerland.
Most interestingly, associations between MHC
class II alleles and susceptibility to papilloma virus
induced tumours have also been demonstrated in
other species such as squamous cell carcinoma in
women (Apple et al. 1994) and tumours induced
by Shope papilloma virus in rabbits (Han et al.
1992).
An association between ELA and
susceptibility to sweet itch, an allergic dermatitis
response to bites of midges and/or black flies, has
been described in Icelandic horses (DW22;
Halldorsdottir et al. 1991) and in some Swiss
Warmblood horse families (DW23; Lazary et al.
1994).
Two studies suggest that the antibody
production against specific antigens is influenced
by the MHC in the horse: in the first study, weak
associations (0.1>P>0.01) between certain ELA
alleles and antibody titres against influenza 1 and
2, equine herpesvirus-1 and ovalbumin were found
(Bodo et al. 1994). In the second study (Eder et al.
2001) a possible association between MHC class I
antigens and mould-specific IgE antibody levels
was investigated. Significant associations (P0.01)
were found between ELA-A8 and low IgE titres
against some recombinant Aspergillus fumigatus
and Alternaria alternata allergens. Furthermore,
ELA-A1 was associated with higher titres and
ELA-A14 with lower IgE titres against mould
extracts (P0.05).
ACKNOWLEDGEMENTS
This work was supported by the Swiss National
Science Foundation grant No. 31-49618.96 and by
the Hans-Sigrist Foundation of the University of
Berne.
REFERENCES
Apple, R.J., Erlich, H.A., Klitz, W., Manos, M.M.,
Becker, T.M. and Wheeler, C.M. (1994) HLA DR-
DQ associations with cervical carcinoma show
papillomavirus-type specificity. Nature Genetics 6,
157-162.
Bailey, E., Marti, E., Fraser, D., Antczak, D. and Lazary,
S. (2000) Immunogenetics. In: The Genetics of the
33
Horse. Eds: A. T. Bowling and A. Ruvinsky. CAB
INTERNATIONAL, Oxon, UK, 123-155.
Bodo, G., Marti, E., Gaillard, C., Weiss, M., Bruckner,
L., Gerber, H. and Lazary S. (1994) Association of
the immune response with the major
histocompatibility complex in the horse. Proc. 7th
int. Conf. equine inf. Dis. Eds: H. Nakajima and J.F.
Wade. R&W Publications (Newmarket) Ltd, pp 143-
151.
Eder, C., Curik, I., Brem, G., Crameri, R., Bodo, I.,
Habe, F., Lazary, S., Slkner, J. and Marti, E. (2001)
Influence of environmental and genetic factors on
allergen-specific immunoglobulin E levels in sera
from Lipizzan horses. Equine vet. J. (in press).
Halldorsdottir, S., Lazary, S., Gunnarsson, E. and Larsen,
H.J. (1991) Distribution of leucocyte antigens in
Icelandic horses affected with summer eczema
compared to non-affected horses. Equine vet. J. 23,
300-302.
Han, R., Breitburd, F., Marche, P.N. and Orth, G. (1992)
Linkage of regression and malignant conversion of
rabbit viral papillomas to MHC class II genes.
Nature 356, 66-68.
Lazary, S., Marti, E., Szalai, G., Gaillard, C. and Gerber,
H. (1994) Studies on the frequency and associations
of ELA antigens in sarcoid and summer dermatitis.
Animal Genetics 25, 75-80.
Otten, N., von Tscharner, C., Lazary, S., Antczak, D.F.
and Gerber, H. (1993) DNA of bovine
papillomavirus type 1 and 2 in equine sarcoids: PCR
detection and direct sequencing. Arch. Virol. 132,
121-131.
34
Immunology in 2001
A SYNDROME OF ANAEMIA, IMMUNODEFICIENCY
AND PERIPHERAL GANGLIONOPATHY IN
FELL PONY FOALS
M. A. Holmes, S. F. E. Scholes*, A. Holliman
and P. D. F. May**
University of Cambridge; *Veterinary Laboratory Agency, Lasswade, Edinburgh;
Veterinary
Investigation Centre, Penrith; **Rowcliffe House Veterinary Hospital, Penrith, UK
35
INTRODUCTION
Fell ponies were used extensively in Cumbria as
draft animals but their numbers decreased
markedly after the Second World War, although
they remained an integral part of fell farming. In
recent years there has been a marked increase in
the number of breeding mares on fell farms
because the breed has become popular for riding
and showing. Recently an unexplained progressive
syndrome of Fell pony foals has been seen.
Despite intensive supportive therapy, foals die
usually between 2 and 3 months of age (Scholes et
al. 1998). At 2 to 3 weeks of age affected foals are
bright and alert but many have diarrhoea, cough
and fail to suck. The latter is associated with
frequent chewing movements, halitosis and a pale
pseudomembranous lingual coating. The diarrhoea
and coughing initially respond to treatment but
then become unresponsive and persist. The foals
become clinically anaemic with dry staring coats,
usually at 4 to 8 weeks of age. They deteriorate
and most die or are destroyed, between one and 3
months of age.
TABLE 1: Summary of clinical pathology in 3 affected Fell ponies (1, 2, 3), a foal with an unrelated
neuropathy (N) and 2 control animals (A, B)
Affected foals Foal Healthy foals Reference
1 2 3 N A B range*
Age 24 42 49 44 46 38 42
Sex F F F M M M F
WBC (10
12
/litre) 2.97 6.39 1.95 3.46 8.23 10.64 13.21
PCV (litres/litre) 0.16 0.27 0.09 0.16 0.35 0.45 0.4
Hb (g/dl) 5.8 9.9 4.3 5.9 11.8 16.0 15.5
MCV (fl) 554.5 42.7 46.2 45.0 41.7 41.8 36.8
MCH (pg) 19.5 15.4 22.1 17.0 14.3 15.0 11.7
MCHC (g/dl) 35.8 36.2 37.8 37.8 34.3 35.9 31.8
WBC (10
9
/
/litre) 6.7 12.3 41.1 29.2 14.2 9.9 14.2
Lymphocytes (10
9
/
/litre) 1.07 1.47 3.28 1.75 2.27 3.16 2.27
Neutrophils (10
9
/
/litre) 5.36 9.1 36.9 23.9 9.94 5.94 10.7
Monocytes (10
9
/
/litre) 0.27 1.72 0.82 3.5 1.7 0.60 0.85
Eosinophils (10
9
/
/litre) 0 0 0 0 0.28 0.20 0.28
Total protein (g/litre) 48 60 so 65 59 59 62
Albumin (g/litre) 23.6 29.7 20.9 29.4 23.4 25.6 27.2
Globulin (g/litre) 24.4 30.3 29.1 35.6 35.6 33.4 34.8
Urea (mmol/litre) 6.1 49.4 114.0 44.3 9.1 6.1 6.0
Creatinine (mmol/litre) 114 414 810 796 79 ND ND
Total bilirubin (mmol/litre) 25.4 24.2 37.8 25.2 29.9 ND ND
Direct bilirubin (mmol/litre) 14.2 8.7 8.5 3.1 11.3 ND ND
*Thoroughbred foals; RBC red blood cells; PCV packed cell volume; Hb Haemoglobin; MCV mean
corpuscular volume; MCH mean corpuscular haemoglobin; MCHC mean corpuscular haemoglobin
concentration; WBC white blood cells; ND not done.
36
Immunology in 2001
CLINICAL PATHOLOGY
Haematological and serum biochemical findings in
3 Fell pony foals with the syndrome of anaemia,
immunodeficiency and peripheral ganglionopathy,
and in 2 healthy control foals and a foal with an
unrelated neuropathy are shown in Table 1.
PATHOLOGY
Bone marrow
The bone marrow of the affected foals was more
densely populated with haemopoietic cells and
contained fewer lipocytes than marrow from
similar sites in the control foals. Late normoblasts
were rare in the femoral bone marrow compared to
an age-matched control bone marrow. Numerous
coarse haemosiderin granules were present in
macrophages in affected foals.
Lymph nodes
There were sparse to moderate numbers of
lymphocytes in the cortices and paracortices of the
lymph nodes, and germinal centres were absent
from all the lymph nodes examined from these
foals. There were macrophages in the sinuses of all
the lymph nodes examined from affected foals and
they were particularly numerous, together with
variable numbers of neutrophils, in inflamed
nodes.
Thymus
The thymic lobules were very small, there was no
clear demarcation between the cortex and medulla,
the epithelial structure was prominent and the
outer areas of the lobules contained variable
numbers of vacuolated macrophages and few
lymphocytes.
Spleen
Foals had small numbers of periarteriolar
lymphocytes and the red pulp was markedly
contracted and contained numerous siderophages;
neither germinal centres nor their stromal support
were detected.
Intestine
No plasma cells were recognised in the small or
large intestinal lamina propria of foals, in which
the tissue preservation allowed a critical
evaluation, and there were no germinal centres in
TABLE 3: Summary of the results of purine enzyme analysis from 2 affected Fell ponies (FPS 1, 2)
and 3 control animals (1, 2, 3)
P5N PNP SAH ADA
Control 1 2.2 1.3 1.5 25
Control 2 1.5 0.6 1.2 30
Control 3 2.6 2.1 1.9 54
FPS1 2.1 6.2 0.7 46
FPS2 1.4 8.1 0.9 52
Human control 13 4500 8.1 12000
Normal range 821 37000 38 820000
TABLE 2: Summary of the results of immunoglobulin analysis from the plasma of 3 affected Fell
ponies (1, 2, 3), a foal with an unrelated neuropathy (N) and 2 control animals (A, B)
Foal lgG lgA lgM lgGa lgGb lgGc
FPS1 23 0.5 2.4 1.6 8.5 2.1
FPS2 14 0.7 3.0 1.8 9.6 1.6
FPS3 11 0.1 1.4 0.9 6.8 2.6
N 18 0.7 0.9 2.1 13.8 2.5
A 12 0.5 1.1 1.6 8.6 3.2
B 13 0.5 0.8 1.9 10.4 3.9
the gut-associated lymphoid tissue (GALT). There
were large numbers of Cryptosporidia on the
apical surfaces of enterocytes and, to a smaller
extent, of enteroblasts in the duodenum and ileum
of some foals. In other foals, the nuclei of some
duodenal enterocytes contained large basophilic
intranuclear inclusion bodies typical of an
adenovirus infection. In contrast, in the control
foals, there were many mature plasma cells in the
deeper lamina propria of the small and large
intestine, particularly in the duodenum and there
were prominent germinal centres in the GALT.
Nervous system
All the peripheral ganglia examined contained
sparse to moderate numbers of chromatolytic
neurones and occasional axonal spheroids.
Chromatolysis was usually central but
occasionally complete, the latter being
accompanied by nuclear pyknosis. Chromatolysis
was not detected in the peripheral ganglia of the
control foal. No abnormality was detected in the
central nervous system.
IMMUNOGLOBULIN ANALYSIS
Isotype concentrations were established using
single radial immunodiffusion and the subisotype
concentrations were measured using a competition
ELISA (Sheoran et al. 1997). The results of the
serum immunoglobulin assays are shown in Table
2. The total immunoglobulin levels in the affected
Fell pony foals were similar to the control foals.
However, the affected foals had relatively higher
levels of IgM. The relative concentrations of IgG
subisotypes in the serum of the affected foals was
also similar to that in the control animals although
the absolute levels were, on average, lower.
MEASUREMENT OF PURINE ENZYME
ACTIVITIES
A proportion of human SCID conditions result
from defects in enzymes involved in purine
metabolism such as adenosine deaminase (ADA)
and purine nucleoside phosphorylase (PNP)
(Markert 1991; Webster 1987). Conventional
biochemical techniques were used to assess ADA,
PNP, SAHH and P5N enzyme levels (Peters et al.
1985; Magnusson and Perryman 1980; Morris and
Simmonds 1985; Rocchigiani et al. 1992)
Evaluation of purine and pyrimidine pathways
was examined by the incorporation of
14
C
radiolabelled substrates. Substrates were incubated
for 14 minutes with cell lysates. The fate of
radiolabelled purines, dexoxyadenosine and
hypoxanthine, and the pyrimidine, orotic acid was
established by running product through an anion
exchange column using HPLC together with an in-
line radiation detector. The incorporation or
absence of
14
C in various pathway products
indicated whether or not the major purine
metabolic pathways were intact (Table 3).
Purine enzyme activities are generally very
low in horses. The PNP levels are higher, if
anything, in affected foals. There were low SAHH
activities in affected foals. ADA levels were
extremely low in all equine samples which
provokes a question as to why horses have such
low ADA levels.
CONCLUSION
These results suggest that this disease is not a
homologue of any well characterised immuno-
deficiency in any other species. There is no
evidence that the syndrome is the same as a SID
condition described in Arabian horses (Shin et al.
1997). Although immunoglobulin was present in
the circulation of older foals, there is no evidence
that this was not derived from the dam via
colostrum. There is a clear need for basic genetic
and epidemiological studies to establish the
heritability and other basic parameters of the
disease. Further studies will concentrate on
characterising the functional immunodeficiency,
bone marrow deficit, and the purine and
pyrimidine enzymology.
37
38
Immunology in 2001
SESSION II :
New technologies in
equine imunity
Chairman: Doug Antczak
39
40
Immunology in 2001
MONOCLONAL ANTIBODIES: WHATS NEXT?
D. P. Lunn
School of Veterinary Medicine, 2015 Linden Drive West, Wisconsin 53706, USA
Progress in producing monoclonal antibodies
(mAbs) for equine immunological study could be
well described by a bell-shaped curve. Early
progress was slow, then there was a rush of progress
charted by 2 workshops. More recently, it has
become increasingly difficult to find the resources
and the more exacting techniques to focus on
specific but pivotal molecules, like IgE and key
cytokines. It is hoped that meetings, such as the
current workshop, will provide further stimulus.
Large workshops with many new anti-equine
reagents are not likely to recur, with the exception
of what might be accomplished with proper access
to the human antibody body panel. For example,
up to 20% of 116 human antibodies cross-reacted
with equine cells in a preliminary study conducted
at the most recent human leucocyte differentiation
antigen workshop. Specific new reagents have
been identified since the most recent equine
workshop, and include antibodies recognising
FcRIII and FcRII, CD38, and B7-2 (CD86). In
the authors laboratory, work has been underway,
in collaboration with David Horohov of Louisiana
State University, to characterise select equine
cytokines further. Biological activity for equine
IL-6 and IL-10 has been demonstrated, and -IFN
is currently being investigated. In addition mAbs
recognising equine IL-2 have been produced, and
-IFN is currently being targeted. These efforts
have progressed slowly, and further advances will
require increased collaborations so that
investigators can combine their efforts. To this end
a webpage is maintained which can be found at:
http://www.vetmed.wisc.edu/research/eirh/. This
lists many equine reagents that became available
after the last equine workshop, and it will be
updated with new information arising from the
present meeting.
41
DETECTION OF INTERFERON-
PRODUCING EQUINE
T LYMPHOCYTES BY FLOW CYTOMETRY: PULMONARY
RESPONSES TO RHODOCOCCUS EQUI CHALLENGE
S. Hines, D. Stone, M. Hines, L. Norton and T. C. McGuire
Washington State University, College of Veterinary Medicine, Pullman, Washington, USA
Rhodococcus equi is an important pulmonary
pathogen of young horses. Previous work in a
mouse model has shown that blocking IFN- in
vivo prevents immune clearance of R. equi from
the lung. Likewise, adoptive transfer of R. equi-
specific CD4+ Th1 cells that produce IFN- is
sufficient for clearance in immunodeficient nude
mice that normally cannot control pulmonary
infection. There is also evidence in mice that
CD8+ T lymphocytes play a role in protective
immune responses. At least part of the effect of
CD8+ lymphocytes may be mediated by IFN-
secretion as well.
The purpose of this study was to develop and
validate a flow cytometric method for measuring
intracellular IFN- in lymphocytes from the horse,
and to apply the technique to the study of equine
rhodococcal pneumonia. Monoclonal antibodies
(Mab) were raised against a synthetic peptide
corresponding to the predicted amino terminus of
equine IFN- (EqIFN-) and shown by
immunoblotting to react with a full length
recombinant EqIFN- molecule. One of these
antibodies was further shown to detect
intracellular IFN- in individual lymphocytes
following ex vivo stimulation with ionomycin and
phorbol 12-myristate 13-acetate (PMA). Briefly,
monensin was added to stimulated cultures to
block intracellular transport and cause cytokine
accumulation in the Golgi apparatus. Upon
harvest, stimulated and unstimulated cells were
fixed with paraformaldehyde, permeabilised with
saponin, and stained with anti-IFN- Mab
followed by a fluorochrome conjugated secondary
antibody. Importantly, the specificity of the IFN-
staining was demonstrated using synthetic peptide
to competitively inhibit binding of the anti-IFN-
Mab. Staining with Mab to CD2, CD8 and/or CD4
prior to fixation allowed for identification and
enumeration of the types of cell producing IFN-.
Following work to optimise the assay, we first
characterised IFN- production by peripheral
blood mononuclear cells (PBMC) from normal
horses (n=5). As described in other species, no
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
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10
1
10
2
10
3
10
4
10
4
10
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10
2
10
1
10
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10
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10
3
10
2
10
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10
0
10
4
10
3
10
2
10
1
10
0
C
D
2
+
C
D
2
+
C
D
2
+
IFN-
IFN-
IFN-
Unstimulated
Stimulated Stimulated + blocked
Fig 1: Example of detecting IFN- producing equine T lymphocytes (PBMC) by flow. Percentages indicated in the right
quadrants of the second panel are percent of the total cells counted. Percentages described in the text are percent IFN-
-positive cells for each indicated cell surface marker (eg %CD8+ cells that are IFN--positive).
Immunology in 2001
42
12.4%
0.2%
significant production of IFN- was detected in
unstimulated cells. However, after stimulation
with ionomycin/PMA, the percentage of CD2+ T
lymphocytes producing IFN- ranged from 13.5%
to 45.7% (Fig. 1). This variation is similar to that
described in PBMC from man and other species.
The percentage of CD8+ lymphocytes producing
IFN- was 17.631.5%, whereas 18.147.2% of
CD4+ lymphocytes were positive for IFN-. As
noted in other species, stimulation with
ionomycin/PMA resulted in down regulation of
surface CD4 expression. Therefore, in subsequent
experiments (on cells from the lung) we used
tricolour fluorescence and defined CD2+/CD8-
cells as putative CD4+ T lymphocytes.
Because all foals are normally exposed to R.
equi in their environment, and only a subset develop
pneumonia, we have hypothesised that most foals
develop protective immune responses. Further,
these antigen-specific responses are hypothesised to
operate throughout life to prevent rhodococcal
pneumonia in adults. In support of this model, work
in our laboratory has shown that adult horses
efficiently clear R. equi bacteria from the lung in
association with an influx of both CD4+ and CD8+
T lymphocytes. The timing and antigen-specific
lymphoproliferation of these cells are compatible
with a local recall response. A better understanding
of the mechanisms of immune clearance in adult
horses would help define the requirements for an
effective vaccine in foals. Using tricolour
fluorescence we determined the phenotype of cells
in bronchoalveolar fluid (BALF) producing IFN-
both before and after challenge with virulent R.
equi. Only a small number of horses have been
tested thus far. Prior to challenge, approximately
10% of CD4+ (CD2+/CD8-) T lymphocytes and
10% of CD2+/CD8+ T lymphocytes in BALF
produce IFN- when stimulated with
ionomycin/PMA ex vivo. Following challenge,
approximately 30% of CD4+ T lymphocytes and
30% of CD2+/CD8+ T lymphocytes in BALF
produce IFN- upon stimulation. Moreover,
clearance of a pulmonary challenge is associated
with a dramatic increase in the absolute numbers of
CD4+ T lymphocytes in BALF that produce IFN-
upon stimulation (examined at Days 7 and 14 post
challenge). In contrast, only small increases in the
absolute numbers of CD2+/CD8+ T lymphocytes
that produce IFN- are observed. Future work will
characterise antigen-specific intracellular IFN-
production associated with clearance of virulent R.
equi. Our long term goal is to use this information
to design a vaccine for use in foals.
43
IL-4 INDUCED CD23 (FCERII) UPREGULATION IN
EQUINE PERIPHERAL BLOOD MONONUCLEAR CELLS
AND ALVEOLAR MACROPHAGES
J. L. Watson, K. A. Jackson and J. L. Stott
Departments of Medicine and Epidemiology and Pathology, Microbiology and Immunology, School of
Veterinary Medicine, University of California, Davis, California 95616, USA
SUMMARY
The objective of this study was to quantify the
induction of equine CD23 transcripts in equine
peripheral blood mononuclear cells (PBMCs) and
alveolar macrophages cultured with recombinant
equine IL-4 (rEQ IL-4). Whole blood was
collected from 4 healthy 3-year-old horses and
PBMCs were purified over Histopaque.
Bronchoalveolar lavage fluid was collected from
one healthy horse and alveolar macrophages were
purified using adherence to plastic for 120 min.
PBMCs and alveolar macrophages were cultured
using 4 media conditions: rEQ IL-4 and LPS, rEQ
IL-4 only, LPS only and no addition. Total RNA
was isolated from cells cultured for 24 and 48 h.
Reverse transcribed mRNA was amplified and
quantified in real-time polymerase chain reaction
(PCR) using a fluorescein labelled internal
TaqMan probe. Relative quantification of the
CD23 signal was done using the signal for
GAPDH, a housekeeping gene, for each sample.
Raw data were analysed using the C
T
method
where the value for each sample was normalised
using the corresponding GAPDH value and all
values for each condition were calibrated using the
untreated control. Without exception, the relative
value for CD23 transcripts from PBMCs cultured
with rEQ IL-4 and LPS were significantly higher
than the untreated controls (P< 0.05). The relative
value for CD23 transcripts from alveolar
macrophages cultured with rEQ IL-4 was
increased more than 1,000-fold when compared to
LPS only or untreated controls. In conclusion, rEQ
IL-4 induced equine CD23 mRNA transcript
upregulation in PBMCs and alveolar macrophages
cultured for 24 and 48 h. These findings support a
role for equine CD23 in allergic, anti-parasitic and
other IL-4 mediated immune responses in horses.
INTRODUCTION
The low-affinity receptor for IgE, CD23 (FceRII),
is expressed on lymphocytes, monocytes,
eosinophils and platelets. CD23 plays a role in the
differentiation and activation of B lymphocytes
and is upregulated, or induced, by interleukin 4
(IL-4). There is evidence that the expression of
CD23 plays an important role in IgE-associated
immune responses, including allergies. Alveolar
macrophages from people with allergic asthma
express CD23 at a high frequency when compared
to non-asthmatics. Horses suffer from a number of
allergic conditions in which CD23 may play a role.
This study provides evidence that 1) CD23 is
present on circulating PBMCs, 2) CD23 can be
upregulated on PBMCs by rEQ IL-4 and 3) CD23
can be induced on equine alveolar macrophages by
rEQ IL-4.
MATERIALS AND METHODS
Cell isolation and culture
Peripheral blood mononuclear leucocytes (PBML)
were harvested from whole blood collected in
ACD by centrifugation over Histopaque. PBMLs
were washed twice in sterile phosphate buffered
saline, resuspended in RPMI at 6 x 10
5
/ml
and transferred to tissue culture flasks (75 cm
2
).
Four culture conditions were used: media
alone, supplementation with lps at 10 g/ml,
supplementation with recombinant equine IL4
rich cell supernatant at 12.5%, or both. Cells were
cultured at 37C and 5% CO
2
for 24 or 48 h.
Harvested cell pellets were washed twice in PBS
and frozen immediately at -80C.
Cells were harvested from BAL fluid by
centrifugation for 20 min at 400 g and washed
44
Immunology in 2001
twice in sterile Hanks Buffered Salt Solution.
BAL cells were resuspended in RPMI at 2 x
10
5
/ml and transferred to tissue culture flasks (25
cm
2
). Cells were incubated at 37C and 5% CO
2
for 2 h and non-adherent cells were rinsed out with
2 washes of sterile HBSS. Adherent cells were
cultured by the same method as for PBMLs.
Total RNA isolation and cDNA synthesis
Total RNA was isolated from each cell pellet using
the Qiagen RNAeasy mini kit. All RNA
extractions were treated with RNAse-free Dnase I
for 30 min. First strand synthesis was performed in
a 20 l volume containing 50 units Superscript II
(Invitrogen Life Technologies), 1.25mM random
hexamers and 1nM dNTPs.
Real-time polymerase chain reaction (RT PCR)
assay
Real-Time TaqMan systems for GAPDH and
CD23 were run in separate wells. The PCR
reactions contained 400 nM of each primer, 80
nM of each probe, commercially available PCR
Mastermix (TaqMan Universal PCR Mastermix,
Applied Biosystems) and 5 l diluted cDNA
sample (5-fold dilution)I in a final volume of 25
ml. The samples were placed in 96-well plates
and amplified in an automated fluorometer (ABI
7700 Sequence Detection System, Applied
Biosystems). Amplification conditions were 2
min at 50C, 10 min at 95C, 40 cycles of 15 s at
95C and 60 s at 60C. Transcript quantification
was done using the C
T
method and is reported
as the n-fold difference relative to a calibrator
cDNA (CD23 transcription in unstimulated
cells).
RESULTS
When PBMCs were cultured with rEQ IL-4 and
LPS for 24 or 48 h CD23 signal could be detected
and the relative value for CD23 transcripts were
significantly higher than the untreated controls,
P<0.05 (Figs 1 and 2). The relative value for CD23
transcripts from alveolar macrophages cultured
with rEQ IL-4 was increased more than 1,000-fold
when compared to LPS only or untreated controls
(Fig 3).
CONCLUSIONS
In conclusion, rEQ IL-4 induced equine CD23
mRNA transcript upregulation in PBMCs and
alveolar macrophages cultured for 24 and 48 h.
These findings support a role for equine CD23 in
allergic, anti-parasitic and other IL-4 mediated
immune responses in horses.
R
e
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a
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i
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m
R
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A

t
r
a
n
s
c
r
i
p
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i
o
n
12
10
8
6
4
2
0
1 2 3 4
IL4/LPS
LPS
Media
Fig 2: IL-4/LPS induced expression of CD23 in equine
PBML at 48 h. Mean values of relative CD23 mRNA
transcripts in PBML cultured with IL4/LPS, LPS alone
or media alone in 4 horses at 48 h.
16
14
12
10
8
6
4
2
0
R
e
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a
t
i
v
e

m
R
N
A

t
r
a
n
s
c
r
i
p
t
i
o
n
1 2 3 4
IL4/LPS
LPS
Media
Fig 3: IL-4/LPS induction of CD23 on equine alveolar
macrophages. Relative CD23 mRNA transcripts detected
on alveolar macrophages cultured with IL4/LPS, LPS
alone, IL4 alone or media alone at 24 and 48 h.
1000
1000
100
10
1 R
e
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a
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i
v
e

m
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t
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a
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s
c
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Fig 1: IL-4/LPS induced expression of CD23 in equine
PBML at 24 h. Mean values of relative CD23 mRNA
transcripts in PBML cultured with IL4/LPS, LPS alone
or media alone in 4 horses at 24 h.
45
IL4/LPS LPS IL4 Media IL4/LPS LPS IL4 Media
24 h 48 h
OPSONISATION BY COMPLEMENT C3 AND IgG AS
MEASURED BY A FLOW-CYTOMETRIC IMMUNOASSAY
G. Grndahl
Department of Large Animal Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala,
Sweden
INTRODUCTION
For a successful phagocytosis by neutrophils, the
microbes must be coated with serum opsonins (eg
antibodies and complement factor C3b), for which
the phagocytes have surface receptors. If the
opsonic capacity is low, the risk for infection
increases, such as in failure of passive transfer of
immunoglobulins in foals.
Knowledge of the mechanisms of complement
activation and deposition is important in studies of
phagocytosis as well as of immune evading
mechanisms of microbes. It is also critical in
immune-mediated and inflammatory diseases, and
in adverse reactions to artificial surfaces in
implants in blood vessels, other soft tissues and
hard tissues, eg catheters and bone plates.
When complement opsonisation is of interest, a
functional coating assay is more valuable than
measuring serum concentrations of the native
proteins, or the haemolytic capacity of serum
complement. However, traditional haemolytic
assays reflect the final result of the full complement
cascade reaction and involve many other steps and
proteins following initial activation and deposition.
Binding of C3 depends on the conversion of
serum C3 to opsonic C3b (Fig 1) by convertases
produced by 2 major complement pathways. In the
classical pathway, antibodies bound to the antigen
initiate the formation of the classical C3
convertase, C4b2a. The alternative pathway, on the
other hand, is promoted without the presence of
antibodies by spontaneously formed C3b which
persists on activating surfaces leading to the
formation of the alternative C3 convertase, C3bBb.
The objective of the present study was to
monitor the deposition of complement C3 and IgG
from equine serum on yeast cells (Saccharomyces
cerevisiae) using a flow cytometric immunoassay.
The effects of serum concentration and incubation
time were monitored. The opsonic coating was
further correlated with the phagocytic capacity
using equine blood neutrophils.
MATERIALS AND METHODS
5 x 10
6
heat-killed yeast cells in PBS
supplemented with 0.75 mM CaCl
2
and 2.5 mM
MgCl
2
(PBS++) were opsonised with pooled adult
horse serum at 37C.
To block the Mg
2+
-requiring convertases of
both complement pathways, 10 mM ethylene
diaminetetraacetic acid was added to non-
supplemented PBS (PBS-EDTA) (Fine et al. 1972).
Heating of serum to 56C for 30 min was also used
to inactivate components within both pathways.
To selectively block the Ca
2+
-dependent
classical pathway, 10 mM ethylene glycoltetraacetic
acid in PBS with 2.5 mM MgCl
2
was used (PBS-
EGTA) (Fine et al. 1972; Kozel 1996). Final serum
concentrations ranged from no serum or 0.7590 %
Fig 1: Schematic view of pathways for conversion of C3
to C3b.
Complement activation
Classical pathway
IgG, IgM
C3
C3b
Classical
C3-Convertase
Mg
2+
Ca
2+
EGTA
EDTA
heat
inhibition
Alternative
C3-Convertase
Alternative pathway
Activating surfaces
Mg
2+
EDTA
heat
46
Immunology in 2001
1000
100
1
1000
100
1
1000
100
1
1000
100
1
M
e
a
n

F
L
1

(
C
3
)
M
e
a
n

F
L
2

(
I
g
G
)
M
e
a
n

F
L
1

(
C
3
)
M
e
a
n

F
L
2

(
I
g
G
)
0.1 1 10 100
Serum concentration %
0.1 1 10 100
Serum concentration %
0 10 20 30
0 10 20 30
Time (min)
Time (min)
a) C3 deposition
b) C3 deposition kinetics
c) IgG deposition
d) IgG deposition kinetics
in the studies of the effect of serum dilution, and the
incubation time was 15 min. To study the deposition
kinetics, 5 or 50 % serum was used and the samples
were incubated for 0, 2, 4, 8, 15 or 30 min.
The opsonic coating of C3 and IgG was
analysed with a flow cytometric immunoassay. A
sheep anti-horse C3 polyclonal antibody (PC261,
The Binding Site Ltd, Birmingham, UK)
conjugated to a fluorochrome, BODIPY FL-CASE
(Molecular Probes, Eugene, Oregon, USA) was
used for detection of C3.
To detect IgG, a biotinylated polyclonal rabbit
anti-horse IgG antibody (308-065-003, Jackson
Immunoresearch Laboratories, Pennsylvania, USA)
was followed by streptavidin-R-phycoerythrin
(RPE) (R0438; DAKO, Glostrup, Denmark).
Phagocytosis assays were performed as
described by Johannisson et al. (1995). The yeast
cells were labelled with fluorescein isothiocyanate
(FITC) and opsonised with equine serum as
described above. EDTA-blood from 3 adult horses
was used, after lysis of erythrocytes with a solution
containing NH
4
Cl. The leucocytes and the
opsonised yeast were mixed with a neutrophil:yeast
cell ratio of 1:5 and incubated for 15 min at 37C
for phagocytosis. Fluorescence of extracellular
yeast particles was quenched with trypan blue. The
percentage of neutrophils with ingested yeast cells
was determined with flow cytometry.
RESULTS AND DISCUSSION
Opsonic coating of yeast with equine C3 and IgG
occurred rapidly. With 50% serum, high C3
deposition was observed as early as at 2 min (Fig
2b). This increased up to 15 min and remained at
the same level at 30 min. When only the alternative
pathway was allowed to be active by addition of
EGTA to 50% serum, or when the serum
concentration was low, at 5%, there was a lag
period of 15 min before C3 was detected (Fig 2b).
The alternative pathway alone could be responsible
for high C3-deposition at 30 min when 50% serum
was used. However, with only 5% serum, the
alternative pathway did not lead to any detectable
C3 opsonisation (Fig 2b), even after 30 min.
Substantial amounts of IgG were also
deposited within the first minutes of incubation,
but the values continued to increase over 30 min
when 50% serum was used (Fig 2d).
Inactivation of both complement pathways
with EDTA or by heating of serum to 56C for 30
min resulted in undetectable levels of deposited
C3 regardless of the incubation time or the serum
concentration (Figs 2a & b). When complement
was inactivated, the fluorescence levels for
attached IgG increased 3- to 6-fold, indicating
spatial competition between C3 and IgG at binding
(Figs 2c,d).
PBS ++
PBS-EGTA
PBS-EDTA
HI
PBS ++ 50%
PBS ++ 5%
PBS-EGTA 50%
PBS-EGTA 5%
PBS-EDTA 50%
PBS-EDTA 5%
HI 50%
HI 5%
PBS ++
PBS-EGTA
PBS-EDTA
HI
PBS ++ 50%
PBS ++ 5%
HI 50%
HI 5%
Fig 2: Deposition of C3 (a, b) and IgG (c, d) on yeast particles; a) and c) The effects of the serum concentration; b)
and d) The kinetics of deposition after opsonisation with 5 or 50% serum.
47
Opsonisation occurred already at low serum
concentrations, with detectable levels of deposited
C3 and IgG with as little as 0.75% serum.
Complement C3 and IgG deposition increased
with the serum concentration, as illustrated in Figs
2a and c. The fluorescence levels indicating IgG
deposition increased 15-fold between levels
observed with 0.75% serum and 90% serum.
Some results indicated that, normally, there is
less IgG than complement C3 on serum-opsonised
yeast cells, but quantitative experiments are
required to confirm this.
When the classical pathway of complement
activation was inhibited with EGTA, there was no
complement activation by the alternative pathway
when <50% serum was used. However, with 50%
serum or more, C3 was deposited also by the
alternative pathway, and at a concentration of 90%
serum, maximal C3 coating was detected with the
alternative pathway alone.
The results from the flow cytometric
immunoassay of opsonin deposition correlated
well with phagocytosis results (Fig 3). Serum
opsonisation was found to be of critical
importance for phagocytosis, with no phagocytosis
of non-opsonised yeast observed. Complement
activation was particularly important at low serum
concentrations. When the opsonising serum
concentration was low at 1.5%, suboptimal
phagocytosis was seen, with recruitment of about
half of the neutrophils in phagocytosis. This
phagocytic activity was dependent on complement
activation by the classical pathway, as there was
almost no phagocytosis with the use of EGTA or
EDTA.
When the serum concentration was increased
to 3%, the percentage of phagocytosing
neutrophils was doubled and the mean
fluorescence per cell, indicating uptake, was also
doubled. Similar magnitudes of phagocytic
activity were achieved with further increases of the
serum concentration (Fig 3).
A somewhat unexpected finding was that the
fluorescence indicating deposition of IgG was
enhanced 3- to 6- fold by complement inactivation.
Spatial competition between C3 and IgG may
account for this. Thus, more sites for antibody
binding may be exposed when complement coating
is diminished on the yeast surface. Masking of cell
surface antigens by C3b and iC3b has been
described (Pangburn 1983). In vivo, turning to more
IgG opsonisation in a situation with only a small
amount of complement would presumably be
beneficial for maintenance of the phagocytic
function. However, attempts to evaluate the impact
of IgG opsonisation on neutrophil phagocytosis of
yeast in the normal situation are not without
technical difficulties. If complement activation in
serum is inhibited, to separate and study the effect
of IgG, more IgG is deposited on the yeast cells, as
discussed, compared to opsonisation with unaltered
serum. This probably leads to an increased Fc-
receptor-mediated phagocytosis in these samples.
It may be concluded that the immunoassay
worked well, and opsonisation of yeast particles,
leading to phagocytosis, occurs at very low serum
concentrations (1.5 %). Further, that opsonisation is
dependent on activation of the classical complement
pathway at this low opsonic level. This is an
important finding for efficient host defence, eg
extravascular phagocytosis at infection sites.
REFERENCES
Fine, D., Marney, S., Colley, D., Sergent, J. and Des Prez,
R. (1972) C3 shunt activation in human serum
chelated with EGTA. J. Immun. 109, 807-809.
Johannisson, A., Grndahl, G., Demmers, S. and Jensen-
Waern, M. (1995) Flow-cytometric investigations of
the phagocytic capacities of equine granulocytes.
Acta vet. Scand. 36, 553-562.
Kozel, T.R. (1996) Activation of the complement system
by pathogenic fungi. Clin. microbiol. Rev. 9, 34-46.
Pangburn, M. (1983) Activation of complement via the
alternative pathway. Fed. Proc. 42, 139-143.
100
80
60
40
20
0
%

p
h
a
g
o
c
y
t
i
c

n
e
u
t
r
o
p
h
i
l
s
1 10 100
% serum
PBS
EGTA
EDTA
Fig 3: Percentage of neutrophils
phagocytosing yeast cells after
various opsonisations.
48
Immunology in 2001
FLOW CYTOMETRIC CHARACTERISATION OF
NEUTROPHIL PHAGOCYTOSIS AND OXIDATIVE
BURST ACTIVITY
S. L. Raidal
Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia
Methods for the evaluation of the functional
integrity of phagocytic cells are important tools for
investigating the causes of, and host response to,
disease. Chemotaxis, phagocytosis (the attachment
and internalisation of foreign particles) and the
intracellular degradation of ingested material are
the principal mechanisms by which phagocytes,
such as neutrophils, fulfil their protective role
within the body.
Flow cytometry methods have been developed
most extensively for the determination of cellular
phenotype, using monoclonal antibodies to cell
surface antigens coupled with fluorescent markers,
and for the evaluation of the nucleic acid content
of cells, using DNA or RNA binding dyes. The use
of this technology also expediates the evaluation of
cell function because it permits rapid evaluation of
large numbers of cells on a cell by cell basis,
providing greater precision than conventional
methods which rely on cell counting or the mean
response of a group of cells. Intra- and
extracellular events can be discriminated and
subpopulations of cells can be identified, negating
the need for prior cell separation, which is time
consuming and may alter cell function (Bertram
and Coignoul 1982; Fearon and Collins 1983;
Macey et al. 1992).
PHAGOCYTOSIS
Flow cytometry has been used to evaluate
phagocytosis by human neutrophils since the early
1980s and techniques have more recently been
described for the evaluation of phagocytic activity
of equine neutrophils (Foerster and Wolf 1990;
Johannisson et al. 1995; Raidal et al. 1998a). The
principle of the phagocytosis assay is illustrated in
Fig 1. Neutrophils are discriminated in mixed
leucocyte preparations following lysis of
erythrocytes, or may be separated by density
gradient techniques. Opsonins must be included in
assays of peripheral blood neutrophil
phagocytosis. Serum (or plasma), purified
immunoglobulins or complement may be utilised.
Fluorescent labelled particles (bacteria, yeast or
latex beads) are added to the cell suspension
following pre-opsonisation or the addition of
opsonins at the time of assay. Cells which interact
with fluorescent particles (by attachment to cell
surface membrane receptors and/or by
internalisation within the cell) have measurably
increased fluorescence and may readily be
differentiated from cells which do not have
cells
bacteria
ethidium bromide trypan
blue
Fig 1: The principle of flow cytometric phagocytosis
assays. Cells are mixed with a source of opsonins for 10
min at 37C or fluorescent particles (latex beads,
bacteria or yeast cells) are opsonised prior to assay.
Aliquots of cell suspension are withdrawn for flow
cytometric evaluation. Fluorescent particles are added
to the cell suspension in an agitating water bath. Cells
able to interact with fluorescent bacteria are
discriminated (because of their increased fluorescence)
from cells unable to do so. Cells which have internalised
bacteria are further differentiated by removing the
fluorescence of externalised particles by quenching
(trypan blue) or counterstaining (eg ethidium bromide).
49
attached or internalised bacteria. The intensity of
fluorescence of positive cells is proportional to the
fluorochrome used, the staining intensity of
labelled particles, the number of particles
interacting with the cell and the amount of protein
or DNA degradation occurring within the cell. If a
pH sensitive fluorochrome is used, fluorescence
intensity is also influenced by the pH of the
phagolysosome.
Attached particles can be differentiated from
internalised particles by quenching or
counterstaining the fluorescence of attached
particles, or by the lysis of attached bacterial cells.
All methods are depended on a process which does
not change the fluorescence of particles inside the
cell membrane, but alters the fluorescence of free
or attached particles. Quenching involves the
extinction of fluorescence and is achieved most
commonly by the addition of trypan blue, which
effectively quenches fluorescein isothiocyanate
(FITC) (Raidal et al. 1998a) and propidium iodide
(Flaminio et al. 1999) fluorescence but is unable to
penetrate the cytoplasmic membrane of viable
cells. Counterstaining to discriminate attached
bacteria may be achieved by using a different
colour fluorochrome to change fluorescence by the
process of resonant energy transfer (Fattorossi et
al. 1989), or by using an antibody to the
fluorochrome label derivatised to an alternate
fluorescent label (Sveum et al. 1986). Lysis of
externalised bacteria has been described in an
assay utilising whole blood (White-Owen et al.
1992).
Estimates of the number of attached and/or
internalised particles per cell have been reported
using direct and indirect methods. Direct methods
derive an estimate of the number of particles per
cell from the mean fluorescent intensity of the
positive cell population. This technique is not
suitable when a pH-sensitive dye, such as FITC, is
used. Comparison of mean fluorescence of
positive cells has been used as an indicator of
phagocytic capacity without quantitation of the
number of organisms per cell. Refinements of this
technique have been described to evaluate protein
degradation within the cell, intracellular killing
(Basse and Bjerknes 1985) and phagolysosome
pH (Basse et al. 1983a; Rothe and Valet 1988).
Indirect methods for the determination of number
of particles per cell are based on subtraction of the
number of particles which have not interacted with
cells at the completion of the assay from the
number of particles initially added to the assay,
divided by the number of cells in the assay (Basse
et al. 1983b; Bjerknes and Basse 1984).
Flow cytometric studies of equine neutrophil
function have utilised fluorescent microspheres
(Foerster and Wolf 1990), bacteria (Johannisson et
al. 1995; Raidal et al. 1997; 1998a; 2000; 2001;
Flaminio et al. 2000; McTaggart et al. 2001) or
yeast particles (Grndahl et al. 1997, 1999).
Commercially prepared latex particles are of
uniform size and staining intensity. Phagocytosis
assays performed with beads give characteristic
multi-peaked positive populations, with each peak
corresponding with an integer number of
associated particles (Fujikawa-Yamamoto and
Odashima 1987; Fredrickson et al. 1992).
However, beads are non-physiological and require
treatment, such as albumin coating, to aid
phagocytosis. Bacterial species utilised in studies
of equine neutrophil phagocytosis include
Staphylococcus aureus (Raidal 1996; Flaminio et
al. 2000; McTaggart et al. 2001), Actinobacillus
spp. (Raidal et al. 1997, 1998a, 2000, 2001), E.
coli (G. Grndahl et al. unpublished data) and
Streptococcus equi ss. zooepidemicus (Raidal
1996). Fluorescent labelled S. zooepidemicus
produced a more heterogeneous fluorescence
histogram than other bacterial species, presumably
due to variation in chain length (Raidal 1996).
Fluorochromes used for phagocytosis assays
bind cellular protein or nucleic acid. FITC and
propidium iodide are used most commonly,
although Lucifer yellow and ethidium bromide
have also been used successfully to label bacteria
for the evaluation of phagocytosis by equine
neutrophils (Raidal 1996).
Assay conditions, such as the particle-
fluorochrome combination, the number of
fluorescent particles, staining intensity of
particles, source and amount of opsonins and flow
cytometer settings must be strictly controlled to
achieve reproducible results. An external standard
should be passed through the flow cytometer prior
to performing repeat assays to ensure machine
settings have not altered. The number of
neutrophils present in the assay has been shown
not to affect the percentage of phagocytosing cells,
within the range of 112 x 10
9
/l (Hed et al. 1987),
although neutrophil numbers are usually adjusted
to a more narrow range (57 x 10
9
/l). The ideal
particle:neutrophil ratio has not been determined.
Increased numbers of fluorescent particles are
associated with an increased percentage of positive
neutrophils and an increased number of particles
50
Immunology in 2001
per cell. Low particle:neutrophil ratios may not
discriminate functional differences between
different cell populations (Miller 1979).
Cells only, or cells plus serum, are used as a
negative control, prior to running a phagocytosis
assay. An aliquot of cells should be removed
immediately after the addition of fluorescent
particles (0 min), mixed with an equal volume of
PBS with 0.2% w/v EDTA and kept on ice, to
arrest phagocytosis. Further aliquots of cells are
removed serially during incubation in a 37C
agitating water bath and similarly mixed with
PBS-EDTA and placed on ice. Evaluation of the
neutrophil population during the assays shows a
progressive increase in the percentage of positive
cells, from a small number at 0 min (usually less
than 5%), to peak usually after 2030 min of
incubation. Extending the assay to 60 min of
incubation demonstrates little further increase in
the percentage of positive cells (the percentage of
positive cells may decrease, presumably
associated with reduced fluorescence of
internalised bacteria due to increased acidity of the
phagolysosome and the degradation of bacterial
protein). Mean fluorescence of the positive cell
population increases initially (more cells in the
brighter fluorescence channels) as the number of
bacteria per cell increases, and may decrease after
2030 min due to internalisation of particles,
protein or NA degradation and the more acidic
environment within the cells.
OXIDATIVE BURST ACTIVITY
The evaluation of neutrophil oxidative burst
activity is performed routinely on human
neutrophils to identify individuals affected by
disorders such as chronic granulomatous disease.
The technique has been utilised for the evaluation
of equine neutrophil function (Adamson and
Slocombe 1995; Raidal et al. 1998b, 2000, 2001;
Flaminio et al. 2000; McTaggart et al. 2001). The
oxidative (or respiratory) burst is a co-ordinated
series of metabolic events initiated when
neutrophils are exposed to appropriate stimuli. In
addition to the action of neutrophil granule
contents, the oxidising agents of the respiratory
burst, such as superoxide, peroxide, hydroxyl
radical, singlet oxygen and halogenated
hydrocarbons, are important for the intra-cellular
killing and digestion of microbes.
Flow cytometric methods for the evaluation of
oxidative burst are based on the measurement of
intracellular oxidative reactions in single cells. The
basic principle of most methods is illustrated in
Fig 2 and involves loading cells with a non-
fluorescent precursor substance which is readily
oxidised to a fluorescent compound by
components of the oxidative burst. After loading,
cells are exposed to a stimulant to induce an
oxidative burst response, the magnitude of which
is proportional to the fluorescence intensity
generated. Most referenced techniques include the
addition of azide or cyanide, to inhibit
mitochondrial oxidation and hence reduce the
amount of oxidation of the substrate prior to the
addition of the stimulant (usually referred to as
auto-oxidation) and increase the magnitude of the
stimulated oxidative burst.
Dihydrorhodamine, hydroethidium and 2',7'-
dichlorofluorescin diacetate are the non-
fluorescent substrates most commonly used in
published techniques. These substrates are
oxidised to the fluorescent derivatives rhodamine
123, ethidium and 2',7'-dichlorofluorescein
respectively. Hydroethidium is oxidised primarily
by the superoxide anion and dichlorofluorescin is
oxidised by peroxide (Bass et al. 1983) and
DCFH-DA
DCF
H2O2
DCFH
Fig 2: The principle of flow cytometric oxidative burst
assays. Cells are mixed for 15 min at 37C in an agitating
water bath with a non-fluorescent precursor, such as 2',7'-
dichlorofluorescin diacetate (DCFH-DA), which freely
permeates the cytoplasmic membrane. Non-specific
esterases within the cytosol cleave the diacetate molecule,
leaving non-fluorescent 2',7'-dichlorofluorescin (DCFH),
which is a polar molecule and hence unable to pass
through the cell membrane. Oxidative metabolism is
stimulated within the cell by the addition of a soluble
stimulant (such as phorbol myristate acetate) or by
phagocytosis. DCFH is oxidised by peroxide to the
fluorescent compound 2',7'-dichlorofluorescein (DCF).
The intensity of fluorescence of each cell is proportional
to the amount of DCF present within the cell and hence
the magnitude of the oxidative response.
51
possibly other reactive oxygen (Rothe and Valet
1990) and nitrogen (Rao et al. 1992) species.
Oxidative burst activity may be initiated by
particulate stimuli, such as opsonised zymozan or
bacteria, or soluble stimulants, including zymozan
activated serum, phorbol esters, a variety of
ionophores, bacterial products (Tarigan et al.
1994; Raidal et al. 1998b) and N-formyl
methionyl peptides (Lehmeyer et al. 1979).
Oxidative activity stimulated by soluble
stimulants is independent of phagocytic function
and such assays do not require the inclusion of an
opsonin. The suitability of phorbol myristate
acetate (PMA) as a stimulant of oxidative burst
activity in equine neutrophils has been reported
(Raidal et al. 1998b). Ethidium bromide (Raidal
et al. 1998b) and propidium iodide-labelled
bacteria (Flaminio et al. 2000) have also been
used to stimulate oxidative burst activity. The
magnitude of the response generated was not as
great as that elicited by PMA stimulation and
peaked after 60 min of incubation. Such
techniques have permitted the simultaneous
measurement of phagocytosis and oxidative burst
activity (Raidal 1996; Smits et al. 1997; Flaminio
et al. 2000), although care must be taken to ensure
appropriate compensation of spectral overlap
between fluorochromes (more difficult in a
dynamic assay where fluorescence intensity
increases over time), and conclusions on the
oxidative capacity of cells must be tempered by
awareness that the magnitude of response
generated is influenced by the efficiency of
phagocytosis (Raidal 1996).
CONCLUSIONS
Flow cytometric techniques are effective tools for
the evaluation of non-specific immunity in
response to a variety of stressors and for the
elucidation of phagocytosis and oxidative burst
events within equine neutrophils. The availability
of fluorescent markers for the evaluation of
calcium flux, other intracellular events and cellular
antigen expression offer new opportunities to
improve understanding of the mechanisms of
health and disease in this species.
ACKNOWLEDGEMENTS
Portions of work completed by the author have
been funded by the New South Wales Racing
Research Fund and the Rural Industries Research
and Development Corporation. The author is
supported by the National Australia Bank.
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Grndahl, G., Johannisson, A. and Jensen-Waern, M.
(1997) Opsonic effect of equine plasma from
different donors. Vet. Microbiol. 56, 227-235.
52
Immunology in 2001
Grndahl, G., Johannisson, A., Demmers, S. and Jensen-
Waern, M. (1999) Influence of age and plasma
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(2000) Effect of single bouts of moderate and high
intensity exercise and training on peripheral blood
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Smits, E., Burvenich, C. and Heyneman, R. (1997)
Simultaneous flow cytometric measurement of
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Sveum, R.J., Chused, T.M., Frank, M.M. and Brown, E.J.
(1986) A quantitiative fluorescent method for
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Tarigan, S., Slocombe, R.F., Browning, G.F. and
Kimpton, W. (1994) Functional and structural
changes of porcine alveolar macrophages induced
by sublytic doses of a heat labile, haemolytic,
cytotoxic substance produced by Actinobacillus
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White-Owen, C., Alexander, J.W., Sramkoski, R.M. and
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2071-2076.
53
RT-PCR DETECTION OF EQUINE CYTOKINES
D. W. Horohov
Louisiana State University, Department of Pathobiological Sciences, School of Veterinary Medicine,
Patterns of cytokine gene expression can be
considered characteristic of various protective and
pathological conditions (Powrie and Coffman
1993). While the initial detection of cytokines in
human medicine and murine models utilised a
variety of bioassays, current methods rely almost
exclusively on commercially available monoclonal
antibodies. Few antibodies are available for equine
cytokine detection and researchers must rely
instead upon bioassays or mRNA detection
techniques to measure cytokine expression in the
horse. The latter approach has been facilitated by
the cloning and sequencing of a number of equine
cytokine genes. A number of methodologies are
available for detecting equine cytokine-specific
mRNA expression including northern blots, in situ
hybridisation (Aggarwal and Holmes 1999),
quantitative RT-PCR (Swiderski et al. 1999a),
competitive PCR (Gigue`re and Prescott 1999) and,
more recently, real time PCR (Leutenegger et al.
1999). While each of these techniques has its
unique characteristics, they all share several
common features. First, they all rely upon the
assumption that cytokines are transcriptionally
regulated. Secondly, vagaries in the process of
RNA isolation and reverse transcription
necessitate the use of internal standards to
compensate for yield differences between samples.
Lastly, the detection of the product, either directly
or following PCR amplification, also varies
between the techniques and ultimately requires the
determination of copy number within the starting
sample.
Protein expression in the cell can be regulated
by various mechanisms including transcriptional,
translational, post-translational or a combination
of all 3 processes. Cytokine gene regulation occurs
primarily at the transcriptional level and is
mediated by DNA-binding proteins interacting
with specific recognition motifs in genetic
promoter and enhancer elements (Ye and Young
1997), although interleukin 1 and other
inflammatory cytokines are also regulated post
transcriptionally (Fenton 1992). As such, detection
of mRNA levels for T cell cytokines is considered
comparable to detecting the protein itself (OGarra
and Vieira 1992). Nevertheless, issues remain
regarding the impact of mRNA half-life and
transcriptional rates on RNA-based detection
procedures.
Selection of a suitable internal control for
RNA detection assays is likewise not without
controversy. Since current RNA isolation
procedures can result in variations, both in RNA
yield and integrity, a housekeeping gene is used
to correct for inter sample variability. A number
of housekeeping genes, including -actin (Suzuki
et al. 2000), glyceraldehyde-3-phosphate
dehydrogenase (GADPH) (Rottman et al. 1996),
hypoxanthine phosphoribosyl-transferase (HPRT)
(Murphy et al. 1993), and peptidyl-prolyl-cistrans
isomerase (Cyclophilin) (Dozois et al. 1997) have
been utilised in the analysis of cytokine mRNA
content. One concern regarding any housekeeping
gene is the effect of activation state on its
expression (Schmittgen and Zakrajsek 2000).
Thus, 1.3- to 3-fold increases in -actin message
have been reported in response to mitogen
stimulation using cloned T cells and normal
human PBMC (McCairns 1984). Increases in
HPRT production in the order of 1020 fold have
been demonstrated in mitogen stimulated human
lymphocytes (Steen et al. 1991) making HPRT a
less favourable internal control for immunological
studies. The primary drawback of -actin is that it
is a moderately abundant transcript (0.1% of
cellular mRNA) whose expression is several
orders of magnitude greater than cytokines, a
Immunology in 2001
54
problem not significantly different for GADPH,
cyclophylin and 18s rRNA which are,
respectively, moderately abundant, slightly less
than moderately abundant transcripts and most
abundant (Spencer and Christensen 1999). While
each selection has its advocate and detractors
(Schmittgen and Zakrajsek 2000), -actin was
chosen as the internal control in our assays
because the equine sequence has been determined
allowing the generation of sequence-specific
oligonucleotide probes and primers. Further,
Swiderski et al. (1999a) have shown that -actin is
an accurate normalisation factor for inter-sample
differences in the RT-PCR assay.
To determine the amount of mRNA in the test
samples amplifications from cloned cDNA of
identical sequence to the wild type target were
used to generate standard curves for the
quantification of PCR products (Fig 1). Standard
curves are used routinely in a variety of scientific
disciplines, including immunology, to evaluate
enzymatically dependent phenomena
characterised by an exponential rise to maximum.
PCR standard curves derived from dilutions of
cloned cDNA sequences have been described
(Melby et al. 1993). Swiderski et al. (1999a)
demonstrated using equine interleukin (IL)-10,
that the quantity of mRNA can be determined via
RT-PCR using standard curves generated from
dilutions of double stranded circular plasmids
encoding the transcript of interest; and that, by
decreasing the cycle number and cDNA volume
used for -actin amplification, as compared to
cytokine amplifications, -actin is an accurate
normalisation factor for the differences that are
inherent in the RT step of the assay. This approach
eliminates the need for synthetic templates
common to competitive PCR methods. This
method is also less labour intensive because there
is no internal standard to construct nor is there a
large series of samples with differing
competitor:target ratios. Most significantly, this
method is performed easily in a single day with
very high sample throughput, from cells to
quantified product in replicates sufficient for
statistical analysis of the data.
This procedure has been utilised to determine
cytokine-specific mRNA levels in a variety of
samples and model systems including the
characterisation of a protective Th2 response to
Strongylus vulgaris (Swiderski et al. 1999b),
demonstration of a Th1 response in equine
recurrent uveitis (Gilger et al. 1999, 2000), and
demonstration of a modified Th2 response in
summer pasture-associated obstructive pulmonary
disease (Horohov 2000). Until monoclonal
antibodies to equine cytokines become readily
available, we and others shall continue to rely
upon these types of procedure to measure cytokine
responses in the horse.
REFERENCES
Aggarwal, N. and Holmes, M.A. (1999) Characterisation
of equine T helper cells: demonstration of Th1- and
Th2-like cells in long-term equine T-cell cultures.
Res. vet. Sci. 66, 277-279.
Dozois, C.M., Oswald, E., Gautier, N., Serthelon, J.P.,
Fairbrother, J.M. and Oswald, I.P. (1997) A reverse
transcription-polymerase chain reaction method to
analyse porcine cytokine gene expression. Vet.
Fenton, M.J. (1992) Review: transcriptional and post-
transcriptional regulation of interleukin 1 gene
expression. Int. J. Immunopharmacol. 14, 401.
Gigure, S. and Prescott, J.F. (1999) Quantitation of
6000
5000
4000
3000
2000
1000
0
L
u
m
i
n
o
s
i
t
y
16 18 160 480 1600 4800 .4 1.2 4 12 40 120 400
Copy number Copy number
(x 1,000) (x 100)
B-actin IFN-
Fig 1: Standard curves. Various
dilutions of -actin and
interferon- (IFN-) plasmid
DNA were amplified in a PCR
reaction and detected using a
fluorescence-based detection
system (QPCR 5000; Perkin
Elmer). The luminosity values are
plotted against the input DNA (in
copy numbers) to yield a standard
curve. Note the difference in input
copy numbers between -actin
and the cytokine reflecting their
relative abundance in cellular
RNA samples.
55
Vet. Immunol. Immunopathol. 67, 1-15.
Gilger, B.C., Malok, E., Cutter, K.V., Stewart, T.,
Horohov, D.W. and Allen, J.B. (1999)
Characterisation of T-lymphocytes in the anterior
uvea of eyes with chronic equine recurrent uveitis.
Gilger, B.C., Malok, E., Stewart, T., Horohov, D.,
Ashton, P., Smith, T. Jaffe, G.J. and Allen, J.B.
(2000) Effect of an intravitreal cyclosporine implant
on experimental uveitis in horses. Vet. Immunol.
Horohov, D.W. (2000) Equine T-cell cytokines.
Protection and pathology. Vet. Clin. North Am.
equine Pract.16, 1-14.
Leutenegger, C.M., von Rechenberg, B. Huder, J.B.,
Zlinsky, K., Mislin, C., Akens, M.K., Auer, J. and
Lutz, H. (1999) Quantitative real-time PCR for
equine cytokine mRNA in nondecalcified bone
tissue embedded in methyl methacrylate. Calcif.
Tissue Int. 65, 378-383.
McCairns, E., Fahey, D., Muscat, G., Murray M. and
Rowe, P.B. (1984) Changes in levels of actin and
tubulin mRNAs upon the lectin activation of
lymphocytes. Mol. cell. Biol. 4, 1754-1760.
Melby, P.C., Darnell, B.J. and Tryon, V.V. (1993)
Quantitative measurement of human cytokine gene
expression by polymerase chain reaction.
J. immunol. Methods 159, 2235-244.
Murphy, E., Hieny, S., Sher, A. and OGarra, A. (1993)
Detection of in vivo expression of interleukin-10
using a semi- quantitative polymerase chain reaction
method in Schistosoma mansoni infected mice.
J. immunol. Methods. 162, 211-223.
OGarra, A. and Vieira, P. (1992) Polymerase chain
reaction for detection of cytokine gene expression.
Curr. Opin. Immunol. 4, 211-215.
Powrie, F. and Coffman, R. (1993) Cytokine regulation
of T-cell function: potential for therapeutic
intervention. Immunol. Today 14, 270-274.
Rottman, J.B., Tompkins W.A. and Tompkins M.B.
(1996) A reverse transcription-quantitative
competitive polymerase chain reaction (RT-qcPCR)
technique to measure cytokine gene expression in
domestic mammals. Vet. Pathol. 33, 242-248.
Schmittgen, T.D. and Zakrajsek, B.A. (2000) Effect of
experimental treatment on housekeeping gene
expression: validation by real-time, quantitative RT-
PCR. J. biochem. biophys. Methods 46, 69-81.
Spencer, W. E., and Christensen, M.J. (1999) Multiplex
relative RT-PCR method for verification of
differential gene expression. Biotechniques 27,
1044-1052.
Suzuki, T., Higgins, P.J. and Crawford, D.R. (2000)
Control selection for RNA quantitation.
Biotechniques 29, 332-337.
Steen, A.M., Sahlen, S. and Lambert, B. (1991)
Expression of the hypoxanthine phosphoribosyl
transferase gene in resting and growth-stimulated
human lymphocytes. Biochim. biophys. Acta 1088,
77-85.
Swiderski, C.E., Klei, T.R. and Horohov, D.W. (1999a)
Quantitative measurement of equine cytokine
mRNA expression by polymerase chain reaction
using target-specific standard curves. J. immunol.
Methods 222, 155-169.
Swiderski, C.E., Klei, T.R., Pourciau, S., Chapman,
M.R., Moore, R., McClure, J.R. and Horohov, D.W.
(1999b) T cell cytokine responses to Strongylus
vulgaris in infected and vaccinated ponies. Proc. 8th
int. Conf. equine inf. Dis. Eds: U. Wernery, J.F.
Wade, J.A. Mumford and O.-R. Kaaden. R&W
Publications (Newmarket) Ltd, pp 206-210.
Ye, J. and Young, H.A. (1997) Negative regulation of
cytokine gene transcription. Faseb J. 11, 825-833.
Immunology in 2001
56
EQUINE CYTOKINES AND ASSOCIATED REAGENTS
L. Nicolson, L. McMonagle, S. Taylor, C. Hopkins, L. Sanders*,
H. van Kuilekom*, N. Scholtes*, D. Argyle, D. Onions and V. Schijns*
Department of Veterinary Pathology, Veterinary School, Bearsden Road, Bearsden, Glasgow G61 1QH,
UK; *Intervet International B.V., Boxmeer, Netherlands
INTRODUCTION
Cytokines are a group of immunologically active
peptides which are being applied increasingly in
therapeutic and prophylactic regimes in
experimental models and in clinical cases. We are
interested in the potential application of cytokines as
vaccine adjuvants and for the treatment of diseases
such as cancer and arthritis in domestic animals. Our
current focus in the horse is on cytokines associated
with Th1 type immune responses interferon- and
interleukins-12 and -18.
EQUINE INTERFERON-
Interferon- (IFN-) has a range of biological
activities including activation of macrophages,
MHC class I and II induction, promotion of NK
cell activity and regulation of other cytokines
(Farrar and Schreiber 1993). Equine IFN- (eIFN-
) was cloned by 2 groups independently in 1994
(Curran et al. 1994; Grunig et al. 1994). We have
expressed eIFN- in bacterial, baculovirus and
mammalian systems. Biological activity has been
detected by vesicular stomatitis virus cytopathic
effect reduction assay for products derived from
bacterial, baculovirus and mammalian expression
systems (manuscript in preparation). No
bioactivity has been associated with bacterial
eIFN- preparations and it is possible that the
purification process inactivates the recombinant
cytokine.
Polyclonal antibody preparations raised
against bacterial recombinant IFN- have been
used to derive a sandwich ELISA with a lower
detection limit of approximately 100 pg/ml for
recombinant eIFN-. This has been applied to the
analysis of upregulation of IFN- in PBMCs
incubated with IL-12 preparations (see below).
Analysis of cell lysates and supernatants from cells
transiently transfected with eIFN- constructs
indicates that secretion of bioactive equine IFN-
occurs in baculovirus and mammalian expression
systems.
INTERLEUKIN-12
Interleukin-12 (IL-12) is a key cytokine in the
development of Th1 responses through its role in
the development of T cells and upregulation of
IFN- production (Gately et al. 1998). Human IL-
12 is a heterodimeric cytokine (p70) formed by
p35 and p40 subunits. Expression of p40 in vitro
and in vivo is associated with formation of
homodimeric p40 molecules which antagonise p70
activity. To circumvent the inhibitory activity
associated with p40 homodimer formation, we
have adopted the approach of Lieschke et al.
(1997) and synthesised equine IL-12 (Nicolson et
al. 1999) as a single chain construct with the p35
and p40 subunits covalently linked through a
glycine/serine-rich peptide. Expression of single
chain equine IL-12 was confirmed by Western blot
using an anti-feline p40 peptide serum or a
commercially available cross reactive bovine p40
MAb. A bicistronic construct in which the p35 and
p40 cDNAs were separated by an IRES element
was generated as a source of p70.
Detection of bioactivity of IL-12 expressed in
baculovirus and mammalian expression systems
was achieved through analysis of IFN- induction
by equine mononuclear cells. Culture supernatants
from cells transfected with single chain or
bicistronic IL-12 constructs or infected with
baculovirus were positive for IFN- induction in
this assay. Levels of induction were reduced by
prior incubation of supernatants with anti-IL-12
antibody preparations. Human recombinant IL-12
57
upregulated IFN- production in this assay.
Bioactivity of baculovirus-expressed eIL-12 was
also verified using a proliferation assay in which
infected culture supernatants were incubated with
equine PBMCs pre-incubated with PHA and IL-2
(McMonagle et al. 2001).
INTERLEUKIN-18
Interleukin-18 (IL-18) is a potent inducer of IFN-
and acts synergistically with IL-12 in this respect
(reviewed in Dinarello 2000). IL-18 is synthesised
as an inactive precursor which requires cleavage of
an N-terminal peptide by caspase-1 for release of
bioactive mature IL-18. Wardlow et al. (1999)
cloned equine caspase-1 cDNA and are currently
investigating secretion and bioactivity associated
with several forms of equine IL-18 constructs in
vitro.
A set of monoclonal antibodies to equine IL-
18 have been recovered. Based on cross reactivity
with feline IL-18, it was surmised that a minimum
of 2 epitopes are recognised. A sandwich ELISA
has been developed with a sensitivity of
approximately 2 ng/ml for detection of bacterially
generated recombinant equine IL-18.
Human myelomonocytic KG-1 cells are
inducible for IFN- production on incubation with
human and murine IL-18, although the sensitivity
of detection of the latter is reduced 100-fold
(Konishi et al. 1997). Incubation of cell lysates
harvested from equine proIL-18 transfected cells
with human recombinant caspase-1 resulted in
detection of an IL-18 protein of a size consistent
with that of predicted mature IL-18. Incubation of
this material with KG-1 cells was associated with
induction of human IFN- as assessed using a
commercial human IFN- ELISA. This is
suggestive of equine IL-18 bioactivity in the KG-1
assay although the sensitivity relative to that of
human IL-18 is unknown. Harvests from cells
transfected with IL-18 constructs with an artificial
signal peptide were also associated with induction
of IFN- by these cells. Preliminary data indicate
that the level of IFN- induction can be
diminished through incubation of cell lysates with
anti-IL-18 monoclonal or polyclonal antibody
preparations prior to application to KG-1 cells.
THE FUTURE
We now have a complement of equine-specific
cytokine and serological reagents and have
verified bioactivity of equine IFN-, IL-12 and IL-
18. The next step will be the assessment of these
cytokines as vaccine adjuvants in the horse.
REFERENCES
Dinarello, C.A. (2000) Interleukin-18, a pro-
inflammatory cytokine. Eur. Cytokine Netw. 2000
11, 483-486.
Curran, J.A., Argyle, D.J., Cox, P., Onions, D.E. and
Nicolson, L. (1994) Nucleotide sequence of the
equine interferon- cDNA DNA Sequence 4, 405-
407.
Farrar, M.A. and Schreiber, R.D. (1993) The molecular
cell biology of interferon- and its receptor. Ann.
Rev. Immunol. 11, 571-611.
Gately, M.K., Renzetti, L.M., Magram, J., Stern, A.S.,
Adorini, L., Gubler, U. and Presky, D.H. (1998) The
interleukin-12/interleukin-12-receptor system:role
in normal and pathologic immune responses. Ann.
Rev. Immunol. 16, 495-521.
Grunig, G., Himmler, A. and Antczak, D.F. (1994)
cloning and sequencing of horse interferon- cDNA.
Immunogenetics 39, 448-449.
Konishi, K., Tanabe, F., Taniguchi, M., Yamauchi, H.,
Tanimoto, T., Ikeda, M., Orita, K. and Kurimoto, M.
(1997) A simple and sensitive bioassay for the
detection of human interleukin-18/interferon--
inducing factor using human myelomonocytic KG-1
cells. J immunol Methods 209,187-191.
Lieschke, G.J., Rao, P.K., Gately, M.K. and Mulligan,
R.C. (1997) Bioreactive murine and human
interleukin-12 fusion proteins which retain anti-
tumour activity in vivo. Nat. Biotechnol. 15, 35-
40.
McMongle, E.L.J., Taylor, S., van Zuileom, H., Sanders,
L., Scholtes, N., Keanie, L.J., Hopkins, C.A., Logan,
N.A., Bain, D. Argyle, D.J., Onions, D.E., Schijns,
V.E. and Nicolson, L. (2001) Production of
biologically active equine interleukin 12 through
expresson of p35, p40 and single chain IL - 12 in
mamalian and baculovirus expression systems.
Equine vet. J. In press.
Nicolson, L., Penha-Goncalves. M.N., Keanie, J.L.,
Logan, N.A., Argyle, D.J. and Onions, D.E. (1999)
Nucleotide sequence of equine interleukin 12 and 18
cDNA. Immunogenetics 50, 94-97.
Wardlow, S., Penha-Goncalves, M.N., Argyle, D.J.,
Onions, D.E. and Nicolson, L. (1999) Nucleotide
sequence of equine caspase-1 cDNA. DNA sequence
10, 133-137.
Immunology in 2001
58
QUANTITATION OF EQUINE CYTOKINE mRNA
EXPRESSION BY RT-PCR
S. Gigu` ere
University of Florida, Department of Large Animal Clinical Sciences, College of Veterinary Medicine,
Gainesville, Florida 32610, USA
Cytokines are hormone-like low molecular weight
proteins produced by various types of somatic
cells in response to a number of inducing stimuli.
Cytokines regulate both the initiation and
maintenance of immune and inflammatory
responses. They also play a role in many important
biological functions such as embryological growth
and development, stem cell growth and
differentiation, tissue repair, endocrine functions,
aging, etc. Over the last 10 years, classification of
murine and, to a lesser extent, human T
lymphocytes into type 1 and type 2 subsets has
provided a useful framework for understanding
immune response in infectious diseases (Mossman
and Sad 1996). Although it remains to be
definitively established whether this paradigm can
be applied to the horse, preliminary studies
suggest the presence of type 1- and type 2-like
cells in equine long-term T-cell culture (Aggarwal
and Holmes 1999). Knowledge of the equine
immune system and understanding of the
pathophysiology of inflammatory and infectious
diseases of the horse and other domestic animal
species has been limited in part by the lack of
species-specific reagents available for measuring
cytokines. Some progress has been made by
adapting bioassays for only a few cytokines that
function in a non-species-specific manner
(Seethanathan et al. 1990; Morris and Moore
1991; Mackay and Lester 1992). However, such
assays are cumbersome and have low specificity,
and some equine cytokines such as IL-2 and IL-4
do not exert activity on murine cells (Dohmann et
al. 2000). The development of immunoassays in
the horse has been limited by the lack of species-
specific monoclonal antibodies and cytokine
standards. Because of this limitation with
bioassays and immunoassays in the horse, and
because of the apparent correlation between
cytokine gene expression and protein production
(Cherwinski et al. 1997), several researchers have
relied on detection of equine cytokine mRNA
expression. Thus the cloning, sequencing and
expression of a number of equine cytokines have
been accomplished (Curran et al. 1994; Grunig et
al. 1994; Kato et al. 1997; Kato et al. 1995; Penha-
Goncalves et al. 1997; Su et al. 1991; Nicolson et
al. 1999). Equine-specific genomic or cDNA
sequences currently available in GenBank include
IL-1 IL-1, IL-1RA, Il-2, IL-4, IL-5, IL-6, IL-8,
IL-10, IL-12 p35, IL-12 p40, IL-18, IFN-, IFN-,
IFN-, IFN, TNF-, and TGF-.
Several techniques are available to detect
mRNA expression. Because it is rapid and
considerably more sensitive than traditional RNA
blot techniques, RT-PCR is increasingly being
used to detect small changes in gene expression
that would otherwise be undetectable. Although
RT-PCR offers many advantages over RNA blot
methods, it can be difficult to obtain quantitative
information. This is mainly because of the
exponential nature of PCR, where small tube-to-
tube variation in amplification efficiency have
dramatic effects on product yield. Furthermore, the
amount of PCR product generated reaches a
plateau after several cycles of amplification
through consumption of necessary components
and the generation of inhibitors, obscuring
differences in the initial amount of target DNA
(Gause and Adamovicz 1995). Truly quantitative
methods are required for accurate comparison of
cytokine expression between groups of animals or
in response to treatment or vaccination. A
quantitative RT-competitive PCR (RT-cPCR) assay
for the measurement of equine cytokines has been
developed (Gigure and Prescott 1999). This assay
proved accurate and reproducible for quantitation
of mRNA expression for cytokines expressed at a
59
high level. However, quantitation of mRNA
expression for cytokines expressed at low levels
such as IL-4 and IL-5 is difficult or sometimes
impossible using RT-cPCR (Gigure et al. 1999).
Also, RT-cPCR requires testing several dilutions
of a competitor DNA or RNA fragment along with
the sample. This is labour intensive and the
amount of sample used for each dilution limits the
number of cytokines that can be measured and
prevents replicate determination of the same
samples. Furthermore, RT-cPCR entails laborious
post PCR processing steps for quantitation of
target and competitor fragments. Extensive
manipulation of post PCR products is also a
significant source for contamination of pre-PCR
samples.
The ability to monitor the real-time
progress of PCR has completely revolutionised
quantitation of DNA and RNA. Real-time PCR
detects specific nucleic acid amplification
products as they accumulate by using a
fluorogenic oligonucleotide probe. Attached to the
hybridisation probe are a reporter fluorescent dye
and a quencher dye. In an intact probe, emission
from the reporter dye is minimal due to the energy
transfer to the quenching dye and negligible
fluorescence is observed. During PCR
amplification, as the newly synthesised DNA
strand reaches the site of probe hybridisation, the
DNA polymerase cleaves the probe, releasing the
reporter dye from the quencher dye. The
fluorescence intensity therefore increases
proportionally to the amount of DNA amplified.
The fluorescence signal is detected in all 96 wells
during every cycle of amplification. Detection of
fluorescence by real-time PCR is several fold more
sensitive than conventional analysis of PCR
products on agarose gel (Lockey et al. 1998). The
high sensitivity of this technique allows the use of
less sample material. Because detection of the
signal depends on hybridisation of a gene-specific
probe, real-time PCR is also more specific than
traditional PCR systems. Rather than monitoring
final amplification of product as done in traditional
PCR, the real-time PCR reactions are
characterised by the point in time when
amplification is first detected rather than the
amount of PCR product accumulated after a fixed
number of cycles. This point is referred to as
threshold cycle (Ct). Detection of the Ct ensures
quantitation during the exponential phase of PCR,
therefore avoiding problems related to detection of
PCR products after the plateau has been reached.
Real-time PCR eliminates the need for a
competitive fragment to be amplified with the
target allowing the measurement of more samples
on the 96-well plate. Real-time quantitation
eliminates post-PCR processing of PCR products,
which not only increases throughout and reduces
the chances for carry-over contamination, but also
removes post PCR processing as a potential source
of error. Besides quantitation of mRNA expression
for cytokines or other proteins, real-time PCR can
also be used for detection of microorganisms,
quantitation of viral load, gene knockout analysis
and single nucleotide polymorphism screening.
REFERENCES
Aggarwal, N. and Holmes, M.A. (1999) Characterisation
Res. vet. Sci. 66, 277-279.
Cherwinski, H.M., Schumacher, J.H., Brown, K.D. and
Mosmann, T. (1987) Two types of mouse helper T
cell clone. III. Further differences in lymphokine
synthesis between Th1 and Th2 clones revealed by
RNA hybridisation, functionally monospecific
bioassays, and monoclonal antibodies. J. exp. Med.
166, 1229-1244.
Curran, J.A., Argyle, D.J., Cox, P., Onions, D.E. and
Nicolson, L. (1994), Nucleotide sequence of the
equine interferon gamma cDNA, DNA Seq. 4, 405-
407.
Dohmann, K., Wagner, B., Horohov, D.W. and Leibold,
W. (2000) Expression and characterisation of equine
interleukin 2 and interleukin 4. Vet. Immunol.
Gause, W.C. and Adamovicz, J. (1995) Use of PCR to
quantitate relative differences in gene expression. In:
PCR Primer a Laboratory Manual. Eds C.W.
Dieffenbach and G. S. Dveksler. Cold Spring Harbor
Laboratory Press, pp 293-311
Gigure, S. and Prescott, J.F. (1999) Quantitation of
Gigure, S., Wilkie, B.N. and Prescott, J.F. (1999)
Modulation of cytokine response of pneumonic foals
by virulent Rhodococcus equi. Infect. Immunol. 67,
5041-5047.
Grunig, G., Himmler, A. and Antczak, D. (1994) Cloning
and sequencing of horse interferon-gamma cDNA.
Immunogenetics 39, 448-449.
Kato, H., Ohashi, T., Matsushiro, H., Watari, T.,
Goitsuka, R., Tsujimoto, H. and Hasegawa, A.
(1997) Molecular cloning and functional expression
of equine interleukin-1 receptor antagonist. Vet.
Kato, H., Ohashi, T., Nakamura, N., Nishimura, Y.,
Watari, T., Goitsuka, R., Tsujimoto, H. and
Hasegawa, A. (1995) Molecular cloning of equine
interleukin-1 alpha and -beta cDNAs. Vet. Immunol.
Immunology in 2001
60
Lockey, C., Otto, E. and Long, Z. (1998) Real-time
fluorescence detection of a single DNA molecule.
Biotechniques 24, 744-746.
MacKay, R.J. and Lester, G.D. (1992) Induction of the
acute-phase cytokine, hepatocyte stimulating
factor/interleukin 6, in the circulation of horses
treated with endotoxin. Am. J. vet. Res. 53, 1285-
1289.
Morris, D.D. and Moore, J.N. (1991) Tumour necrosis
factor activity in serum from neonatal foals with
presumed septicemia. J. Am. vet. med. Ass. 199,
1584-1588.
Mosmann, T.R. and Sad, S. (1996) The expanding
universe of T-cell subsets: Th1, Th2 and more.
Immunol. Today 17, 138-146.
Nicolson, L., Penha-Gonzales, N.M., Keanie, J.L.,
Logan, N.A., Argyle, D.J. and Onions, D. E. (1999)
Cloning and sequencing of horse interleukin-12
and interleukin-18 cDNAs. Immunogenetics 50,
94-97.
Penha-Goncalves, M.N., Onions, D.E. and Nicolson, L.
(1997) Cloning and sequencing of equine
transforming growth factor-beta 1 (TGF beta-1)
cDNA. DNA Seq. 7, 375-378.
Seethanathan, P., Bottoms, G.D. and Schafer, K. (1990)
Characterisation of release of tumour necrosis
factor, interleukin-1 and superoxyde anion from
equine white blood cells in response to endotoxin.
Am. J. vet. Res. 51, 1221-1225.
Su, X.Z., Morris, D.D. and McGraw, R.A. (1991)
Cloning and characterisation of gene TNF alpha
encoding equine tumour necrosis factor alpha. Gene
107, 319-321.
61
TYPE I INTERFERON AND INTERLEUKIN-6 IN NASAL
SECRETIONS AND SERUM FROM PONIES INFECTED
WITH EQUINE INFLUENZA A2 (H3N8)
E. Wattrang, D. M. Jessett*, P. Yates*, L. Fuxler
and D. Hannant
*
Unit of Comparative Medicine and Physiology, Department of Large Animal Clinical Sciences,
Division
of Immunology, Department of Veterinary Microbiology, Swedish University of Agricultural Sciences,
Uppsala, Sweden; *Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK
Influenza viruses infect many species including
birds, swine, man and horses in which the virus is
an important respiratory pathogen. In most
microbial infections, the early immune and
inflammatory responses are crucial not only for
elimination of infectious agents but also for severity
of clinical signs. In human, murine and porcine
models of influenza, the virus induces production of
a number of proinflammatory cytokines such as
type I interferons (IFNs), tumour necrosis factor-,
interleukin-1 (IL-1), IL-6, IL-8 and other monocyte-
attracting chemokines (Julkunen et al. 2001; Van
Reeth 2000). Many of these have wide ranging
effects, eg type I IFNs have direct and indirect
antiviral effects, induce pyrexia and are important in
the regulation of ensuing immune responses
towards a Th1 phenotype (Bogdan 2000). In horses,
very few data exist on cytokine production in
response to influenza infections, but IFN activity
has been detected in nasal secretions during a
natural influenza outbreak (Jensen-Waern et al.
1998). The present study aimed to monitor
production of type I IFN and IL-6 during an equine
influenza A2 virus infection in the natural host. The
effects of 2 European-type influenza strains,
Newmarket/2/93 and Sussex/89, were compared.
The latter is considered to be the more pathogenic.
Twenty Welsh mountain ponies, seronegative to
influenza by SRH, were infected with equine
influenza A2 by nebulised aerosol on Day 0. Group
A (n=10); were infected with Sussex/89 and Group
B (n=10) with Newmarket/2/93. Virus excretion and
clinical signs were monitored and rectal
temperatures recorded daily until Day 10. Serum
samples were collected for cytokine determination
on Days 010 and nasal secretions sampled (with
plain cotton buds) on Days 110. IFN activity was
detected with a VSV-inhibition bioassay on MDBK
cells. IL-6 activity was determined with the IL-6
dependent murine cell-line B9.
Virus was isolated from nasal swabs from all
experimentally infected ponies, with maximum
excretion on Days 2 and 3. There was no difference
between the 2 groups in the onset of virus excretion
or the amount of virus excreted. Group A tended to
excrete virus for a longer period (until Day 7
compared to Day 6 in Group B). The experimental
infection induced coughing and nasal discharge
from Day 2 in both groups. However, throughout
the experiment, more ponies coughed in Group A
than Group B. Onset of nasal discharge was earlier
in Group A, where more ponies displayed
discharge in the initial phase of the experimental
infection (until Day 6). In the second week of
infection there was no difference in the number of
ponies with nasal discharge in the 2 groups.
Increased rectal temperature was seen post
infection in both groups. All Group A ponies had
pyrexia ( 38.9C) on Day 2 and/or Day 3. In
Group B, 6 ponies had pyrexia on Day 2. On
average, Group A ponies were pyrexic from Days 2
to 4 inclusive, whereas Group B ponies were
pyrexic on Day 2 only.
IFN was detected in nasal secretions from all
ponies in Group A on at least one sampling
occasion between Days 2 and 6 (Table 1). In
contrast, only 2 ponies in Group B displayed IFN
in nasal secretions. No IFN was detected in serum
samples collected throughout the study.
IL-6 activity was detected in nasal secretions
from all experimental ponies. Ponies infected
with Sussex/89, however, showed a markedly
higher response with peak levels on Day 3.
Newmarket/2/93-infected ponies showed low but
detectable levels from Day 2 onwards (Fig 1).
In serum, Group A showed a low IL-6 response
from Day 1, whereas Group B displayed occasional
IL-6 positive serum samples only (Table 2).
Taken together, both equine influenza A2
infections elicited local IFN and IL-6 responses. In
Immunology in 2001
62
the case of IL-6, low systemic levels of the cytokine
were also observed, probably due to leakage into
the circulation and a reflection of the local IL-6
production. Infection with Sussex/89 induced IFN
production in all ponies and markedly higher IL-6
responses compared to Newmarket/2/93. Whether
these differences in cytokine production are due to
strain differences in cytokine inducing capacity or
secondary to other viral virulence factors needs to
be addressed. Nevertheless, the different cytokine
production induced by the 2 strains may contribute
to the difference in pathogenicity. Further, the
marked production of IL-6 in nasal secretions
emphasises the importance of this cytokine in
regulating and stimulating local antibody responses
to mucosal infections. This supports the potential of
exogenous IL-6 (eg, as DNA or recombinant
protein) to enhance local immune responses to
mucosally-administered vaccines.
ACKNOWLEDGEMENTS
This study was supported by the Swedish Council
for Forestry and Agricultural Research and the
Linna and Axel Ericsson Fund. We thank Sarah
Flatt and Zoe Swann for excellent technical
assistance and Caroline Fossum for discussion and
scientific support.
REFERENCES
Bogdan, C. (2000) The function of type I interferons in
antimicrobial immunity. Curr. Opin. Immunol. 12,
419-424.
Jensen-Waern, M., Persson, S.G.B., Nordengrahn, A.,
Merza, M. and Fossum, C. (1998) Temporary
suppression of cell-mediated immunity in
standardbred horses with decreased athletic
capacity. Acta vet. scand. 39, 24-34.
Julkunen, I., Meln, K., Nyquist, M., Pirhonen, J.,
Sareneva, T. and Matikainen, S. (2001)
Inflammatory responses in infuenza A virus
infection. Vaccine 19, S32-S37.
Van Reeth, K. (2000) Cytokines in the pathogenesis of
influenza. Vet. Microbiol. 74, 109-116.
TABLE 1: No. of ponies with IFN in nasal secretions (Positives), and range of IFN levels after
experimental infection with equine influenza A2 Sussex/89 (n=10) or Newmarket/2/93 (n=10) on Day 0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Sussex/89
Positives 0 4 6 5 4 1 0 0 0 0
U/ml - 464 464 4128 448 24 - - - -
Newmarket/2/93
Positives 0 0 2 0 0 0 0 0 0 0
U/ml - - 4512 - - - - - - -
TABLE 2: No. of ponies with IL-6 in serum (positives), and range of IL-6 levels after experimental
infection with equine influenza A2, strain Sussex/89 (n=10) or Newmarket/2/93 (n=10) on Day 0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Sussex/89
Positives 1 5 4 3 6 6 4 8 5 2
U/ml 2 320 119 215 518 614 211 213 17 24
Newmarket/2/93
Positives 0 4 2 0 0 1 0 1 1 0
U/ml - 212 0.35 - - 3 - 2 1 -
63
0 1 2 3 4 5 6 7 8 9 10 11
300
250
100
150
100
50
0
I
L
-
6

(
U
/
m
l
)
Newmarket/2/93
Sussex/89
Fig 1: The levels of IL-6 (meansd) in nasal secretions
from ponies infected with equine influenza A2 strain
Sussex/89 (n=10) or Newmarket/2/93 (n=10) on Day 0.
Immunology in 2001
64
GENOMIC EXPLORATION OF ORTHOPAEDIC
INFECTION USING cDNA MICROARRAYS
E. M. Santschi, A. Rink* and C. W. Beattie*
University of Wisconsin,2015 Linden Drive West, Madison, Wisconsin 53706-1102; *University of
Nevada-Reno, Nevada, USA
Our goals are to develop practical strategies to
prevent the establishment of infection in equine
fractures contaminated with bacteria and to
eradicate orthopaedic infection quickly and
economically. We believe the inability of the local
immune system to control infection is the primary
reason for treatment failure. We have developed an
in vivo model of porcine implant-associated
orthopaedic infection with Staphylococcus aureus
to analyse the genomics of the local immune
response using DNA microarrays.
Microarrays represent high throughput
technology to determine differential gene
expression. They are an important experimental
approach to identifying and interpreting changing
levels of individual transcripts in various biological
systems. Several thousand discrete DNA sequences
(known genes or expressed sequence tags) can be
printed (arrayed) on a glass slide using an arrayer
robot. Messenger RNA is isolated from cell
populations from 2 tissues of interest and labelled
with fluorescent dyes (typically red and green). The
labelled samples are mixed and hybridised
(allowed to bind to sequence complements) to the
arrayed DNA spots. Using a scanner, the ratio of
colours is measured, giving a comparative analysis
of the relative abundance of transcripts in both
samples. This allows simultaneous, rapid
monitoring of many differentially expressed genes.
Although establishment of the procedure initially
requires a substantial investment, processing of
individual samples is relatively inexpensive.
DNA microarrays have been used to explore
the genomes of several model organisms (3-15,000
genes) and provide vital information about the
relationship between gene expression and the
subsequent effect of the gene product on the
physiology of the organism. It is believed that
further use of DNA microarrays will generate
crucial information about gene function in more
complex organisms by identifying and analysing
patterns of co-expression and degree of sequence
similarity (identification of orthologous genes).
Microarrays have been used in man to
investigate disease-related gene activity and to
profile diseases. In orthopaedic infection,
inflammation is a complex process caused by many
different cell types in the bone, surrounding tissues
and circulation. Surveying gene expression in cells
from these sites will provide insight to the
development of pathology (absolutely and
temporally), and an opportunity to identify target
genes for treatment intervention. We anticipate a
differential expression of genes between non-
infected and infected fractures. Monitoring local
and systemic immune responses will elucidate the
immune response to orthopaedic implants and
infection and could identify host genetic markers
for infection that facilitate diagnosis of implant-
associated orthopaedic infection.
We have cloned and sequenced 10,000 ESTs
from 5 immune tissues (lymph node, spleen,
peripheral blood cells, thymus and bone marrow) in
pigs with implant associated orthopaedic infection.
Annotated transcripts and ESTs were used to
construct a cDNA microarray. This resulted in the
construction of microarrays, identification and
radiation hybrid mapping of the proeasome/
ubiquitin system and a first generation EST
radiation hybrid map of the porcine genome.
Our work with pigs and horses is ongoing and
we will evaluate the use of heterologous cDNA
microarrays to monitor gene expression in equine
orthopaedic infection using peripheral blood cells,
the cell population accessible with minimal
invasion. We also hope to construct an equine DNA
microarray to evaluate the specific genes
transcribed during equine orthopaedic infection.
65
TABLE 1: Circulating EHV-1 specific CTL frequencies before and after challenge infection in 5 groups
of ponies
Group Status Mean se CTL frequency/10
6
PBMC Outcome of n
Pre-infection Post vaccination Post infection*
pregnancy
1 Primed 0.50.6 - 4.16.2 na 5
2 Multiply infected 45.340.0 - 32.434.3 na 3
3 Primed, pregnant ud - 1.41.0 5 aborted 5
4 vaccs Primed, pregnant 2.73.6 2.02.6 5.75.5 4 foaled 5
4 controls Primed, pregnant 0.91.4 0.30.2 2.52.1 4 aborted 4
5 Multiply infected, 4.70.6 - 5.02.6 3 foaled 3
pregnant
*Post infection samples: Groups 2 & 3 sampled at 3 weeks post infection, Groups 4 & 5 sampled at 7 weeks
post infection; na = not applicable; ud = undetectable
THE POWER OF LIMITING DILUTION ANALYSIS
J. H. Kydd, E. Wattrang*, G. P. Allen
, T. ONeill and D. Hannant

Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK; *Unit for
Comparative Medicine & Physiology, Department of Large Animal Clinical Sciences, PO Box 7018,
SE-750-07, Uppsala, Sweden;
Gluck Equine Research Centre, Department of Veterinary Science,

University of Kentucky, Lexington, Kentucky 40546-0099, USA
Limiting dilution analysis (LDA) can be used to
quantitate the in vitro activity of a responding cell
type within a mixed leucocyte population. Using a
linear concentration range, aliquots of cells are
cultured in microwells under optimum conditions
so that an individual immune cell can be amplified
into a measurable clone of cells and the well
scored for the presence or absence of a
precursor/memory cell. Large numbers of
replicates (at least 24 wells) are required and the
results are reported as the fraction of negative
wells. The negative log of the fraction of non-
responding cultures is linearly proportional to the
mean number of precursor cells per culture with
the experimental plot passing through the origin.
By interpolating the intercept of this line with 37%
non-responding cultures, the frequency of
precursor cells can be estimated. The preferred
statistical method to calculate the frequency is
currently the Jacknife version of Maximum
Likelihood. LDA can be applied to any randomly
distributed cellular or subcellular system and
provides a longitudinal measure of events within
the individual. To date, lymphocyte frequencies
that have been successfully investigated using
LDA in several species include antigen specific B
or T cells, cytotoxic lymphocyte precursors
(CTLp) and cytokine producing cells.
CTL frequencies were determined in horses in
an effort to identify a correlate of immunity to
equine herpesvirus-1 (EHV-1). The working
hypothesis was that high frequencies of EHV-1
specific CTL correlate with reduced clinical and
virological signs after experimental infection and
furthermore protect pregnant mares from virus
induced abortion. A total of 25 ponies in 5 groups
were used to test this hypothesis (Table 1).
Primed ponies had received no experimental
infections but were assumed to have had at least
one natural field infection with EHV-1. Multiply
infected ponies had received at least 3
experimental infections with EHV-1 (strain Ab4/8)
on an annual basis (ONeill et al. 1999 for details).
Groups 1 and 2 were adult non-pregnant animals
and Groups 3, 4 and 5 consisted of pregnant mares.
The Group identities were as follows: Group 1: 5
primed ponies; Group 2: 3 multiply infected
ponies; Group 3: 5 primed pregnant mares; Group
66
Immunology in 2001
4: 9 pregnant mares divided into 5 vaccinates
(inactivated EHV-1 in carbomer adjuvant
vaccinated im at 5, 7 and 9 months of gestation)
and 4 controls; Group 5: 3 multiply infected
pregnant mares. All ponies were infected
intranasally with Ab4/8, a virulent strain of EHV-
1, then monitored over a 2 week period for
clinical, serological and virological signs of
infection. Group 4 mares were challenged 4 weeks
after V3. Nasopharyngeal swabs and peripheral
sera and mononuclear cells (PBMC) were
collected from all ponies before infection, after
vaccination but before infection (Group 4 only)
and following infection. Seroconversion of
complement fixing antibodies, pyrexia,
nasopharyngeal virus shedding and cell associated
viraemia occurred in all ponies except the immune
Group 2 animals and Group 5 pregnant mares (see
below).
CTL were induced from PBMC in the
presence of Mitomycin C-treated PBMC as
antigen presenting cells, infectious virus and
immune equine serum in 96 well plates for 7 days.
Wells were then split 3 ways and different
radioisotopically labelled target cells added.
Target cells comprised autologous or heterologous
EHV-1 infected pokeweed mitogen lymphoblasts
or mock infected autologous lymphoblasts.
Following a 4 h incubation, supernatants were
harvested and gamma emissions counted.
Previous data show that the cytotoxic activity
measured in this assay is MHC class I restricted,
specific for EHV-1 and mediated by CD8+
lymphocytes (ONeill et al. 1999). The results
(Table 1) suggest that frequencies of EHV-1
specific CTL are lower in primed but susceptible
animals (Group 1) which exhibit the normal
clinical and virological signs of disease after
experimental intranasal infection with Ab4/8
(ONeill et al. 1999) than CTL frequencies in
multiply infected, immune ponies (Group 2).
Three groups of pregnant mares were infected
with EHV-1 and their frequencies of CTL
measured. The 5 primed mares in Group 3 all had
undetectable levels of CTL prior to experimental
infection and all aborted. For these mares, CTL
frequencies increased 3 weeks after infection.
Group 4 control mares had a low mean CTL
frequency immediately before infection and all
aborted but frequencies increased at 7 weeks after
infection. Vaccinates in Group 4 had a higher CTL
frequency than controls both before and after
vaccination and the mean frequency also increased
at the 7 week sampling point. Of 5 vaccinated
mares, 4 foaled. The Group 4 vaccinated mares
had higher CTL frequencies before vaccination
and challenge infection than the controls although
the difference was not significant. There was no
evidence that vaccination with inactivated virus
increased the CTL frequency. Group 5 contained 3
multiply infected mares which had higher
frequencies of CTL before infection and these
CTL numbers were maintained up to the 7 week
sampling point after infection. All 3 of these mares
foaled. Neither clinical, serological nor virological
signs of infection could be detected among Group
5 animals.
Comparison of foaling vs aborting mares using
Students t test illustrated that successful foaling
correlated directly with significantly higher
frequencies of CTL both before (P<0.01) and after
(P<0.01) infection. The duration of nasal virus
shedding was also significantly shorter (P<0.02) in
mares which foaled. There was no significant
difference in duration of cell associated viraemia
between the foaling and aborting groups.
These results suggest that a high frequency of
EHV-1 specific CTL (precursors and memory
cells) before infection correlates directly with
reduced nasal shedding of infectious virus and
successful foaling in pregnant mares. In
conclusion, LDA is a powerful technique,
applicable to many systems and it has enabled the
identification of a potential correlate of immunity
to EHV-1 infection in horses.
ACKNOWLEDGEMENTS
The financial assistance of the Equine Virology
Research Foundation, Horserace Betting Levy
Board and Fogarty International Research
Fellowship is gratefully acknowledged.
REFERENCES
ONeill, T., Kydd, J.H., Allen, G.P., Wattrang, E.,
Mumford, J.A. and Hannant, D. (1999)
Determination of equid herpesvirus 1-specific,
CD8+, cytotoxic T lymphocyte precursor
frequencies in ponies. Vet. Immunol. &
67
FLOW CYTOMETRIC TECHNIQUES IN CLINICAL
INVESTIGATION
B. R. Rush, M. J. B. F. Flaminio*, E. G. Davis and M. J. Wilkerson
College of Veterinary Medicine, Kansas State University, Kansas; *The James A. Baker Institute for
Animal Health, Cornell University, Ithaca, New York 14853, USA
Flow cytometric analysis has become a valuable
tool for clinical investigation of
immunosuppressive, immune-mediated and
neoplastic disease in horses. Flow cytometry
provides rapid assessment of proportions of cellular
populations and, in some instances, can evaluate
cellular function. Clinical application of flow
cytometric analysis is relatively common in human
medicine and is used predominantly for evaluation
of immunosuppressive and neoplastic disorders.
Flow cytometric determination of lymphocyte
subpopulations allows the equine clinician to
detect immunosuppressive disorders, monitor the
response to immunostimulant therapy and
characterise lymphoid neoplasias. In human
medicine, determination of CD4+ and CD8+
lymphocytes is performed routinely in patients
with HIV to monitor disease progression and
direct prophylactic therapy. Lymphoid neoplasias
are characterised to predict the biological activity
of the tumour.
Phagocytosis and oxidative burst activity of
equine leucocytes may be determined
simultaneously via flow cytometry. In this
technique, propidium iodide (PI)-labelled
Staphylococcus aureus is used to measure uptake
of bacteria by equine phagocytes and oxidative
burst activity is measured by oxidation of
dehydrorhodamine 123. The advantage of the
simultaneous technique is to provide independent
and combined assessment of phagocytosis and
oxidative burst. In addition, this assay allows the
use of autologous heat-inactivated sera to evaluate
opsoniation capacity of antibody separately.
Application of this technique includes
identification of inherited neutrophil function
disorders, prognosis and treatment of septicaemia,
and effects of exercise and long distance transport
on phagocyte function.
Immune-mediated hemolytic anaemia can be
diagnosed using flow cytometric direct
immunofluorescent assay. This rapid, quantitative
assay is more sensitive than the Coombs test for
detection of membrane-bound antibody and can
identify the isotype-specific immunoglobulin
(IgG, IgA, IgM). IgG is the most common isotype,
although IgM and IgA have been implicated in
clinical cases of immune-mediated haemolytic
anaemia in horses. The DIF technique can detect
small numbers of immunoglobulin-bound red
blood cells and is independent of the prozone
effect. Serial direct immunofluorescent assay
allows the equine clinician to monitor response to
immunosuppressive therapy.
The flow cytometric direct immunofluorescent
assay has been inconsistent for diagnosis of
immune-mediated thrombocytopenia in horses.
The technique has been reliable in select horses,
but has produced apparent false negative and false
positive results in others. Flow cytometry has been
valuable for detection of reticulated (regenerative)
platelets in horses with thrombocytopenia using
thiazole orange. Thiazole orange permeates the
cell membrane, binds nucleic acids and emits
fluorescence detected by the flow cytometer.
Immature platelets contain increased amounts of
nucleic acids, predominantly mRNA, which is
detected by the flow cytometer. The technique is a
simple, non-invasive alternative to bone marrow
aspirate/biopsy for characterisation of
thrombocytopenia in horses as regenerative vs
non-regenerative.
In human medicine, DNA cell cycle analysis
via flow cytometry is used to characterise
neoplastic and metaplastic tissues and predict their
biological behaviour. In some cases, cell cycle
analysis provides more powerful prognostic
information than any other parameter. Cell cycle
68
Immunology in 2001
analysis evaluates the cellular population.
Propidium iodide binds to cellular DNA at all
stages of the cell division cycle, and the intensity
of PI emission is directly proportional to its DNA
content. Cells in G
0
and G
1
have a diploid
chromosomal number (32 pairs), and dividing
cells (G
2
and M-phase) have double DNA content
(tetraploid). During DNA synthesis (S-phase), the
DNA content increases progressively until
tetraploidy is achieved. Single colour flow
cytometric analysis produces a DNA histogram,
and the major fluorescent intensity peaks
correspond to specific phases of the cell cycle.
Many malignancies have abnormal chromosomal
content (aneuploidy), which will appear as an
aberrant peak in the cell cycle histogram. The
aggressive nature of a tumour is evaluated by
determining the percentage of cells in S-phase and
documenting aneuploidy. In human medicine,
these flow cytometric parameters are used to
determine prognosis (5 year survival) and assist
with the development of a treatment plan. Cell
cycle analysis can predict the likelihood of
response to specific therapies (radiation,
endocrine, chemotherapy) and disappearance of
aneuploidy indicates a favourable response to
treatment.
In veterinary medicine, cell cycle analysis has
been evaluated to differentiate inflammatory/
granulomatous conditions from equine and canine
lymphoma. Peripheral blood, body cavity effusion,
fine needle aspirate and biopsy samples can be
evaluated via cell cycle analysis. Formalin-fixed
samples can be evaluated but paraffin-embedded
samples are more difficult to process and interpret.
Cell cycle analysis is more sensitive than
cytological evaluation for detection of neoplastic
cells in effusions and peripheral blood, although
not all equine lymphoma patients have circulating
neoplastic lymphoblasts or exfoliative neoplastic
cells in malignant effusion. Therefore, false
negative findings are common with peripheral
blood and effusive samples in horses. Cell cycle
analysis of fine-needle aspirate and biopsy
samples of equine lymphoma have demonstrated
marked aneuploidy (hyperploidy) and increased S-
phase activity. Most human lymphomas
demonstrated aneuploidy, primarily hyperploidy,
although some are diploid. Occasionally,
hypoploidic human and canine lymphomas are
observed. The majority of canine lymphomas are
diploid but approximately 25% of canine
lymphomas demonstrate aneuploidy
(hyperploidy). All human and canine lymphomas
have increased S-phase activity. Our laboratory
has not identified any diploid equine
lymphosarcomas. Normal values for cell cycle
analysis of equine lymph nodes have been
determined in our laboratory. Reactive
(inflammatory) lymph nodes demonstrate a
moderate increase in S-phase activity with
minimal to no aneuploidy. DNA cell cycle analysis
of effusion, tissue and blood provides a rapid
indication of the likelihood of malignancy in
horses. The technique has proved valuable in
differentiation of neoplastic conditions from
inflammatory and granulomatous masses.
Identification of occult neoplasias using peripheral
blood or effusion requires the presence of
neoplastic cells in the sample; negative findings
should not be interpreted as definitive evidence of
an inflammatory process.
69
Based on previously reported flow cytometric
methods for the analysis of phagocytic function in
human, bovine and equine phagocytes (Hasui et al.
1989; Perticarari et al. 1991; Smits et al. 1997;
Raidal et al. 1998a,b), a simple, rapid and precise
methodology was developed to evaluate
simultaneously phagocytosis and oxidative burst
activity (OBA) in horse cells. The technique has
been described by Flaminio et al. (2000, 2001).
Briefly, propidium iodide-labelled Staphylococcus
aureus (PI-Sa) is used to measure uptake of
bacteria by equine phagocytes. Trypan blue is
added to quench fluorescent signals imparted by
non-phagocytised bacteria or extracellular bacteria
that adhere non-specifically to the surface of the
phagocytes. Oxidation of non-fluorescent
dihydrorhodamine 123 (DHR) by reactive oxygen
intermediates to the green fluorescent rhodamine
123 (RHO) is measured after phagocytosis of
bacteria. The log red (PI, FL2) fluorescence is
proportional to the number of ingested bacteria.
The log green (RHO, FL1) fluorescence is
proportional to the OBA activity. In stimulated
cells, fluorescence imparted by the conversion of
DHR to RHO is proportional to the level of
activation of phagocytes following ingestion of
opsonised and non-opsonised bacteria.
Opsonised reactions have greater mean
fluorescence values than non-opsonised reactions
(Basse and Bjerknes 1984). Phagocytic activity is
improved by the presence of opsonins, which may
function via C3b receptors (for complement) or
Fc-receptors (for immunoglobulins). This activity
is dependent on the amount of serum added to the
bacteria, because opsonisation with 10% horse
serum results in lower phagocytic and OBA
compared to 20%, 30% and 40% serum (Flaminio
et al. 2000). In addition, phagocytosis activity is
reduced when heat-labile opsonins (which include
complement) are inactivated (Basse and Bjerknes
1984). Immunoglobulins and complement reveal
synergistic interaction in phagocytosis and
oxidative burst activity. One of the advantages of
this assay is the possibility of incubation of
leucocytes with a standard source of pooled sera
for opsonisation, which reduces the variability in
phagocytic response caused by individual
differences in opsonic activity (Grndahl et al.
1997). In addition to evaluating individual
phagocytosis and cellular oxidative activity, this
technique allows the use of autologous and heat-
inactivated sera to characterise opsonisation
capacity (complement x antibody).
Foal neutrophil function is dependent on the
maturation of humoural opsonic factors rather than
on maturity of neutrophil function (Grndahl et al.
1999; Flaminio et al. 2000). For this reason, foals
may have decreased neutrophil activity in vivo in
the first few months of age compared to adult
horses. This effect becomes more evident by the
benefits of plasma therapy in neonates, which
SIMULTANEOUS ANALYSIS OF PHAGOCYTOSIS AND
OXIDATIVE BURST ACTIVITY OF EQUINE
PHAGOCYTES BY FLOW CYTOMETRY
M. J. B. F. Flaminio, B. R. Rush*, E. G. Davis*, W. Shuman
and M. Wilkerson
James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University,
Ithaca, New York 14850; *Department of Clinical Sciences and
Department of Diagnostic
Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan;
Kansas 66506-5606, USA
Fig 1: Mean fluorescence of phagocytosis (FL2) and
oxidative burst activity (FL1) of healthy foal phagocytes
from 24 h to 4 months of age. Phagocytes were opsonised
with 40% normal adult serum. Adult phagocytic function
reference value is depicted by the continuous line.
24 h 2 w 3 w 1 m 2 m 3 m 4 m
3000
2500
2000
1500
1000
500
0
M
e
a
n

f
l
u
o
r
e
s
c
e
n
c
e
Oxidative burst
Phagocytosis
70
Immunology in 2001
significantly increases opsonic capacity of foals
sera (Grndahl et al. 1999).
The application of this flow cytometric
technique allowed the evaluation of phagocytosis
and oxidative burst activity in foals, as well as the
opsonic capacity of their serum. Blood samples
from 10 foals (6 females, 4 males) of mixed breeds
were collected in heparinised tubes and tubes with
no additives at 24 h (post colostrum ingestion),
weekly for the first month of life and monthly up
to 4 months of age. All foals in this group had
adequate transfer of maternal immunoglobulins.
The results demonstrate no age-related differences
in the phagocytic capacity of foal neutrophils
when adult serum was used for opsonisation,
indicating that equine neonate phagocytes are
competent (Fig 1). However, adult horse
phagocytes had inferior function when sera from
foals of different ages were used for opsonisation.
Moreover, normal foal serum showed age-related
improvement of opsonic activity, whereas heat-
inactivated serum did not show changes (Fig 2).
Simultaneous analysis of phagocytosis and
oxidative burst activity by flow cytometry is a
rapid and sensitive method to evaluate phagocyte
function in horses. Potential applications of this
technique include identification of inherited
neutrophil function disorders, prognosis and
treatment evaluation of septicaemia, and effects of
exercise and transportation on phagocyte function.
REFERENCES
Basse, C.F. and Bjerknes, R. (1984) The effect of serum
opsonins on the phagocytosis of Staphylococcus
aureus and zymosan particles, measured by flow
cytometry. Acta Path. Microb. Immunol. Scand. 92,
51-58.
Characterisation of peripheral blood and pulmonary
leucocyte function in healthy foals. Vet. Immunol.
Simultaneous flow cytometric analysis of
phagocytosis and oxidative burst activity in equine
leucocytes. Vet. Res. Comm. In press.
Grndahl, G., Johannisson, A. and Jensen-Waern, M.
(1997) Opsonic effect of equine plasma from
different donors. Vet. Microbiol. 56, 227-235.
Grndahl, G., Johannisson, A., Demmers, S. and Jensen-
Waern, M. (1999) Influence of age and plasma
treatment on neutrophil phagocytosis and CD18
expression in foals. Vet. Microbiol. 65, 241-254.
Hasui, M., Hirabayashi, Y. and Kobayashi, Y. (1989)
Simultaneous measurement by flow cytometry of
phagocytosis and hydrogen peroxide production of
neutrophils in whole blood. J. immunol. Methods
117, 53-58.
Perticarari, S., Presani, G., Mangiarotti, M.A. and Banfi,
E. (1991) Simultaneous flow cytometric method to
measure phagocytosis and oxidative products by
neutrophils. Cytometry 12, 687-693.
Raidal, S.L., Bailey, G.D. and Love, D.N. (1998a) The
flow cytometric evaluation of phagocytosis by
equine peripheral blood neutrophils and pulmonary
alveolar macrophages. Vet. J. 156, 107-116.
Raidal, S.L., Bailey, G.D. and Love, D.N. (1998b) Flow
cytometric determination of oxidative burst activity
of equine peripheral blood and bronchoalveolar
lavage-derived leucocytes. Vet. J. 156,117-126.
Smits, E., Burvenich, C. and Heyneman, R. (1997)
Simultaneous flow cytometric measurement of
phagocytic and oxidative burst activity of
polymorphonuclear leucocytes in whole bovine
blood. Vet. Immunol. Immunopathol. 56, 259-
269.
24 h 2 w 3 w 1 m 2 m 3 m 4 m
Foal sera in different ages
24 h 2 w 3 w 1 m 2 m 3 m 4 m
Foal sera in different ages
160
140
120
100
80
60
40
20
0
1000
900
800
700
600
500
400
300
200
100
0
M
e
a
n

f
l
u
o
r
e
s
e
c
e
n
c
e

(
F
L
2
)
M
e
a
n

f
l
u
o
r
e
s
e
c
e
n
c
e

(
F
L
2
)
PHAGOCYTOSIS
OXIDATIVE
BURST
Fig 2: Mean fluorescence of phagocytosis (FL2) and oxidative burst activity (FL1) of adult phagocytes measured by flow
cytometry. Phagocytes were opsonised with 40% normal or heat-inactivated sera from foals from birth to 4 months of age.
Normal serum
Heat-inactivated serum
71
SESSION III :
Inflammation
Chairman: Paul Lunn
72
Immunology in 2001
73
CHRONIC AIRWAY DISEASE IN THE HORSE
N. E. Robinson
Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, Michigan
48823, USA
Chronic airway disease in the horse is also known
as equine chronic obstructive pulmonary disease
(COPD). The latter name is inappropriate because
human COPD is largely a disease of smokers and
is generally unresponsive to therapy with
corticosteroids or bronchodilators. By contrast,
equine chronic airway disease is very responsive to
these modes of treatment. A recent workshop
(Robinson 2001) recommended that 2 phenotypes
of equine chronic airway disease be recognised,
heaves or recurrent airway obstruction (RAO) in
middle-aged horses and inflammatory airway
disease (IAD) in young performance horses in
training. RAO is a severe inflammatory obstructive
airway disease of middle-aged and older horses
(Robinson et al. 1996). It is induced by exposure
of susceptible horses to inhaled organic dust,
generally from hay. A similar syndrome, summer
pasture-associated obstructive pulmonary disease
(SPAOPD) occurs in the southern United States
during the summer months (Seahorn and Beadle
1993) and is believed to be due to inhalation of
spores from molds that grow in lush pasture. When
horses feed, they push their noses into the hay and
other feeds and thereby expose themselves to high
levels of dust in their breathing zone (Woods et al.
1993). Hay dust is a mixture of mold spores,
forage mites, particulates and endotoxins (Woods
et al. 1993; Vandenput et al. 1997; McGorum et al.
1998). All of these agents can either induce or
exacerbate airway inflammation. When dust
exposure is reduced either by returning the horse
to pasture or by feeding it a low-dust diet such as
silage or pelleted feed, airway function improves
within a few days (Vandenput et al. 1998; Jackson
et al. 2000).
Radiolabelling of inflammatory cells has
shown that when an RAO-susceptible horse is
exposed to hay dust, neutrophils invade the lungs
and airways within 46 h. Airway obstruction
develops concurrently (Fairbairn et al. 1993b). The
most intense inflammatory response
(peribronchiolar accumulation of lymphocytes and
intraluminal accumulation of neutrophils) occurs at
the bronchioles (Derksen et al. 1985b; Kaup et al.
1990a,b; McGorum et al. 1993b), but functional
changes also occur in the larger airways (Robinson
et al. 1999). Bronchoalveolar lavage demonstrates
that the principal inflammatory cell in the airway
lumen is the neutrophil (Derksen et al. 1985b;
McGorum et al. 1993b). Concurrent with the onset
of inflammation and airway obstruction, non-
specific airway hyperresponsiveness develops
(Derksen et al. 1985a; Klein and Deegen 1986).
Non-specific airway hyperresponsiveness is an
exaggerated bronchospasm occurring in response
to a wide variety of stimuli such as inflammatory
mediators, neurotransmitters or changes in
osmolarity. This airway hyperresponsiveness
makes the horse particularly prone to
bronchospasm during attacks of airway
inflammation. Even brief exposures of susceptible
horses to hay dust can initiate airway
hyperresponsiveness that can persist for days
(Fairbairn et al. 1993a). Administration of
bronchodilators to horses with acute attacks of
heaves demonstrates that obstruction is due
primarily to bronchospasm (Murphy et al. 1980;
Robinson et al. 1993; Derksen et al. 1996).
However, following maximal bronchodilation,
some airway obstruction persists, probably due to
accumulation of mucus and exudates, and
remodelling of the airway wall. Recent
investigations by our group have demonstrated
persistent increases in measures of intraluminal
mucus even after horses have been at pasture for
several weeks. Twenty-four hours after such horses
are stabled, the mucus becomes extremely
viscoelastic and less readily cleared, contributing to
mucus accumulation (Gerber et al. 2000).
Bronchospasm in horses with heaves results
from facilitation of parasympathetically mediated
smooth muscle contraction (Robinson et al. 1993;
Duvivier et al. 1997) by inflammatory mediators
and decreased inhibition of contraction by
inhibitory prostanoids and the iNANC nervous
system (Yu et al. 1994). In vitro experiments have
demonstrated that activation of neutrophils has no
effect on the response of smooth muscle to
cholinergic stimulation, but mast cell-derived
mediators such as histamine, serotonin and
leucotriene D4 greatly facilitate smooth muscle
contraction and could be responsible for the
cholinergically mediated bronchospasm of heaves
(Olszewski et al. 1999).
Measurements of inflammatory mediators in
blood and BALF have demonstrated increases in
histamine, serotonin, some prostanoids,
thromboxane, isoprostanes and 15-HETE (Gray et
al. 1989, 1992; Watson et al. 1992; McGorum et
al. 1993a; Kirschvink et al. 1999). However, use of
specific blockers has not identified a unique role
for any mediator in RAO. This is very similar to
the situation in asthma and other inflammatory
obstructive airway diseases. The importance of
inflammation as a cause of airway obstruction is
demonstrated by the fact that therapy with
corticosteroids, especially dexamethasone,
reverses many of the functional consequences of
RAO (Lapointe et al. 1993; Rush et al. 1998).
Even though breeding of affected and
unaffected horses has shown that there is a genetic
predisposition to chronic airway disease (Marti et
al. 1991), it is unlikely that heaves is a result of
over-expression of a single trait. Elevated levels of
IgE in bronchoalveolar lavage fluid support the
involvement of a type-1 hypersensitivity reaction
(Halliwell et al. 1993), probably a local allergic
response to thermophilic molds. However, the
immediate (within 15 min) airway obstruction that
usually accompanies the release of mast cell-
derived mediators during a type-1 reaction is never
observed in horses. Attempts to define the
predominant T-cell population in blood and
bronchoalveolar lavage fluid have identified
differences in the total number and ratio of CD4+
and CD8+ T-cells (McGorum et al. 1993a; Watson
et al. 1997), but inconsistent findings prevent
definitive conclusions at this time.
There are 2 possible neutrophil chemotactic
factors involved in heaves: leucotriene B4 and IL-
8. The horse lung produces larger amounts of
leucotriene B4 than of cysteinyl leucotrienes and
leucotriene B4 is chemotactic for neutrophils in
horses (Marr et al. 1998; A. Lindberg, unpublished
data). However, elevated levels of leucotriene B4
have not been detected in BALF. Furthermore,
unpublished data from experiments that blocked
leucotriene synthesis and leucotriene receptors
suggest that leucotriene B4 is not important in the
pathogenesis of heaves. Elevated levels of IL-8 are
associated with acute exacerbations of heaves
(Franchini et al. 2000) suggesting that IL-8 is a
chemotactic factor for neutrophils. Increased
expression of NF-B in bronchial epithelial cells
and granulocytes parallels airway obstruction
(Bureau et al. 2000b). NF-B is probably
activating genes encoded for IL-8, ICAM-1 and
other pro-inflammatory cytokines. Persistent
expression of NF-B by IB may be due to an
imbalance between high levels of IL-1- and
TNF-mediated IB degradation and low levels
of IB synthesis (Bureau et al. 2000a). As
granulocytes undergo apoptosis, secretion of IL-
1- and TNF decreases, IB degradation ceases
and newly synthesised IB binds and inactivates
NF-B, thus turning off inflammation.
IAD (Moore et al. 1995) affects 2530% of
horses in training in the USA (Sweeney et al.
1992). In the UK, racehorses spend about 30% of
that time in training with IAD and each bout can
last for several weeks (Burrell et al. 1996).
Epidemiological investigations have implicated
streptococcal infections of the larger airways in
about 50% of horses with IAD (Wood et al. 1993;
Burrell et al. 1996; Chapman et al. 2000) and
equine rhinovirus in another small percentage. In
some horses, no infectious cause can be identified
and environmental factors are thought to be the
aetiological agents. It is highly likely that in young
horses, as in young people, there can be numerous
causes of airway inflammation: bacterial, viral,
allergic and environmental. Neutrophils are the
predominant inflammatory cell, but occasional
horses have increased numbers of eosinophils or
mast cells (Viel 1997; Hare and Viel 1998). The
relevance of these different cell types to the
aetiology and treatment of IAD is currently
unknown. At present, there is no evidence to
connect IAD with RAO in the old horse and there
is no way to identify a young horse that will
develop RAO later in life (Robinson et al. 2001).
Further advances in understanding the
pathogenesis of both RAO and IAD require in-
Immunology in 2001
74
depth investigation of the inflammatory process in
horse airways and its immunological basis. The
goal should be early identification of markers that
will predict which horses will develop heaves later
in life. Once such horses are identified, they could
be managed to prevent exposure to dusts and the
development of severe lung disease.
REFERENCES
Bureau, F., Delhalle, S., Bonizzi, G., Fivez, L., Dogn,
S., Kirschvink, N., Vanderplasschen, A., Merville,
M.-P., Bours, V. and Lekeux, L. (2000a)
Mechanisms of persistent NF-B activity in the
bronchi of an animal model of asthma. J. Immunol.
165, 5822-5830.
Bureau, F., Bonizzi, G., Kirschvink, N., Delhalle, S.,
Desmecht, D., Merville, M.-P., Bours, V. and
Lekeux, P. (2000b) Correlation between nuclear
factor-B activity in bronchial brushing samples and
lung dysfunction in an animal model of asthma. Am.
J. Respir. Crit. Care Med. 161, 1314-1321.
Burrell, M.H., Wood, J.L.N., Whitwell, K.E., Chanter,
N., Mackintosh, M.E. and Mumford, J.A. (1996)
Respiratory disease in thoroughbred horses in
training: the relationships between disease and
viruses, bacteria and environment. Vet. Rec. 139,
308-313.
Chapman, P.S., Green, C., Main, J.P.M., Taylor, P.M.,
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75
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Immunology in 2001
76
VARIATION OF NON-INFECTIOUS AIRWAY
DISEASE PHENOTYPE WITH AGE
L. Viel
Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Ontario N1G 2W1,
Canada
Horses of all ages can be afflicted with non-
infectious airway disease (NIAD) that manifests
with varying clinical, physiological and
pathological findings. The common feature is
obstruction of the airways due to accumulation of
secretions, thickening of the airway wall and, in
advanced cases, loss of radial traction at the level of
the terminal airways. Longitudinal epidemiological
studies have not yet been conducted to assess the
relationship between disease and age, and this
abstract outlines the 3 major phenotypes associated
with NIAD: horses with heaves, poorly performing
young horses and foals.
In general, the clinical signs observed in NIAD
vary with the severity of the condition and age of the
animal. Intermittent cough at rest or the beginning
of exercise, early signs of fatigue during exercise
and a persistently elevated respiratory rate post
exercise are often seen in foals and athletic horses.
The detection of crackles and wheezes on
auscultation and significantly expanded lung field
on percussion are additional features. Nasal
discharge, nasal flaring and abdominal expiratory
effort are mostly observed in older horses, or those
with heaves, but can occur in foals with severe
airway inflammation. A diagnostic aid to
compliment clinical examination is endoscopy of
the airway. This allows clinicians not only to score
the amount of mucopus in the large airways, but
also to recognise mucosal oedema and lumen
closure due to bronchoconstriction. To assess the
severity of disease quantitatively, from mild to
severe, indices for clinical and endoscopic scoring
represent important tools for direct correlation
between endoscopic findings and other pulmonary
physiological and pathological parameters (Hare et
al. 1994; Tesarowski et al. 1996).
There is consistent evidence that pathological
lesions in NIAD are localised in the non-
cartilagenous small airways or bronchioles (Viel
1985; Winder 1988). The characteristic histological
changes consist of epithelial cell hyperplasia and
metaplasia, peribronchiolar inflammatory cell
infiltrate, lymphoid cell hyperplasia, smooth
muscle hypertrophy and airway lumen occlusion
by mucopus casts. Fibrosis and alveolar thickening
may be observed in advanced cases of heaves. It is
of interest that, in a study where histological
parameters were assessed semi-quantitatively
between normal and mild-to-severe cases of NIAD,
4 significantly different subgroups could be
identified (Table 1). The data indicated that degree
of lesion severity was closely associated with age
of the animal. Further, horses affected with NIAD
had significant pathological small airway changes
prior to detection of abnormal pulmonary function
parameters such as altered transpulmonary pressure
and dynamic compliance.
TABLE 1: Pathology and lung function in horses with NIAD
Group (n) 1 (11) 2 (40) 3 (7) 4 (4)
Age 50.8 61.8 81.6 121.5
Histological score 393 904 1575 2225
DPpl (cm H
2
0) 51 61 73 157
Cdyn (L/cm H
2
0) 2.10.2 2.10.4 1.80.4 1 0.2
From: (Viel 1985)
77
Airway obstruction resulting from severe
bronchiolitis is compounded further by
accumulation of excessive inflammatory cells and
mucus within the airway lumen. In well-
characterised cases of heaves in horses >10 years
old, a significantly elevated mixed inflammatory
cell population is retrieved from bronchoalveolar
lavage (BAL). Neutrophils frequently account for
over one third of the total inflammatory cell
differential in such cases, and play a pivotal role in
the pronounced airway hyperreactivity. The
massive influx and persistence of neutrophils
within the lumen of the airways is reflective of
heavey horses in a state of disease exacerbation.
Exacerbation can be documented further by a 35%
drop in dynamic compliance following aerosol
exposure to low levels of histamine (<8 mg/ml).
In contrast to heaves in older horses, athletic
horses aged 25 years show a remarkable degree of
airway hyperreactivity to inhaled doses of histamine
as low as 5 mg/ml (Hare et al. 1998) and 6 mg/ml
(Hoffman et al. 1998). BAL fluid from such horses
demonstrates the presence of increased eosinophils
and mast cells, as well as a slight increase in the
number of neutrophils and lymphocytes.
Little emphasis has been placed on the concept
that frequently encountered airway inflammatory
disorders may reflect persistent inflammation
rather than ongoing bacterial or viral infection.
This is best exemplified by discussing respiratory
disease in foals. In general, clinicians assume that
purulent exudate at the nares or seen on
endoscopic examination of a foal directly implies
an active bacterial infection (eg Streptococcus
zooepidemicus). However, these exudates may
actually reflect ongoing, sterile airway
inflammation despite resolution of the underlying
infectious process. Cytologically, airway
inflammation is indicated by large numbers of
neutrophils identified in BAL fluid (Hoffman et al.
1993). In a report by Lakritz et al. (1993) on 1- to
7-month-old foals with acute bronchointerstitial
pneumonitis and/or respiratory distress, 9 of 10
treated aggressively with corticosteroids survived,
supporting the theory that airway inflammation
plays an important role in the pathophysiology of
this condition rather than simply the persistence of
an infectious agent. Further, pathological findings
in foals submitted for necropsy from the same
study indicated the presence of subclinical chronic
inflammatory small airway disease. It is unknown
if these airway changes are transient or permanent
in nature, but they typically respond well to
corticosteroid therapy. Physiologically, there is
supporting evidence that affected foals of the same
age group as the one described by Lakritz have
changes in their pulmonary function parameters,
including airway hyperresponsiveness to
aerosolised histamine and the presence of reduced
arterial oxygen tension (hypoxemia) (Hoffman et
al. 1999).
In summary, although all 3 phenotypes of
NIAD share some common abnormalities in their
clinical, physiological and pathological findings,
our understanding of NIAD in general is hampered
by the lack of a cohesive, integrated approach in
studying these phenotypes.
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Immunology in 2001
78
79
BIOLOGY OF AIRWAY NEUTROPHILS
B. McGorum and T. Brazil*
Department of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, Easter Bush,
Roslin, Midlothian EH25 9RG; *Ridgeway Veterinary Group, Upper Lambourn Road, Lambourn,
Berkshire RG17 8QG, UK
Neutrophils have a critical role as a first line of
defence against host insult from pathogens and
inhaled agents and, consequently, individuals with
neutrophil dysfunction may develop life-
threatening pneumonia. Because neutrophils are
almost absent from the normal lung, to facilitate
this protective role, peripheral blood neutrophils
must be primed and recruited to the lung by
chemoattractants and chemokines such as
interleukin-8 (IL-8), macrophage inflammatory
protein-2 (MIP-2), tumour necrosis factor-
(TNF-), leucotriene B4 (LTB4) and bacterial
products (Sibille and Reynolds 1990).
Transmigration of neutrophils from the pulmonary
vasculature through the basement membrane into
the airways is facilitated by proteinases including
elastase and metalloproteinases (MMPs). Once
recruited, neutrophils may be activated by
inflammatory and bacterial agents, resulting in a
respiratory burst (a marked increase in production
of reactive oxygen species) and degranulation,
with release of a wide array of antimicrobial
agents including proteases and cationic proteins, in
addition to inflammatory mediators and cytokines.
Unfortunately neutrophils contain more than 30
potentially histotoxic agents which may stimulate
further recruitment, activation and degranulation
of inflammatory cells, promote mucus
hypersecretion, increase pulmonary vascular
permeability and ultimately cause host tissue
damage (Haslett et al. 1989). Neutrophil mediated
host damage is considered to be important in the
pathogenesis of human acute respiratory distress
syndrome and chronic obstructive pulmonary
disease. Consequently, the function of neutrophils
must be regulated tightly, and the potentially
detrimental effects of neutrophil contents are
limited by several control mechanisms including
inactivation of neutrophil products by
antiproteinases and antioxidants, and the removal
of intact apoptotic neutrophils by macrophages
and by mucociliary clearance.
Equine heaves (previously termed chronic
obstructive pulmonary disease) is characterised
by a neutrophilic endobronchitis, with
bronchoalveolar lavage fluid (BALF) neutrophil
numbers being increased by up to 50-fold. Airway
neutrophil recruitment can be induced in heaves
horses by hay/straw exposure, and by inhalation of
aqueous mould extracts and endotoxin. There is
evidence to support involvement of IL-8, MIP-2,
LTB4 and platelet activating factor (PAF) in
equine pulmonary neutrophil recruitment
(Franchini et al. 1998; Marr et al. 1998; Brazil
1999). In people with grain dust-induced
occupational asthma, IL-8 has been implicated as
a prime candidate for induction of neutrophil
recruitment (Park et al. 1998). Increased levels of
active MMP-9 in airways of heaves horses is
consistent with the crucial role of MMPs in the
transmigration of inflammatory cells through the
basement membrane into the airways (Raulo et al.
2001). Indeed, consistent with this role, the
magnitude of airway neutrophilia was closely
correlated with the BALF concentrations of
pro-MMP-9 and active MMP-9, but not with
the BALF MMP-2 concentration (Nevalainen et
al. 2001). Activation of MMP-9, which is a pre-
requisite to its biological effect, may be mediated
by either oxidants or proteinases, and there is
evidence for both reactive oxygen species (ROS)-
and protease-mediated MMP-9 activation in
heaves (Nevalainen et al. 2001).
Although neutrophils are the predominant
inflammatory cell in the airways of heaves affected
horses, their presence per se does not necessarily
imply involvement in airway obstruction. Indeed
Fairbairn et al. (1993) demonstrated that in some
Immunology in 2001
80
heaves horses, airway dysfunction preceded
detectable pulmonary neutrophil sequestration.
Furthermore, although inhaled endotoxin (Pirie et
al. 2001) and Faenia rectivirgula (Derksen et al.
1988) induced a marked neutrophilia, this was not
always associated with concomitant airway
dysfunction.
Peripheral blood neutrophils are primed
following antigen challenge in heaves horses, as
indicated by increased basal and agonist
stimulated superoxide production (Marr et al.
1997; Brazil 1999). Furthermore, the neutrophils
within the air spaces are primed and activated, as
evidenced by their increased respiratory burst in
response to the bacterial tripeptide fMLP, and by
the increased BALF concentrations of neutrophil
degranulation products including neutrophil
elastase and MMP-9 (Brazil 1999; Raulo et al.
2001; Nevalainen et al. 2001). In addition, the
finding that heaves is associated with oxidative
stress (Art et al. 1999), and that the degree of
oxidative stress is correlated with the airway
neutrophil count, is consistent with increased
production of ROS by neutrophils, although these
may also be derived from other activated
leucocytes such as macrophages.
Although the neutrophil elastase present in
BALF from heaves horses is inactivated, probably
due to inhibition by proteinase inhibitors such as
alpha-1-proteinase inhibitor (API), it may
contribute to histotoxic damage by acting locally
within the immediate pericellular micro-
environment prior to interaction with inhibitors
(Brazil 1999). In contrast, as much of the MMP-9
in BALF is present in the active form, it may have
a more global effect leading to damage and
remodelling of the airway epithelium, basement
membrane and pulmonary interstitium. Horses
with heaves may have minor degrees of pulmonary
emphysema, with the severity of histological
changes relating to the degree and extent of
clinical disease (Kaup et al. 1990). The extent of
airway remodelling is considerably less that that
which characterises human chronic obstructive
pulmonary disease. This inter-species difference
may, in part, reflect improved efficacy of the
equine antiproteinase screen. In this respect,
several potentially important interspecies
differences in the nature and function of one of the
major proteinase inhibitors, namely API, have
been identified. In contrast to human API, which is
encoded by a single gene, equine API is encoded
by 4 allelles producing 4 proteins termed Spi 1-4.
To date 22 haplotypes have been identified, but
heaves does not appear to be associated with any
particular haplotype. Most of the API in equine
BALF is derived from plasma API. API levels in
normal equine BALF are 23-fold higher than
those in man, and are increased further in horses
with heaves (Milne et al. 1994). As equine Spi 3,
which comprises a significant proportion of the
equine serpin shield, is resistant to the oxidative
inactivation that affects human API, the horse may
have adequate antiprotease activity in local
environments that are rich in ROS. Furthermore,
and again in contrast to the situation in man, the
equine API complex is not chemoattractant for
equine neutrophils, and hence is unlikely to lead to
further pulmonary recruitment of neutrophils
(Scudamore et al.1993). The effect of neutrophil
products on airway smooth muscle function is as
yet unclear, and is likely to be multifactorial
(Olszewski et al. 1995; Art et al. 1999). Although
neutrophil derived products, including elastase,
contribute to goblet cell degranulation in antigen
challenged guinea pigs (Agusti et al. 1998), it is
not yet known whether they contribute to the
mucus hyper-secretion in heaves.
In the resolution phase of heaves there is an
increase in the proportion of neutrophils undergoing
apoptosis (programmed cell death) in the airspaces.
These neutrophils are removed subsequently via
phagocytosis by airway macrophages (Brazil 1999),
or cleared via the mucociliary escalator. As
apoptosis results in down-regulation of a number of
neutrophil functions including chemotaxis,
phagocytosis and granule release, this process
serves to limit further host tissue damage and
promote resolution of inflammation (Whyte et al.
1993). However, although apoptosis undoubtedly
contributes to clearance of neutrophils from the
lungs of heaves horses, the constitutive rate of
neutrophil apoptosis appears to be reduced during
an exacerbation of heaves (Brazil 1999). This
inhibition of apoptosis probably results from the
inhibitory effects of pro-inflammatory agents
including cytokines, which may enhance neutrophil
longevity via upregulation of the nuclear factor-
kappa B transcription factor (Bureau et al. 2000).
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1258.
81
Art, T., Kirschvink, N., Smith, N. and Lekeux, P. (1999)
Indices of oxidative stress in blood and pulmonary
epithelial lining fluid in horses suffering from
recurrent airway obstruction. Equine vet. J. 31, 397-
401.
Brazil, T.J. (1999) Pulmonary Neutrophil Recruitment,
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Kaup, F.J., Drommer, W., Damsch, S. and Deegen, E.
(1990) Ultrastructural findings in horses with COPD
II: pathomorphological changes of the terminal
airways and alveolar region. Equine vet. J. 22, 349-
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Marr, K.A., Foster, A.P., Lees, P., Cunningham, F.M. and
Page, C.P. (1997) Effect of antigen challenge on the
activation of peripheral blood neutrophils from
horses with COPD. Res. vet. Sci. 62, 253-260.
Marr, K.A., Lees, P., Page, C.P. and Cunningham F.M.
(1998) Inhaled leucotrienes cause broncho-
constriction and neutrophil accumulation in horses.
Res. vet. Sci. 64, 219-224.
Milne, E.M., Pemberton, A.D., McGorum, B.C., Dixon,
P.M., Scudamore, C.L. and Miller, H.R.P. (1994)
Quantitation of alpha-1 proteinase inhibitor in
pulmonary epithelial lining fluid of horses with
COPD. Res. vet. Sci. 57, 262-264.
Nevalainen, M., Raulo, S., Brazil, T.J., Pirie, R.S., Sorsa,
T., McGorum, B.C. and Maisi, P. (2001) Inhalation
of organic dusts and lipopolysaccharide increases
gelatinolytic metalloproteinase activity in the lungs
of heaves affected horses. Equine vet. J. (In Press).
Olszewski, M.A., Robinson, N.E., Zhu, F.U., Zhang,
X.Y. and Tithof, P.F. (1999) Mediators of
anaphylaxis but not activated neutrophils augment
cholinergic responses of equine small airways.
Am. J. Physiol.-Lung Cell. Molec. Physiol. 20,
522-529.
Park, H.S., Jung, K.S., Hwang, S.C., Nahm, D.H. and
Yim, H.E. (1998) Neutrophil infiltration and release
of IL-8 in airway mucosa from subjects with grain
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Allergy 28, 724-730.
Pirie, R.S., Dixon, P.M., Collie, D.D. and McGorum,
B.C. (2001) Pulmonary and systemic effects of
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horses. Equine vet. J. 33, 311-318.
Raulo, S.M., Sorsa, T., Tervahartiala, T., Pirila, E. and
Maisi, P. (2001) MMP-9 as a marker of
inflammation in tracheal epithelial lining fluid
(TELF) and in bronchoalveolar fluid (BALF) of
COPD horses. Equine vet. J. 33, 128-136.
Scudamore, C.L., Pemberton, A., Watson, E.D. and
Miller, H.R.P. (1993) Neutrophil chemotaxis in the
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neutrophil elastase and equine API. Brit. vet. J. 149,
331-338.
Sibille, Y. and Reynolds, H.Y. (1990) Macrophages and
polymorphonuclear neutrophils in lung defence and
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Immunology in 2001
82
CYTOKINE IMMUNOREGULATORY ELEMENTS IN RAO
J-P. Lavoie and D. W. Horohov*
Dpartement de Sciences Cliniques, Facult de Mdecine Vtrinaire, Universit de Montral,
Canada; *Pathobiological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA
Recurrent airway obstruction (RAO) of horses,
also known as heaves, and chronic obstructive
pulmonary disease (COPD), is a condition
affecting the equine lung in Europe and the United
States. Two forms of this disease have been
identified based on seasonal occurrences, RAO
and summer pasture-associated obstructive
pulmonary disease (SPAOPD), although clinical
signs and cellular composition of bronchoalveolar
lavage fluid suggest a common aetiopathogenesis.
Clinical signs of heaves result from small airway
inflammation leading to terminal airway
obstruction, primarily due to bronchospasm,
mucus plugs and airway remodelling affecting the
bronchiolar walls. The finding that exacerbation of
heaves could be provoked by exposure to dusty
hay led researchers to postulate that heaves is an
allergic reaction to inhaled moulds and fungi.
However, to date, the immunological events
leading to airway inflammation in heaves remain
ill defined.
Recent findings using molecular tools and
murine animal models have highlighted the
complex interactions involved in the modulation of
allergic lung disease. It is now clear that most cells
present within the lung tissue, including epithelial
cells, myocytes and nerve endings in addition to
traditional inflammatory cells, contribute to this
modulation. Among these, T cells (CD4+ cells,
more specifically of Th2 cells) play a central role
in allergic airway inflammation. Cytokines
produced by Th2 cells, such as interleukin (IL)-4,
IL-5 and IL-13 have been implicated in allergic
inflammation. Th1 cells are important for cell-
mediated immunity by their production of INF-
and other cytokines. Although a polarisation of T
helper cells into predominantly Th1 and Th2
subsets has not been investigated thoroughly in
horses, in vitro culture of equine CD4+ T cells
following chronic allergen stimulation has induced
a Th2 cytokine profile characterised by an
increased expression of IL-4 mRNA and a
decreased expression of IL-2 mRNA (Aggarwal et
al. 1999).
Lower airway inflammation, reversible airway
obstruction and bronchial hyperresponsiveness are
characteristic of heaves in horses and human
asthma. As asthma is associated with a Th2-type
cytokine expression in bronchoalveolar (BAL)
cells (Robinson et al. 1992) and because the 2
diseases share many similarities, we postulated
that heaves also has a Th2 type cytokine
expression in BAL cells. Using in situ
hybridisation, we found that BAL cells from
horses with heaves chronically exposed (months)
to dusty hay have increased expression of IL-4 and
IL-5 mRNA and decreased expression of INF-
compared to controls, which is consistent with a
Th2 type cytokine response (Lavoie et al. 2000).
IL-4 promotes the development and the growth of
Th2 cell phenotype and is essential for the
induction of B-cell isotype switching to IgE
antibody production (Lasky and Brody 1997). The
increase in IL-4 mRNA expression in heaves is
consistent with the elevated levels of antigen
specific IgE in serum and BAL of horses with
heaves (Halliwell et al. 1993; Schmallenbach et al.
1998; Eder et al. 2000). The results of IL-5 mRNA
expression in the present study are less clear. An
increased expression of IL-5 mRNA is usually
associated with tissue eosinophil migration, which
is not a common feature in heaves. The presence of
IL-5 within the airway lumen may not be sufficient
by itself to induce a local eosinophilia, as
suggested by the results of a study in a murine
model, showing that circulating but not local IL-5
was associated with pulmonary eosinophilia
(Wang et al. 1998). It is also possible that IL-5
83
mRNA expression may not have been associated
with protein production in the present study, as it
occurred in human bronchitis (Jeffery 1999).
However, when using RT-PCR on BAL cells of
horses with heaves compared to controls, no
consistent findings on cytokine mRNA expression
were found in 3 preliminary reports (Lavoie et al.
1999; Ainsworth et al. 2000; Giguere et al. 2000)
and IL-5 mRNA was not detected in 2 of them
(Lavoie et al. 1999; Giguere et al. 2000).
Quantitative PCR analysis of horses affected with
SPAOPD demonstrated elevated levels of mRNA
for IL-4 and IL-13 during that time of the year
(summer) when clinical symptoms were present
(Horohov 2000). Control animals exhibited a bias
towards Th1 cytokine (interferon-) production in
their lungs. We also failed to detect elevated levels
of mRNA for IL-5 in the lungs of affected horses.
While half of the affected animals returned to a
Th1 bias in their mRNA profile in the winter, the
others exhibited elevated signs of IL-4 and IL-13
mRNA, suggesting a more chronic condition in
these animals. The discrepancies in results
between studies clearly indicate that more work
will be required before a definite conclusion can
be reached concerning the contribution of Th1/Th2
type cytokines to heaves and SPAOPD.
A number of additional cytokines and
chemokines likely to be implicated in the
modulation of airway inflammation in heaves are
being investigated. Of particular interest, the role
of cytokines secreted by epithelial cells,
neutrophils and macrophages may play a crucial
role in airway remodelling and secretions involved
in heaves. It has been shown that BAL fluids of
heavey horses have increased chemotoactic
activity for neutrophils (Franchini et al. 1998,
2000) and that IL-8 mRNA expression (Lavoie et
al. 1999) and IL-8 concentration (Franchini et al.
2000) are also increased.
REFERENCES
Aggarwal N. and Holmes MA. (1999) Characterisation
Res. vet. Sci. 66, 277-279.
Ainsworth, D.M., Appleton, J.A., Antczak, D.F., Baker,
J.M., Santiago, M. and Aviza, G. (2000) Immune
responses in horses with recurrent airway
obstruction. Am. J. Resp. crit. care Med. 161, A842.
Eder, C., Crameri, R., Mayer, C., Eicher, R., Straub. R.,
Gerber, H., Lazary, S. and Marti, E. (2000) Allergen-
specific IgE levels against crude mould and storage
mite extracts and recombinant mould allergens in
sera from horses affected with chronic bronchitis.
Franchini, M., Gilli, U., Akens, M.K., Fellenberg, R.V.
chronic obstructive pulmonary disease (COPD). Vet
Immunol. Immunopathol. 66 53-65.
Franchini, M., Gilli, U., von Fellenberg, R. and Bracher,
V.D. (2000) Interleukin-8 concentration and
neutrophil chemotactic activity in bronchoalveolar
lavage fluid of horses with chronic obstructive
pulmonary disease following exposure to hay. Am. J.
vet. Res. 61 1369-74.
Giguere, S., Viel, L. and Lee, E. (2000) Cytokine
messenger RNA (mRNA) expression in
bronchoalveolar cells from normal horses and horses
with heaves, and effect of therapy with inhaled
fluticasone proprionate. 18th Annual Veterinary
Medical Forum, ACVIM, Seattle, Washington,
USA. 710.
Halliwell, R.E.W., McGorum, B.C., Irving, P. and Dixon,
pulmonary disease. Vet. Immunol. Immunopathol.
38, 201-215.
Horohov, D.W., (2000) Equine T-cell cytokines,
protection and pathology. Vet. clin. North Am.
equine Pract. 16, 1.
Jeffery, P.K. (1999) Differences and similarities between
chronic obstructive pulmonary disease and asthma
Clin. exp. Allergy 29, 14-26.
Lasky, J.A. and Brody, A.R. (1997) Interleukins involved
in the pathogenesis of chronic airway inflammation.
Res. Immunol. 148, 39-47.
Lavoie, J., Maghni, K., Desnoyers, M,, Taha, R., Martin,
J.G. and Hamid, Q. (2000) Bronchoalveolar cells
from horses with heaves express a Th2-type
cytokine profile. 18th Annual Veterinary Medical
Forum, ACVIM, Washington, USA. 751.
Lavoie, J.P., Maghni, K., Desnoyers, M., Hamid, Q. and
Martin, J.G. (1999) Expression of cytokine
messenger mRNA in bronchoalveolar cells of horses
with heaves. In A. T. Society, International
Conference, San Diego, USA.
Robinson, D.S., Hamid, Q., Ying, S., Tsicopoulos, A.,
Barkans, J., Bentley, A.M., Corrigan, C., Durham,
S.R. and Kay, A.B. (1992) Predominant Th2-like
bronchoalveolar T-lymphocyte population in atopic
asthma. N. Engl. J. Med. 326, 298-394.
Schmallenbach, K.H., Rahman, I., Sasse, H.H.L., Dixon,
P.M., Halliwell, R.E.W., McGorum, B.C., Crameri,
R. and Miller, H.R.P. (1998) Studies on pulmonary
and systemic Aspergillus fumigatus-specific IgE and
IgG antibodies in horses affected with chronic
obstructive pulmonary disease (COPD). Vet.
Wang, J., Palmer, K., Lotvall, J., Milan, S., Lei, X.F.,
Matthaei, K.I., Gauldie, J., Inman, M.D., Jordana,
M. and Xing, Z. (1998) Circulating, but not local
lung, IL-5 is required for the development of
antigen-induced airway eosinophilia. J. clin. Invest.
102, 1132-1141.
Immunology in 2001
84
IgG SUBTYPES AND CLUES TO THE IMMUNO-
PATHOLOGY OF RECURRENT AIRWAY
OBSTRUCTION (HEAVES)
D. M. Ainsworth, J. A. Appleton*, D. F. Antczak*, M. A. Santiago and G. A. Aviza
Department of Clinical Sciences; *Department of Microbiology & Immunology and James A Baker
Institute for Animal Health; Cornell University, Ithaca, New York 14853, USA
Heaves is an inflammatory condition of the small
airways of middle-aged and older horses stabled
on straw and fed hay. The ailment is life-long, it is
debilitating, and it has a negative effect on the
athletic usefulness of susceptible horses (Aviza et
al. 2001). The pathogenesis of heaves currently
remains unknown but it is widely believed to
represent an immune-mediated disease triggered
by exposure to environmental moulds or dust
found in hay and bedding. The moulds implicated
most commonly are Aspergillus fumigatus, Faenia
rectivirgula and Thermoactinomyces vulgaris.
Understanding the nature of this immune-
mediated disease of heavey horses becomes
important for therapeutic and prophylactic
interventions. This is especially applicable to
North American show horses for which
environmental management changes are not
feasible and for which the administration of
glucocorticoid therapy is not permissible.
Studies demonstrating elevated broncho-
alveolar lavage fluid (BALF) and serum IgE levels
against A. fumigatus and F. rectivirgula in affected
horses provide data suggesting that heaves is a
type 2 (T helper cell2) immune response (Eder et
al. 2000; Halliwell et al. 1993; Schmallenbach et
al. 1998). In contrast, studies demonstrating: 1) a
lack of correlation between antigen-specific IgE
levels and expression of the disease (Dixon et al.
1996; Eder et al. 2000); 2) a lack of early phase
BALF histamine release in naturally challenged
heavey horses (McGorum et al. 1993); and 3) a
lack of development of pulmonary eosinophilia in
heavey horses (Derksen et al. 1985), provide data
supporting the hypothesis that heaves is a Th-1
immune response.
One approach to investigating the type of
immune responses existing within the respiratory
tract of heavey horses would be to measure the
pulmonary and systemic antibody responses to a
nebulised novel foreign antigen. Currently,
measurement of equine BALF and serum IgE is
confounded by the lack of readily available
reagents. However, in most species, type 2
cytokines promote B lymphocyte switching not
only to IgE, but also to selected IgG sub-isotypes
(Coffman et al. 1993; Snapper et al. 1997). In the
horse, the IgG sub-isotype most likely to be
associated with a Th-2 response is IgG(T) whereas
the IgG sub-isotypes most commonly associated
with a Th-1 response are IgGa and IgGb (Nelson
et al. 1998). Thus, using the context of a natural
challenge (NC) environment (dusty hay, indoor
stabling on straw), we sought to determine if the
chronic inflammatory events existing within the
respiratory system of heavey horses would bias
IgG subtype formation in response to a nebulised
foreign antigen, keyhole limpet hemocyanin
(KLH). The aim was to establish whether, in
healthy and affected horses, the KLH sub-isotypes
formed reflect a Th-1 or Th-2 immune response.
Pulmonary function testing and BALF
neutrophil populations were used to assign horses
to control or heaves-affected groups. There was no
difference in the median age of the 2 groups.
Baseline sera and BALF fluid (Sample 1) were
obtained on Day 14 of natural challenge
environment. Horses were then nebulised 4 times
(at 3 day intervals) with a 0.1% solution of KLH in
saline. Sera and BALF were collected 18 and 32
days later (Samples 2 and 3). KLH-specific
antibody levels were measured using an ELISA
developed and adapted using isotype-specific
mouse monoclonal antibodies. The 4 sub-isotypes
quantified were IgGa, IgGb, IgG(T) and total IgG.
Differences between the 2 groups were detected
using non-parametric analysis with P<0.05
selected for significance.
85
We found that anti-KLH IgG levels were
significantly greater in BALF and sera from
control horses compared to diseased horses.
Differences in sub-isotypic responses between the
2 groups were also evident. BALF anti-KLH IgGb
levels in control horses exceeded those of diseased
horses, but BALF anti-KLH IgG(T) levels in
diseased horses were not significantly increased
relative to controls.
In contrast to the prevailing notion that horses
with heaves have hyperreactive immune responses,
our data suggest that diseased horses mount a
weak IgG response compared to healthy horses.
Differences in IgG levels between the 2 groups
might be attributed to down-regulation of type 1
cytokines. However, as the anti-KLH IgG(T)
levels of the heavey horses failed to exceed those
of the controls, it seems unlikely that heaves
reflects a Th-2 response.
Differences in the antibody responses between
the 2 groups of horses may also be attributed to: 1)
the large numbers of neutrophils in the lungs of
diseased horses that would bind available IgGa and
IgGb via Fc receptors (McGuire et al. 1973);
and/or 2) protease-induced destruction of the
antigen in the lungs of heavey horses (Koivunen et
al. 1996). Based upon the results of our study, we
were unable to conclude whether the pulmonary
immune environment of heavey horses reflects a
Th-1 or Th-2 process. (This work was supported
by the Harry M. Zweig Memorial Fund for Equine
Research).
REFERENCES
Aviza, G.A., Ainsworth, D.M., Eicker, S.W., Santiago,
M.A., Divers, T.J. and Perkins, G.A. (2001)
Outcome of horses diagnosed with and treated for
heaves. Equine vet. Educ. In press.
Coffman, R.L., Lebman, D.A. and Rothman, P. (1993)
Mechanisms and regulation of immunoglobulin
isotype switching. Adv. Immunol. 54, 229-270.
Derksen, F.J., Scott, J.S., Miller, C.D., Slocombe, R.F.
and Robinson, N.E. (1985) Bronchoalveolar lavage
in ponies with recurrent airway obstruction (heaves).
Am. Rev. resp. Dis. 132, 1066-1070.
Dixon, P.M., McGorum, B.C., Marley, C., Halliwell,
R.E.W., Matthews, A.G. and Morris, J.R. (1996)
Effects of equine influenza and tetanus vaccination
on pulmonary function in normal and chronic
obstructive pulmonary disease affected horses.
Equine vet. J. 28, 157-160.
Eder, C., Crameri, R., Mayer, C., Eicher, R., Straub, R.,
Gerber, H., Lazary, S.and Marti, E. (2000) Allergen-
specific IgE levels against crude mould and storage
mite extracts and recombinant mould allergens in
sera from horses affected with chronic bronchitis.
Halliwell, R.E.W., Mcgorum, B.C., Irving, P. and Dixon,
pulmonary disease. Vet. Immunol. Immunopathol.
38, 201-215.
Koivunen, A.-L., Maisi, P., Fang, W. and Sandholm, M.
(1996) Inhibition of the protease activity in
tracheobronchial aspirates of horses with chronic
obstructive pulmonary disease. Am. J. vet. Res. 57,
603-607.
McGorum, B.C., Dixon, P.M. and Halliwell, R.E.W.
(1993) Phenotypic analysis of peripheral blood and
bronchoalveolar lavage fluid lymphocytes in control
and chronic obstructive pulmonary disease affected
horses, before and after natural (hay and straw)
challenges. Vet. Immun. Immunopath. 36, 207-222.
McGuire, T.C., Crawford, T.B. and Henson, J.B. (1973)
The isolation, characterisation and functional
properties of equine immunoglobulin classes and
subclasses. Proc. 3rd int. Conf. equine inf. Dis. Eds:
J.T. Bryans and H. Gerber, H. Karger, Basel, Paris.
pp 364-381.
Nelson, K.M., Schram, B.R, McGregor, M.W., Sheoran,
A.S., Olsen, C.W. and Lunn D.P. (1998) Local and
systemic isotype-specific antibody responses to
equine influenza virus infection versus conventional
Schmallenbach, K.H., Rahman, I., Sasse, H.H.L., Dixon,
P.M., Halliwell, R.E.W., McGorum, B.C., Crameri,
R. and Miller, H.R.P. (1998) Studies on pulmonary
and systemic Aspergillus fumigatus-specific IgE and
IgG antibodies in horses affected with chronic
obstructive pulmonary disease (COPD). Vet.
Snapper, C.M., Marcu, K.B. and Zelazowski, P.
(1997) The immunoglobulin class switch: beyond
accessibility. Immunity 6, 217-223.
Immunology in 2001
86
EFFECTS OF DUST AND ENDOTOXIN EXPOSURES ON
LUNG FUNCTION AND AIRWAY CYTOLOGY OF
HORSES
L. L. Coutil, M. A. Hunt* and F. S. Rosenthal*
School of Veterinary Medicine; *School of Health Sciences, Purdue University, West Lafayette,
Indiana, USA
The purpose of the study was to assess the effects
of airborne dust and endotoxin exposures on lung
function and airway cytology in horses maintained
in environmentally controlled rooms. Four horses
were housed for 2-week periods in each of 3
environments: low dust (LD, pelleted feed and
wood shavings), medium dust (MD, pelleted feed
and straw) and high dust (HD, mouldy hay and
straw). Lung mechanics, airway responsiveness
and bronchoalveolar lavage fluid cytology were
assessed at the end of each exposure period. Total
and respirable dust and endotoxin levels were
monitored in the horses breathing zone for each
environment.
Dust exposure levels were significantly greater
in HD than in MD or LD environments for both
total dust (6.84 1.93, 2.12 0.18, 1.46 0.69
mg/m
3
, respectively) and respirable dust (1.85
1.11, < 0.003, 0.182 0.244 mg/m
3
, respectively).
Endotoxin exposure levels in respirable dust were
significantly higher in the HD than in MD and LD
environments (9.21 2.07, 4.88 3.39, 1.50
1.86 ng/m
3
, respectively). No significant
differences in lung function were detected in
horses after exposure to HD, MD and LD
environments. There was a trend for an increase in
airway responsiveness to histamine challenge
when horses were placed in the HD environment.
The percentage of neutrophils in the broncho-
alveolar lavage fluid was significantly higher in
HD than in MD and LD environments (15.8 4.9,
3.0 1.83, 2.8 2.1 %, respectively).
We conclude that horses placed for 2 weeks in
HD environment exhibited significant airway
inflammation as a result of high exposure levels to
respirable dust and endotoxin. However, the effect
of dust and endotoxin exposure levels on lung
function was marginal.
87
SEPTICAEMIA AND ENDOTOXAEMIA IN HORSES
J. N. Moore
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia,
Athens, Georgia 30362, USA
More than 100 years ago, it was discovered that
heat stable extracts of Gram-negative enteric
bacteria were highly toxic. Based on the
assumption that these toxins were released from
the interior of the bacteria upon their death, the
term endotoxin was coined. The results of
subsequent biochemical studies, however,
determined that these endotoxins do not arise from
the interior of the bacteria, but are lipopoly-
saccharide (LPS) components of the outer
membrane of enteric bacteria. Furthermore, it was
determined that LPS is comprised of 3 structural
units: an outer polysaccharide component, a core
oligosaccharide region and the innermost portion,
lipid A, which affords the molecule its pro-
inflammatory activities.
Although the gastrointestinal tract has a very
efficient mucosal barrier to restrict LPS to the
intestinal lumen, gastrointestinal diseases which
compromise this mucosal barrier often result in
movement of LPS into the bloodstream. Using the
highly sensitive limulus amoebocyte lysate assay
to detect LPS, it has been determined that 3545%
of horses presented to veterinary colleges with
colic are endotoxaemic, that most endotoxaemic
horses have intestinal strangulation obstruction or
severe inflammatory intestinal diseases and that
prognosis for survival is inversely correlated with
the presence of LPS in circulation. The situation is
equally serious in neonatal foals that develop a
nidus of bacterial infection, such as an infected
umbilical remnant. In a recent clinical study of 34
neonatal foals with presumed septicaemia, LPS
was detected in the plasma of 50% of septic foals
sampled and derangements in hemostasis and
fibrinolysis were correlated with the presence of
endotoxin in circulation. Thus, endotoxaemia is
associated with the primary causes of death in
horses of all ages.
The clinical evidence cited above has led a
variety of workers to investigate the effects of LPS
in horses, either by studying the effects of the
toxins in vivo or in vitro. The results of numerous
studies performed in a variety of species, including
the horse, indicate that endotoxic shock is initiated
when the hosts mononuclear phagocytes interact
with LPS and synthesise pro-inflammatory
mediators. Under experimental conditions, serum
concentrations of the cytokine tumour necrosis
factor (TNF) increase to peak values within 2 h
after onset of endotoxaemia; and serum TNF
activity has been associated with the onset of signs
of abdominal pain, fever and leucopenia. In
clinical studies of horses with colic and foals with
septicaemia, increased serum concentrations of
TNF activity have been identified and associated
with mortality rate.
Similarly, numerous studies have documented
the role of lipid-derived mediators, principally
cyclo-oxygenase products of arachidonic acid
metabolism, in endotoxaemia in horses. The
results of these studies suggest that many of the
early haemodynamic and behavioural effects of
endotoxaemia are due to thromboxane A
2
, and
prostaglandins E
2
, F
2
and I
2
. As a logical
extension of these studies, beneficial effects of
potent non-steroidal anti-inflammatory drugs have
been reported in horses and ponies with
endotoxaemia in experimental studies. These
results form the basis for the use of these drugs in
clinically ill horses and foals with conditions
characterised by endotoxaemia
Alterations in coagulation and fibrinolytic
factors in endotoxaemia have been identified,
either by in vitro studies with isolated monocytes
or peritoneal macrophages, or by measuring these
factors in plasma from horses and foals with
naturally occurring diseases characterised by
Immunology in 2001
88
endotoxaemia. In clinical studies of horses with
colic, significant decreases in plasma
antithrombin-III activity, protein C and
plasminogen activity were identified. Expression
of tissue factor by monocytes isolated from horses
with colic was significantly greater than
expression by monocytes from healthy control
horses. Similarly, the results of a recent study of
neonatal foals with presumed septicaemia
indicated that coagulation times tend to be
prolonged in endotoxaemic foals, compared to
values for age-matched healthy foals and septic
foals in which endotoxin is not present in the
plasma. These findings indicate the presence of a
hypercoagulable state in endotoxaemic horses
with colic and foals with septicaemia.
Although there is convincing evidence that
endotoxaemia occurs in horses and foals and
causes the synthesis of pro-inflammatory
mediators that lead to many of the complications
encountered in clinical practice, the molecular
mechanisms by which endotoxin exerts its effects
in horses are largely unknown. This is not the case
for other species, for which it is known that LPS in
the circulation binds rapidly to a unique plasma
protein named lipopolysaccharide binding protein
(LPS-binding protein). LPS-binding protein,
which is synthesised by hepatocytes as part of the
acute phase response of inflammation, has a strong
affinity for the lipid A portion of endotoxin, as
well as for CD14, a glycosylphosphatidylinositol-
anchored receptor on the surface of mononuclear
phagocytes. When LPS and LPS-binding protein
interact with CD14, these cells become activated
and produce a variety of pro-inflammatory
mediators. Alternatively, LPS and LPS-binding
protein may interact with a soluble form of CD14
in circulation and then activate cells lacking
membrane-bound CD14.
Because CD14 lacks a transmembrane
component, the endotoxin signal must be
transduced through something other than CD14.
The results of more than 250 published studies
since 1998, when the observation was first made,
indicate that the endotoxin signal is transmitted to
the interior of the cell via the Toll-like 4
transmembrane receptor. With these new findings
in mind, potential new therapeutic interventions
that warrant study include mechanisms to interfere
with the binding of LPS to LPS-binding protein, or
CD14 or to prevent the intracellular responses
initiated by activation of Toll-like receptor 4.
89
PATHOPHYSIOLOGY OF ENDOTHELIN-1 IN
EQUINE ACUTE LAMINITIS
A. S. Holm, S. C. Eades, C. S. Venugopal, J. L. Oliver and R. M. Moore
Equine Health Studies Program, Departments of Comparative Biomedical Sciences, Pathobiological
Sciences and Veterinary Clinical Sciences; School of Veterinary Medicine, Louisiana State University,
Baton Rouge, Louisiana, USA
The purpose of the studies reported here was to
address the hypothesis that an imbalance between
endothelium-derived vasodilator (nitric oxide,
NO) and vasoconstrictor (endothelin 1, ET-1)
substances is the initiating factor in the
development of acute laminitis in horses. Although
the pathophysiology of this debilitating,
excruciatingly painful and potentially career-
ending and life threatening disease of the soft
tissues of the foot is not completely understood,
there is substantial evidence suggesting that local
digital haemodynamic alterations play a role in its
initiation. Acute laminitis is characterised
pathophysiologically by decreased pre-to-post
capillary resistance ratio and increased capillary
pressure subsequent to increased venoconstriction;
increased laminar interstitial pressure and oedema
formation; microvascular compression;
thrombosis and formation of cellular (platelet-
platelet and platelet-neutrophil) aggregates; and
decreased digital blood flow and laminar perfusion
which ultimately leads to laminar ischaemia,
inflammation and necrosis. Our current lack of a
complete understanding of acute laminitis
prohibits effective therapeutic and preventive
measures from being developed and employed in
the clinical management of this disease.
Laminitis occurs in adult horses and often
occurs secondarily to diseases typically associated
with endotoxaemia. Although experimental
administration of endotoxin has never been
demonstrated to cause laminitis, its administration
is associated with appreciable decreases in digital
blood flow and laminar perfusion. Additionally,
endotoxin may cause endothelial damage and
result in decreased NO and increased ET-1
synthesis and release. Endotoxaemia has been
associated with alterations in plasma and tissue
NO and ET-1 concentrations in other species. NO
is synthesised constitutively by endothelial NO
synthase and causes vascular smooth muscle
relaxation, decreased neutrophil adhesion and
decreased platelet aggregation and adhesion. ET-1
is a peptide synthesised by the endothelium and is
the most potent vasoconstrictor substance known;
it is also a mitogen and stimulates platelet
aggregation. Because of the physiological and
pathophysiological properties of NO and ET-1, an
imbalance between these substances (decreased
NO and increased ET-1) could account for
initiation and propagation of many of the above
mentioned alterations known to occur with acute
laminitis.
Our laboratory began investigating this
hypothesis using an in vitro study of the effects of
ET-1 and non-selective ET antagonists on
contractile properties of palmar digital artery and
veins collected from normal, non-laminitic horses.
In this study, we investigated the hypotheses that
ET-1 would cause a marked contraction of arteries
and veins, that the response would be greater in
veins, and that this contraction could be prevented
by pre-treatment with ET antagonists. The results
demonstrated that ET-1 causes a slowly
developing, but sustained and profound, dose-
dependent contraction of arteries and veins.
Digital veins were much more sensitive and
responded to a magnitude 3.5 times greater than
that observed in arteries. Pre-treatment of vessels
with either of the antagonists caused a dose-
dependent blockade of the contraction, although
the B-2 antagonist (PD145065) was more effective
than the B-1 antagonist (PD142893) and caused
complete blockade.
A preliminary in vitro vessel study in 4 horses
with naturally acquired laminitis suggested that
digital veins were more sensitive to the effects of
ET-1 than in normal horses. The B-2 antagonist
Immunology in 2001
90
was effective in greatly reducing the contraction in
at least some arteries and veins of these horses.
To further evaluate the effects of ET-1 and ET
antagonists on the response of the normal digital
vasculature of horses, we evaluated these
substances in an in vivo study in conscious horses
with a surgically implanted ultrasonic digital flow
probe around the lateral palmar digital artery. We
investigated the hypotheses that ET-1 infusion into
the arterial circulation of horses would cause a
dose-dependent reduction in digital blood flow and
that this could be prevented and/or reversed with
administration of the ET antagonist B-2 when
infused into the digital arterial circulation as pre-
treatment or after ET-1, respectively. This study
confirmed that ET-1 infusion into the digital artery
caused a dose-dependent reduction in digital
arterial blood flow. Additionally, at higher doses,
ET-1 caused digital pain that was marked in most
horses at the highest concentration (10-6 M). Pre-
treatment with a 10-5 M concentration of B-2
effectively blocked the ET-induced decrease in
digital blood flow. In another experiment in the
same horses, a dose-response study of B-2
infusion was performed after digital blood flow
was decreased by pre-treatment with ET-1. There
was a dose-dependent improvement in blood flow
and at the highest B-2 concentration (10-5 M),
there was a significant improvement in blood flow.
To assess the effects of ET-1 on the digital
microvasculature, we have performed a
preliminary study of the digital Starling forces in 3
horses. In this study, we infused ET-1 at a dose
comparable to 10-6 M into the digital arterial
circulation. ET-1 caused a decrease in digital blood
flow due to an increase in vascular resistance; the
increased vascular resistance was associated with a
30% increase in post capillary resistance. This
suggests venous constriction is an important
component of the increase in digital vascular
resistance and decreased digital blood flow
observed with ET-1 infusion. Capillary pressure
increased from 36 to 52 mm Hg.
ET-1 immunohistochemical staining of
formalin-fixed, paraffin-embedded laminar tissue
specimens from laminitic and normal horses
demonstrated intense staining of the arteriolar and
venous endothelium and the laminar epithelium
and stroma of the sensitive laminae in laminitic
horses, but not in normal horses. This information
corresponds with previous reports of increased
ET-1 expression in the laminar tissue of horses
with naturally acquired chronic laminitis and in
horses with experimentally induced acute
laminitis.
Collectively, the findings from these studies
suggest that a local increase in digital vascular and
laminar ET-1 could account for many of the
alterations that occur in acute laminitis. Therefore,
the role of ET-1 in the initiation and propagation of
acute laminitis is currently being investigated both
in the carbohydrate overload and black walnut
extract models of experimentally induced acute
laminitis. These studies currently focus on
measuring digital venous plasma ET-1
concentrations; measuring systemic and digital
haemodynamics; monitoring clinical signs;
evaluating ET-1 immunohistochemical staining of
specimens of laminar tissue and digital arteries and
veins; and measuring digital Starling forces in
horses given either B-2 or a saline control solution
after induction of laminitis.
The information gained from these studies
provide important information on the potential role
of ET-1 in the pathophysiology of acute laminitis,
and the potential therapeutic implications for ET
antagonists in horses with naturally acquired
disease.
91
IMMUNOMODULATION OF EVENTS IN SEPSIS:
ROLE OF P-SELECTIN/P-SELECTIN LIGAND SYSTEM
B. J. Darien
School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison,
Wisconsin 53706, USA
Acute intestinal crises in the horse are life-
threatening disorders with intestinal ischaemia or
inflammatory bowel disease being the most
common. In these disorders, the absorption of
endotoxin from enteric bacteria causes the
adherence of leucocytes to vascular walls, release
of inflammatory mediators, loss of fluid from the
vasculature, thrombosis of vessels and necrosis of
the intestinal mucosa. For these reasons, fatality
rates for horses with acute abdominal crises often
approaches 70%.
Adhesion molecules that coordinate signalling
pathways and production of cytokines are central
to the recruitment and activation of leucocytes at
sites of inflammation. Recently, several in vivo and
in vitro experiments have studied microvascular
injury during intestinal ischaemia/reperfusion
injury and endotoxin-mediated acute lung injury in
the horse and pig (Darien et al. 1991, 1993, 1995,
1996, 1997, 1998; Triplett et al. 1996; McAnulty
et al. 1997; Kruse-Elliott et al. 1998). One
mechanism central to mediating tissue necrosis is
leucocyte adhesion, transendothelial cell migration
and inflammatory mediator biosynthesis (Figs 1
and 2). To this end, we have studied extensively
the biosynthesis of 2 chemokines central to
leucocyte recruitment, interleukin-8 (IL-8) and
monocyte chemotactic protein-1 (MCP-1) in
human monocytes following adhesion to P-
selectin via its natural ligand, P-selectin
glycoprotein ligand-1 (PSGL-1).
Despite recent progress in understanding its
molecular aspects and development of novel
Fig 1: P-selectin is an adhesion molecule that mediates
rapid, interactions between leucocytes and endothelial
cells called rolling, the early phase of adhesion. This
contact is a pre requisite for firm adhesion (late phase
adhesion), extravasation of PMNs (inflammation), and
tissue injury. P-selectin is stored in platelets and
endothelial cells and expressed on their surface in
response to various stimuli, such as endotoxin. Most
recently, studies using blocking antibodies or a soluble
form of the P-selectin ligand have shown that the
binding of P-selectin to its ligand, P-selectin
glycoprotein ligand-1 (PSGL-1) is important in
preventing ischemia/reperfusion injury.
Monocytes
Neutophils
Platelets
Endolethial cells
LPS
Inflammation
CD11b/CD1 ICAM-
PSGL- P-
Early phase, platelet-endolethial cell adhesion and
platelet-leucocyte aggregation
Late phase leucocyte and platelet
adhesion to endolethial cells
Inflammation & thrombosis
Irriversible tissue injury
Fig 2: A TEM image from an experimentally induced
720 ascending colon volvulus pony model showing a
venule in the lamina propria of the large colon after 2 h
of no-flow ischaemia. Note that there is endothelial cell
disruption and platelet adhesion, documenting the
initiation of early phase adhesion.
Immunology in 2001
92
therapeutics, there remains high mortality in
horses with severe abdominal crises such as
ascending colon volvulus (ACV), salmonella, and
Potomac horse fever. Central to the systemic
inflammatory response to these disorders, the
pathogenesis of intestinal injury and dysfunction is
the activation of leucocytes and vascular cells
(Darien et al. 1991, 1993, 1995; Chang 1992;
Welbourn and Young 1992). The interaction of
activated leucocytes with endothelial cell adhesion
molecules plays a major role in the release of
soluble mediators, including interleukin-8 (IL-8)
and monocyte chemotactic protein-1 (MCP-1),
which amplify the systemic inflammatory
response (Lorant et al. 1993; Granger and Kubes
1994; Lukacs et al. 1995) As such, leucocytes
which are recruited for host defence in the early
stages of acute inflammatory processes, such as
ischaemia, reperfusion and septic shock, are also
implicated in the vascular injury and tissue damage
of the inflammatory processes of
ischaemia/reperfusion injury, multiple organ
failure and death (Weyrich et al. 1995; Diacovo et
al. 1996).
P-selectin is a member of a family of
transmembrane adhesion molecules expressed on a
variety of cell types which mediates rapid transient
interactions between leucocytes and activated
platelets and endothelial cells called rolling. P-
selectin is stored in -granules in platelets and
Weibel-Palade bodies in endothelial cells and
expressed on the surface of these cells in response
to various stimuli, such as endotoxin (LPS),
thrombin and tumour necrosis factor- (TNF-).
P-selectin binds to leucocytes via glycoproteins
expressing sialyated and fucosylated glycans, such
as P-selectin glycoprotein ligand-1 (PSGL-1). This
transient contact is generally believed to be a pre-
requisite for firm adhesion and subsequent
extravasation of circulating monocytes and
neutrophils, which is central to the pathogenesis of
vascular injury during endotoxa and inflammatory
or ischaemic intestinal disorders. While the P-
selectin system has been shown to play an
important role in regulating vascular inflammation
in man, cats, pigs, cows and mice, there is a
paucity of information about equine P-selectin.
As monocytes are central to a number of
pathophysiological responses caused by the
overproduction of immune mediators, modulation
of leucocyte adhesion has been a goal for the
design of many therapeutics. Anti-P-selectin
therapy has shown a great deal of potential for the
amelioration of a variety of these disease states.
One of the most promising P-selectin agonists,
rPSGL1-Ig, is a recombinant, soluble form of
PSGL-1 truncated after the P-selectin binding
domain and fused to the Fc region of a human
immunoglobulin (Ig). As this molecule has been
shown to prevent inflammation in a number of
primary and secondary vascular disorders (eg
myocardial infarcts and ischaemia/reperfusion
injury, respectively), we hypothesised that
rPSGL1-Ig can block both signalling events and
release of chemotactic cytokines in human
monocytes in an in vitro setting. Indeed, we have
seen that rPSGL1-Ig can block the PSGL-1
mediated activation of the mitogen activated
protein kinases (MAPKs) which has been
implicated in interleukin-8 (Il-8) release in
neutrophils. As such, we examined cytokine
production in monocytes induced both by PSGL-1
ligation and by activated platelets. We have found
rPSGL1-Ig to be effective in blocking both Il-8 and
monocyte chemotactic protein 1 (MCP-1)
production, suggesting that activated platelets bind
to monocytes and induce signalling events central
to immune mediator biosynthesis. Our results
demonstrate the efficacy of this drug in modulating
inflammatory reactions.
Ascending colon volvulus (ACV) of 4 to 6 h
results in the adhesion of neutrophils to the injured
endothelium, release of inflammatory mediators
and loss of fluid, thrombosis of vessels and
necrosis of the tissue (Chang 1992; Welbourn and
Young 1992). P-selectin is an adhesion molecule
that mediates rapid interactions between
leucocytes and endothelial cells called rolling
(the early phase of adhesion; Darien et al. 1991,
1993). This transient contact is a pre-requisite for
firm adhesion (late phase of adhesion),
extravasation of PMNs (inflammation), and tissue
injury (Darien et al. 1995; Lukacs et al. 1995). P-
selectin is stored in platelets and endothelial cells
and expressed on their surface in response to
various stimuli, such as endotoxin (Lorant et al.
1993; Granger and Kubes 1994; Diacovo et al.
1996). Most recently, studies using P-selectin
blocking antibodies or using the human
recombinant P-selectin ligand, P-selectin
glycoprotein ligand-1 (PSGL-1) fused to an IgG1
Fc molecule (referred to as the chimera rPSGL1-
Ig) have been shown to prevent
ischaemia/reperfusion injury (Elstaad et al. 1995;
Lorant et al. 1995; Mazzone et al. 1993; Weyrich
et al. 1995). In the authors laboratory, it has been
93
shown that rPSGL1-Ig prevents PSGL-1-mediated
signal transduction and blocks IL-8 and MCP-1
biosynthesis. Given the high incidence of life
threatening colic and inflammatory bowel disease
in the horse, and the importance of the P-selectin
system in other species, we believe the P-selectin
system should be investigated in the horse.
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Chang, S.-W. (1992) Endotoxin-induced lung vascular
injury: Role of PAF, TNF- and neutrophils. Clin.
Res. 40, 528-536.
Darien, B.J., Potempa, J., Moore, J.N. and Travis, J.
(1991) Antithrombin III activity in horses with colic:
An analysis of 46 cases. Equine vet. J. 23, 211-214.
Darien, B.J., Sims, P.A., Stone, W.C., Schilly, D.A.,
Dubielzig, R.R. and Albrecht, R.M. (1993)
Ischemia/reperfusion injury of the ascending colon
in ponies: A correlative study utilizing
microvascular histopathology and corrosion casting.
Scanning Microsc. 4, 1311-1320.
Darien, B.J., Stone, W.C., Dubielzig, R.R., Clayton,
M.K. (1995) Morphologic changes of the ascending
colon during experimental ischemia and reperfusion
in ponies. Vet. Path. 32, 280-288.
Darien, B.J., Stone, W.C., Hart, A.P., Schilly. D.R. and
Clayton, M.K. (1996) Systemic hemostatic
alterations during no-flow ischaemia and
reperfusion of the ascending colon in ponies. Comp.
Haematol. Int. 6, 63-60.
Darien, B.J., Saban, M.R.M., Hart, A.P., MacWilliams,
P.S., Clayton, M.K. and Kruse-Elliott, K.T. (1997)
Morphometric analysis of oleic acid-induced
permeability pulmonary edema: Correlation with
gravimetric lung water. Shock 8, 61-67.
Darien, B.J., Fareed, J., Centgraf, K.S., Hart, A.P.,
MacWilliams, P.S., Clayton, M.K., Wolf, H. and
Kruse-Elliott, K. (1998) Low molecular weight
heparin ameliorates the pulmonary hemodynamics
and pathomorphologic effects of endotoxin in a
porcine model of acute lung injury. Shock 9, 274-
281.
Diacovo, T.G., Roth, S.J. and Springer, T.A. (1996)
Neutrophil rolling, arrest, and transmigration across
activated, surface-adherent platelets via sequential
action of P-selectin and the 2-integrin
CD11b/CD18. Blood 88, 146-157.
Elstaad, M.R., La Pine, T.R., Cowley, F.S., McEver, R.P.,
McIntyre, T.M., Prescott, S.M. and Zimmerman,
G.A. (1995) P-Selectin regulates platelet-activating
factor synthesis and phagocytosis by monocytes. J.
Immunl. 155, 2109-2122.
Granger, D.N. and Kubes, P. (1994) The microcirculation
and inflammation: modulation of leucocyte-
endothelial cell adhesion. J. Leucoc. Biol. 55, 662-
675.
Johnson, C.J., Aga, M., Bertics, P.J. and Darien, B.J.
(2000) Signaling events in human monocytes via the
platelet P-selectin ligand. J endotoxin Res. 6, 107,
(abstr.).
Kruse-Elliott, K.T., Kamal, C., Grossman, J.E.,
Tomasko, S., Kamke, C. and Darien, B.J. (1998)
Low molecular weight heparin alters porcine
neutrophil responses to platelet activating factor.
Shock 10, 198-202.
Lorant, D.E., Topham, M.K., Whatley, R.E., McEver,
R.P., McIntyre, T.M., Prescott, S.M. and
Zimmerman G.A. (1993) Inflammatory roles of P-
selectin. J. clin. Invest. 92, 559-570.
Lorant, D.E., McEver, R.P., McIntyre, T.M., Moore,
K.L., Prescott, S.M. and Zimmerman, G.A. (1995)
Activation of polymorphonuclear leucocytes reduces
their adhesion to P-selectin and causes redistribution
of ligands for P-selectin on their surfaces. J. clin.
Invest. 96, 171-182.
Lukacs, N.W., Strieter, R.M., Elner, V., Evanoff, H.L.,
Burdick, M.D. and Kunkel, S.L. (1995) Production
of chemokines, IL-8 and MCP-1, during monocyte:
endothelial cell interactions. Blood 86, 2767-2773.
Mazzone, A., De Serev, Ricevuti, G., Mazzucchelli, I.,
Fassati, G., Pasotti, D., Bramucci, E., Angoli, L.,
Marsico, F., Specchia, G. and Notario, A. (1993)
Increased expression of neutrophil and monocyte
adhesion molecules in unstable coronary artery
disease. Circulation 88, 358-363.
McAnulty, J.F., Stone, W.C. and Darien, B.J. (1997) The
effects of ischemia and reperfusion on mucosal
respiratory function, adenosine triphosphate,
electrolyte, and water content in the ascending colon
of ponies. Vet. Surg. 26, 172-181.
Triplett, E.A., Kruse-Elliott, K.T., Hart, A.P., Schram,
B.R., MacWilliams, P.S., Cooley, A.J., Clayton,
M.K. and Darien, B.J. (1996) SK&F 86002, a dual
cytokine and eicosanoid inhibitor, attenuates
endotoxin induced cardiopulmonary dysfunction in
the pig. Shock 6, 357-364.
Welbourn, C.R.B. and Young, Y. (1992) Endotoxin,
septic shock and acute lung injury: neutrophils,
macrophages and inflammatory mediators. Br. J.
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Weyrich, A.S., McIntyre, T. M., McEver, R.P., Prescott,
S.M. and Zimmerman, G.A. (1995) Monocyte
tethering by P-selectin regulates monocyte
chemotactic protein-1 and TNF- secretion. J. clin.
Invest. 95, 2297-2303.
JOINT DISEASE IN THE HORSE
C. W. McIlwraith
Orthopaedic Research Laboratory, Colorado State University, Fort Collins, Colorado 80523, USA
Non-infective arthritis is an extremely common
problem in the horse. It is nearly always
considered traumatic in origin with a spectrum of
injury ranging from traumatic synovitis and
capsulitis through ligamentous injury, meniscal
injury, osteochondral fracture, subchondral bone
disease, to osteoarthritis (progressive degradation
of articular cartilage; McIlwraith 1996). Although
examination of the synovial membrane in all these
entities will reveal accumulation of lymphocytes
and plasma cells as a common reaction, it has been
found that this is a non-specific reaction and does
not imply an immune basis (McIlwraith 1996).
Rheumatoid arthritis has never been described in
the horse but isolated cases of lupus erythematosus
have been described.
On the other hand, considerable work in the
last 10 years has been undertaken on the molecular
basis of joint disease in the horse; and cytokines
and inflammatory mediators have been identified.
Inflammatory mediators associated with
destruction of hyaluronan (HA) in synovial fluid
as well as articular cartilage degradation include:
1) the cytokines interleukin-1 and TNF-alpha;
2) metalloproteinases and aggrecanase;
3) prostaglandins E
2
; and
4) free radicals.
Metalloproteinases are considered to play a major
role in the degradation of the extracellular matrix.
Matrix metalloproteinases that have been
incriminated in osteoarthritis include collagenase
1 (MMP-1), collagenase 2 (MMP-8), collagenase
3 (MMP-13), stromelysin 1 (MMP-3), and 2
gelatinases (MMP-2 and MMP-9; Caron et al.
1992; McIlwraith 1996; Clegg et al. 1997a,b).
Evidence for varying roles of these MMPs in
equine articular cartilage degradation is
accumulating. Up-regulation of MMP-1 has been
demonstrated with in situ hybridisation studies on
equine synovial membrane and cartilage in
diseased equine joints in our laboratory. On the
other hand, recent evidence accumulated in both
human and equine studies indicate that the primary
collagenase involved in the degradation of type II
collagen of articular cartilage may, in fact, be
collagenase 3 (MMP-13) (Caron et al. 1992).
Consideration of the breakdown of proteoglycan is
focused on stromelysin (also called proteo-
glycanase or MMP-3) as well as an enzyme
aggrecanase recently identified and classified as
an ADAMTS. Stromelysin cleaves proteoglycans
between the G1 and G2 domain of aggrecan at the
asparagine (341) - phenylalanine (342) bond
(Flannery et al. 1992). It has a wide variety of
substrates including the various proteoglycans as
well as type II collagen in the non-helical site and
molecular cloning and cartilage gene expression of
equine stromelysin I (MMP-3) has been described
by Balkman and Nixon (1998). In addition to
cleavage of aggrecan at the MMP site, cleavage
between the G1 and G2 domains also occurs at the
GLU373-ALA374 site and this has been attributed
to aggrecanase (Sande et al. 1991). Equine matrix
metalloproteinases 2 and 9 have been
characterised in the horse (Clegg et al. 1997a,b)
and can cause further cleavage to the 1/4 and 3/4
fragments of type II collagen generated by
collagenase cleavage. All metalloproteinase is
secreted as latent proenzymes that are activated
extracellularly. Metalloproteinases are inhibited by
2 tissue inhibitors of metalloproteinase known
commonly as TIMP (TIMP-1 and TIMP-2)
(McIlwraith 1996). Pioneering work with equine
metalloproteinases has been done in separate
laboratories (Caron et al. 1996; Clegg et al.
1997a,b; Richardson and Dodge 1997; Balkman
Immunology in 2001
94
and Nixon 1998) with collagenase -3 and
stromelysin sequences cloned (Caron et al. 1996;
Balkman and Nixon 1998). Metalloproteinases as
well as PGE
2
and NO have also been involved in
other joint conditions such as subchondral cystic
lesions in horses. It was demonstrated that the
fibrous tissue of subchondral cystic lesions
released NO, PGE
2
and neutral MMPs into the
culture media suggesting that these may be
partially responsible for the maintenance, slow
healing rate and expansion of these lesions (Von
Rechenberg et al. 2000). Later work has shown
active bone resorption by the fibrous tissue of
subchondral cystic lesions.
Prostaglandins (primarily E group) were
produced in inflamed joints and can cause a
decrease in the proteoglycan content of the
cartilage matrix. The presence of PGE
2
and
synovial fluid from inflamed joints has been
demonstrated in the horse and PGE
2
is used as an
objective index of the level of synovitis in the
authors laboratory (Frisbie et al. 1997; Kawcak et
al. 1997). Oxygen-derived free radicals including
superoxide anion, hydroxyl radicals and hydrogen
peroxide may be released from injured joint tissues
and their increase in the synovial fluid of cases of
equine joint disease have been demonstrated
(Dimock et al. 2000).
Interleukin-1-like activity was first
demonstrated in inflamed equine joints by Morris
et al. (1990). An equine IL-1 containing extract
was produced by May et al. (1990) and the ability
of this extract to degrade equine cartilage matrix
demonstrated. Billinghurst et al. (1995) first
demonstrated induction of intra-articular TNF
during acute inflammatory responses in equine
arthritis. Earlier it had been demonstrated that
recombinant human TNF-alpha, like IL-1, causes
cartilage degradation (Saklatvala 1986). It was
presumed due to stimulating the chondrocyte to
produce matrix-degrading enzymes (Bunning and
Russell 1989). However the role of TNF in
individual clinical arthritides remains unclear.
Based on inhibition studies it appears that IL-1 is
the principal cytokine responsible for articular
cartilage degradation and TNF-alpha contributes
more to clinical morbidity and pain. IL-1 and TNF-
alpha have been demonstrated using RT-PCR in the
synovial membrane of inflamed equine joints.
Interleukin-1 acts through receptors on
chondrocytes causing release of MMPs and PGE
2
and these findings are now being incorporated into
the development of biological therapies. The
complete gene sequences for equine IL-1 alpha, IL-
1 beta and IL-1 receptor antagonist were described
by Howard et al. (1998a,b). There are 2 types of
TNF (P55 and P75). Both have been identified in
synovial tissue and greater numbers of receptors
are seen in joints affected by RA in comparison to
OA synovial tissue (Deleuran et al. 1992). These
receptors can be shed and act as soluble inhibitors
of TNF (TNF binding proteins). Increased serum
concentrations of soluble TNF receptors (sTNF-R)
have been detected in patients with RA and OA in
comparison to healthy controls (Cope et al. 1992).
Traditionally the treatment of equine joint
disease has been based on non-steroidal anti-
inflammatory drugs and corticosteroids (ie
symptomatic suppression of inflammation). Such
anti-inflammatory therapy does have biological
benefits in some instances as demonstrated with
triamcinolone acetonide by Frisbie et al. (1997).
Other novel therapies have included the use of
intravenous hyaluronan (Kawcak et al. 1997).
More recently, biological therapies aimed at
specifically addressing critical mediators have
been investigated. The use of gene therapy with an
adenoviral-IRAP vector has proven quite
successful. The equine IL-1 receptor antagonist
(IRAP) sequence defined by Howard et al. (1998b)
was used. Good transduction was demonstrated in
vitro and in vivo and a single injection of adeno-
IRAP markedly suppressed experimental
osteoarthritis in the horse (Frisbie and McIlwraith
2000). Challenges remain with this therapy as
repeat dosing is not possible with the current
vector combination. A clinical trial is about to
commence in horses. The use of MMP inhibitors
has also been evaluated. In vitro work by R.C.
Billinghurst (New York Academy of Science)
implies that the inhibitor BAY 12-9566 apparently
inhibits equine aggrecanase as well as MMPs, at
least implied by its inhibition of IL-1 mediated
degradation of articular cartilage. This material is
currently being tested in an in vivo canine model of
osteoarthritis and a clinical trial in the horse is
being planned. Other biological methods are
potentially capable of treating equine joint disease,
including promotion of beneficial cytokines or
inhibition of deleterious ones. In addition to IL-1
receptor antagonist, techniques with TNF soluble
receptors IL-4 and IL-10 deserve attention.
Beneficial effects from anabolic cytokines such as
IGF-1 in cartilage healing have also been implied
with in vitro work, and gene therapy with IGF-1 is
currently being investigated.
95
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Balkman, C.E. and Nixon, A.J. (1998) Molecular cloning
and cartilage gene expression of equine stromelysin
1 (matrix metalloproteinase 3). Am. J. vet. Res. 59,
30-36.
Bunning, R.A.D. and Russell, R.G.G. (1989) The effect
of tumour necrosis factor alpha and gamma-
interferon on the resorption of human articular
cartilage and on the production of prostaglandin E
and of caseinase activity by human articular
chondrocytes. Arth. Rheum. 32, 780-784.
Caron, J.P., Bowker, R.M., Abhold, R.H., Toppin, D.S.,
sonea, I.M. and Vex, K.D. (1992) Substance P in the
synovial membrane and fluid of the equine
metacarpal joint. Equine vet. J. 24, 364-366.
Caron, J.P., Tardit, G. and Martel-Pelletier, J. (1996)
Modulation of matrix metalloproteinase 13
(collagenase 3) gene expression in equine
chondrocytes by equine interleukin 1 and
corticosteroids. Am. J. vet. Res. 57, 1631-1634.
Clegg, P.D., Burke, R.M. and Coughlin, A.R. (1997a)
Characterisation of equine matrix metallo-
proteinase 2 and 9; an identification of the cellular
sources of these enzymes in joints. Equine vet. J.
29, 335-342.
Clegg, P.D., Coughlin, A., Rigg, C.M. and Carter, S.D.
(1997b) Matrix metalloproteinases 2 and 9 in equine
synovial fluids. Equine vet. J. 29, 343-348.
Cope, A.P., Aderka, M.D., Engelmann, H. Gibbons, D.,
Jones, A.C., Brennan, F.M., Maini, R.N. Wallach, D.
and Feldman, M. (1992) Increased levels of soluble
tumour necrosis factor receptors in the sera and
synovial fluid of patients with rheumatic diseases.
Arth. Rheum. 35, 1160-1169.
Deleuran, B.W., Chew, C.-Q., Field, M. Brennan, F.M.,
Mitchell, T., Feldman, M. and Maini, R.N. (1992)
Localisation of tumour necrosis factor receptors in
the synovial tissue and cartilage-pannus junction in
patients with rheumatoid arthritis. Arth. Rheum. 35,
1170-1178.
Dimock, A.N., Siciliano, P.D. and McIlwraith, C.W.
(2000) Evidence supporting an increased presence
of reactive oxygen species in the equine joint.
Equine vet. J. 32, 439-443.
Flannery, C.R., Gordy, J.T., Lark, M.W., Neen, P.J. and
Sande, J.D. (1992) Identification of a stromelysin
cleavage site within the interglobular domain of
human aggrecan: evidence for proteolysis at this site
in vivo. 38th Ann. Mtg. orthop. res. Soc. 84.
Frisbie, D.D., Kawcak, C., Trotter, G.W., Powers, B.E.,
McIlwraith, C.W. and Walton, R.M. (1997) Effects
of triamcinolone acetonide on an in vivo equine
osteochondral fragment exercise model. Equine vet.
J. 29, 349-359.
Frisbie, D.D. and McIlwraith, C.W. (2000) Evaluation of
gene therapy as a treatment for equine traumatic
arthritis. A species with clinical disease. Clin.
orthop. Rel. Res. 3795, 5273-5287.
Howard, R.D., McIlwraith, C.W., Trotter, G.W. and
Nyborg, J. (1998a) Cloning of equine interleukin-1
alpha and equine interleukin-1 beta and
determination of their full length CDNA sequences.
Am. J. vet. Res. 59, 704-711.
Howard, R.D., McIlwraith, C.W., Trotter, G.W. and
Nyborg, J.F. (1998b) Cloning of equine interleukin-
1 alpha and equine interleukin-1 receptor antagonist
and determination of their full-length cDNA
sequence. Am. J. vet. Res. 59, 712-716.
Kawcak, C.E., Frisbie, D.D., McIlwraith, C.W., Trotter,
G.W., Gillette, S.M., Powers, B.E. and Walton, R.M.
(1997) Effects of intravenous administration of
sodium hyaluronate on carpal joints in exercising
horses after arthroscopic surgery and osteochondral
fragmentation. Am. J. vet. Res. 58, 1132-1140.
McIlwraith, C.W. (1996) General pathobiology of the
joint in response to injury. In: Joint Disease in the
Horse. Eds: C.W. McIlwraith & G.W. Trotter, W.B.
Saunders Co, Philadelphia, USA.
May, S.A., Hook, R.E. and Lees, P. (1990) The
characterisation of equine interleukin-1. Vet
Immunol. Immunopath. 24, 169-175.
May, S.A. (1995) Tumour necrosis factor, equine arthritis
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Morris, E.A., McDonald, B.S., Webb, A.C. and
Rosenwasser, L.J. (1990) Identification of
interleukin-1 in equine osteoarthritic joint effusion.
Am. J. vet. Res. 51, 59-64.
Richardson, D.W. and Dodge, G.R. (1997) Cloning of
equine type II procollagen and the modulation of its
expression in cultured equine artricular
chondrocytes. Matrix Biol. 16, 59-64.
Saklatvala, J. (1986) Tumour necrosis factor alpha
stimulates resorption and inhibits synthesis of
proteoglycan in cartilage. Nature 322, 547-549.
Sande, J.D., Neen, P.J., Boynton, R.E. and Flannery, C.R.
(1991) Metabolism of aggrecan and cartilage
explants. Identification of a major cleavage site
within the interglobular domain. J. Biochem. 266,
8683-8685.
Von Rechenberg, B., Guenther, H., McIlwraith, C.W.,
Leutenegger, C., Frisbie, D.D., Akens, M.K. and
Auer, J.A. (2000) Fibrous tissue of subchondral
cystic lesions in horses produce local mediators in
neutral metalloproteinases and cause bone
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Immunology in 2001
96
CHONDROCYTIC RESPONSE TO JOINT
INFLAMMATION AND CORTICOSTEROIDS
J. MacLeod
James A Baker Institute for Animal Health, Cornell University, Hungerford Hill Road, Ithaca,
New York 14853-6401, USA
Articular cartilage is an avascular, aneural tissue
which covers the articulating surfaces of bones. It
functions to distribute load and minimise friction
associated with weight-bearing and joint
movement. Articular cartilage is relatively
acellular, with chondrocytes comprising only
12% of the biomass. These cells, however, are
responsible for the synthesis and maintenance of
the extracellular matrix which constitutes the bulk
of cartilaginous tissues. It is the molecular
components and organisation of this extracellular
matrix which is responsible for the specialised
biomechanical and functional properties of
articular cartilage. In osteoarthritis, structural
matrix integrity is compromised leading to
articular cartilage degeneration and a progressive
loss of joint function. We have investigated how
synovitis and corticosteroid therapy affect
cartilage matrix protein synthesis by articular
chondrocytes, testing the hypothesis that changes
in the expression of specific matrix proteins
contribute significantly to molecular mechanisms
that link synovitis and corticosteroid therapy to
long-term degenerative changes in articular
cartilage.
Comparing gene expression in articular
cartilage between normal and mildly inflamed
equine carpi, it is evident that chondrocytes
respond to acute joint inflammation by increasing
the synthesis of matrix proteins. Although mild
synovitis of 8 days duration did not result in any
detectable histopathological changes in the
articular cartilage, steady state type II procollagen
mRNA increased 5-fold and aggrecan mRNA 2-
fold. This anabolic response of chondrocytes to
synovitis probably reflects compensatory matrix
protein synthesis in order to maintain cartilage
homeostasis. Joint inflammation elevates
expression of matrix proteases. In response to the
resulting increase in the rate of matrix protein
degradation, the upregulation of matrix protein
synthesis by chondrocytes attempts to maintain an
overall balance and structural matrix integrity.
Corticosteroids are potent anti-inflammatory
agents used widely for the therapeutic management
of both acute and chronic arthritic diseases.
Although these agents reduce expression of matrix
metalloproteinases, and are clearly helpful in
relieving the symptoms of joint inflammation,
experimental evidence suggests that corticosteroids
damage articular cartilage independent of the
disease process. Pathological changes described in
articular cartilage of previously normal joints
following intra-articular methylprednisolone
acetate (MPA) or betamethasone administration
include: loss of basophilia and decreased safranin
O staining intensity, chondrocyte necrosis and
hypocellularity, decreased proteoglycan content,
decreased collagen synthesis, increased water
content, and delayed healing of experimentally-
induced osteochondral defects. These degenerative
changes alter the composition of articular cartilage
and appear to render the tissue more susceptible to
mechanical injury both in vitro and in vivo. The
mechanism of the steroid injury, however, remains
unclear.
For in vivo studies, the carpi of young adult
ponies were injected with MPA at 0.1 mg/kg and
articular cartilage collected 24 h later. For in vitro
studies, non-calcified full-thickness shavings of
articular cartilage were collected from weight-
bearing areas of the stifle, shoulder, elbow and
carpal joints of horses 1824 months of age. These
joints had no gross evidence of osteoarthritis or
other joint abnormality. The extracellular matrix
was removed by an overnight collagenase
digestion and articular chondrocytes isolated.
Chondrocytes used for total RNA isolation were
97
seeded in 75 cm
2
culture flasks at an initial density
of 5 x 10
6
cells/flask (6.7 x 10
4
cells/cm
2
).
Chondrocytes used to assess methylprednisolone-
induced cytotoxicity were seeded in 962 mm
2
(6
well) culture plates at a density of 2.1 x 10
4
cells/cm
2
. The cultures were maintained in Ham's
F12 medium supplemented with 10% (v/v) FBS,
L-ascorbic acid (50 g/ml), L-glutamine (300
g/ml), -ketoglutaric acid (30 g/ml), HEPES
(125 mM), penicillin (20 units/ml), and
streptomycin (20 g/ml). Changes in gene
expression were assessed by gel electrophoresis of
total chondrocyte RNA, followed by Northern blot
hybridisation using equine-specific cDNA probes.
In vivo steady-state mRNA levels of type II
procollagen and aggrecan core protein were
suppressed 6-fold and 2-fold, respectively, by a
single intra-articular injection of MPA. In vitro,
type II procollagen expression was suppressed in
articular chondrocytes by corticosteroids in a dose-
dependent fashion as medium methylprednisolone
concentrations were increased from 1 x 10
1
to 1 x
10
8
pg/ml. Steady-state levels of type II
procollagen mRNA decreased to less than 10% of
control values at medium methylprednisolone
concentrations 10
5
pg/ml ( 0.2 M). A parallel
change in type I procollagen (2) mRNA did not
occur, with low levels of expression remaining
relatively stable in all samples. Cytotoxicity was
observed as methylprednisolone levels were
increased further from 1 x 10
8
to 1 x 10
9
pg/ml.
The clinical significance and pathogenesis of
detrimental corticosteroid effects on articular
cartilage, so-called steroid-induced arthropathy, is
debated. Suppression of phenotypic markers of
chondrocyte differentiation as it relates to matrix
protein expression clearly has the theoretical
potential to compromise the structural integrity of
cartilage over time. Corticosteroid-induced
chondrocyte necrosis, especially if superimposed
on the loss of chondrocytes that occurs during the
normal progression of osteoarthritis, would also
exacerbate a degenerative process. As with any
clinical intervention, however, detrimental side
effects of intra-articular corticosteroids need to be
assessed in the context of their therapeutic benefits
that include suppression of matrix
metalloproteinase activities and pain relief. The
issue of symptomatic pain relief is further
complicated by its potential to encourage
premature overuse of a structurally and
metabolically compromised joint. Knowledge of
how synovitis and corticosteroids (independently
and in combination) affect chondrocyte function
and matrix protein synthesis is relevant to
understanding the pathogenesis of osteoarthritis
and improving therapeutic strategies for joint
inflammation.
Immunology in 2001
98
REGULATION OF INDUCIBLE NITRIC OXIDE
EXPRESSION AND ACTIVITY IN EQUINE
CHONDROCYTES
J. T. Tung, P. J. Venta and J. P. Caron
Michigan State University, East Lansing, Michigan, USA
Osteoarthritis (OA) is a well known problem for
athletic horses. The pathophysiological hallmark of
the disease is a chondrocyte-mediated digestion and
progressive loss of important cartilage matrix
components. Interleukin-1 (IL-1) and other
proinflammatory mediators promote the
degradation of the matrix by inducing the synthesis
of proteolytic enzymes, the most important of
which are believed to be the matrix
metalloproteinases (MMPs). Another inflammatory
mediator implicated in OA pathophysiology is nitric
oxide (NO). Nitric oxide is hypothesised to
contribute to a number of the deleterious influences
of IL-1 on cartilage metabolism including
augmented expression and activation of MMPs and
reduced synthesis of the natural receptor antagonist
protein for IL-1 (Murrell et al. 1995; Pelletier et al.
1996). In a canine OA model, treatment with a
selective inhibitor of inducible nitric oxide synthase
(iNOS), resulted in a significant reduction of
arthritis lesions of cartilage and synovial membrane
and significantly reduced MMP activity in cartilage
extracts (Pelletier et al. 1999). These data suggest
that inhibition of NO release may attenuate the
progression of OA by reducing MMP synthesis in
cartilage. The hypothesis tested in this study was
that iNOS expression and/or activity of IL-1-
stimulated equine chondrocytes are influenced by
commonly used anti-arthritis compounds.
RT-PCR methods were used to amplify a
portion of the equine iNOS message to prepare an
RNA probe. Northern blot analyses were used to
quantify the expression of iNOS in first passage
monolayer cultures of equine articular
chondrocytes propagated in the presence or
absence of recombinant equine interleukin-1
(reIL-1), dexamethasone, (10
-6
M and 10
-5
M),
and polysulphated glycosaminoglycan (PSGAG),
hyaluronan, and phenylbutazone, each at
concentrations of 10 and 100 g/ml. Nitrite
concentrations in conditioned media of similarly
treated cells were used to quantify iNOS activity
indirectly. Comparison of means for relative
expression of iNOS as well as media
concentrations of nitrite were performed using a
2-way ANOVA (blocked by horse) followed by a
Dunnetts test. A P value of <0.05 was considered
significant.
A 497-bp fragment of equine iNOS cDNA,
amplified by use of RT-PCR, had approximately
88% sequence identity with the human cDNA
sequence and 92% identity of the deduced amino
acid sequence indicating that the cloned fragment
is a portion of the corresponding equine iNOS
message. Recombinant equine IL-1 increased the
expression of iNOS in a dose-dependent manner.
This result was paralleled by increased
concentrations of nitrite in the culture media of
reIL-1-treated cells. Dexamethsone and PSGAG
significantly reduced iNOS gene expression and
conditioned media nitrite concentrations in
cytokine-stimulated cultures (Figs 1 and 2).
Hyaluronan and phenylbutazone had no
statistically significant effect on the expression or
activity of iNOS.
Although the role of NO in the physiology and
pathobiology of equine vascular reactivity and
neuromuscular transmission in a number of organ
systems has been the subject of recent research, its
role in equine OA pathogenesis has received little
study. We prepared an RNA probe to characterise
an inducible member of the NOS family that
corresponds to a similarly-sized cDNA
demonstrated in cytokine-stimulated human
chondrocytes. Results of subsequent experiments
using reIL-1-stimulated cultures complement
previous reports describing NO synthesis by
equine chondrocytes in response to stimulation
99
Fig 2: Effects of anti-arthritis preparations on reIL-1 stimulated iNOS activity by normal chondrocytes. Treatments
include negative and positive (IL-1) controls and treatments of dexamethasone, polysulphated glycosaminoglycan
(polysulphated GAG), hyaluronan and phenylbutazone. IL-1-stimulated control and treated cultures have cross-hatched
bars and non-stimulated controls have clear bars. Inducible NOS activity was determined indirectly using nitrite
concentrations using the Greiss reaction in conditioned media following a 24 h incubation. Values are mean ( se)
expression from 3 experiments. Inducible NOS activity of reIL-1-stimulated iNOS in chondrocytes treated with
dexamethasone (10
-6
M and 10
-5
M) and polysulphated glycosaminoglycan (10 g/ml and 100 g/ml) (marked by
asterisks) was significantly (P<0.05) less than that of reIL-1-stimulated (positive control) cells.
Fig 1: Effects of anti-arthritis preparations on reIL-1 stimulated iNOS expression by normal chondrocytes. Treatments
include negative and positive (IL-1), controls and treatments of dexamethasone, polysulphated glycosaminoglycan
(polysulphated GAG), hyaluronan and phenylbutazone. IL-1-stimulated control and treated cultures have cross-hatched
bars and non-stimulated controls have clear bars. Inducible NOS expression was calculated as the ratio of the intensity
of the iNOS bands to the intensity of the ethidium bromide-stained 28S ribosomal RNA band of the electrophorectic gel.
Values are mean ( se) expression from 3 experiments. Relative expression of reIL-1b-stimulated iNOS in chondrocytes
treated with dexamethasone (10
-6
M and 10
-5
M) and polysulphated glycosaminoglycan (10 g/ml and 100 g/ml)
(marked by asterisks) was significantly (P<0.05) less than that of reIL-1-stimulated (positive control) cells.
Control Dexamethasone Polyunsulphated
GAG
Hyaluronan Phenylbutazone
Treatment
16
14
12
10
8
6
4
2
0
N
i
t
r
i
t
e

(
n
M
)
* *
* *
2500
2000
1500
1000
500
0
R
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
Control Polyunsulphated
GAG
Hyaluronan Phenylbutazone
*
* * *
INOS expresssion
Treatment
Immunology in 2001
100
with proinflammatory mediators. We also provide
data indicating that chondrocyte iNOS expression
and activity are influenced by corticosteroids and
polysulphated glycosaminoglycans.
Northern blot analyses indicated that
expression of our chondrocyte iNOS was inhibited
by dexamethasone at 10
-6
10
-5
M. In other
species, a number of isoforms of NOS have been
described and vary in their sensitivity to
glucocorticoids (Palmer et al. 1992, 1993). Many
iNOS isolated from mammalian chondrocytes are
insensitive to corticosteroids at modest doses.
However, parallelling our findings, bovine iNOS
expression is inhibited with high doses of
dexamethasone (Murrell et al. 1996) These
findings may suggest a limited influence of
endogenous glucocorticoids on iNOS expression
but that pharmacological doses are inhibitory.
PSGAG was observed to reduce reIL-1-
induced expression of iNOS. Although
polysulphated glycosaminoglycans are generally
considered effective for the symptomatic treatment
of OA in man and animals, their mechanism(s) of
action in pain relief and disease modification
(chondroprotection) remain to be confirmed.
Based on our results, it is possible that, in addition
to other potential effects, the chondroprotective
potential of PSGAG may be due to their ability to
inhibit the expression of iNOS. It has been shown
that heparinoids, molecules of similar structure to
polysulphated glycosaminoglycan, are capable of
influencing gene expression in connective tissue
mesenchyme. One recent report indicates that
heparin down-regulates MMP expression in
human gingival fibroblasts (Gogly et al. 1998) and
it has been shown that heparin and pentosan
polysulphate, a sulphated glycosaminoglycan of
plant origin, down-regulate phorbol-induced
cancer cell proliferation by interfering with the
binding of transcription factor to gene promoter
AP-1 sites (Busch et al. 1992).
NO is considered an important mediator in the
pathophysiological processes of arthritis and an
inducible NOS is expressed by equine chondrocytes.
Results of this study provide previously unreported
data on the modulation of equine iNOS expression
and activity. These data support a role for NO in the
pathogenesis of equine OA, and suggest that pre-
translational regulation of the iNOS gene
by dexamethasone and polysulphated
glycosaminoglycans appears to contribute to the
cartilage-sparing properties of these compounds.
REFERENCES
Busch, S.J., Martin, G.A., Barnhart, G.L., Mano, M. and
Jackson R.L. (1992) Trans-repressor activity of
nuclear glycosaminoglycans on fos and jun/AP-1
oncoprotein-mediated transcription. J. cell. Biol.
116, 31-42.
Gogly, B., Hornebeck,W., Groult, N., Godeau, G. and
Pellat, B. (1998) Influence of heparin(s) on the
interleukin-1-beta-induced expression of
collagenase, stromelysin and tissue inhibitor of
metalloproteinase-1 in human gingival fibroblasts.
Biochem. Pharmacol. 56, 1447-1454.
Murrell, G.A.C., Jang, D. and Williams, R.J. (1995)
Nitric oxide activated metalloprotease enzymes in
articular cartilage. Biochem. Biophys. Res. Commun.
206, 15-21.
Murrell, G.A., Doland, M.M., Jang, D., Szabo, C.,
Warren, R.F. and Hannafin, J.A. (1996) Nitric oxide:
an important articular free radical. J. Bone Joint
Surg. 78, 265-274.
Palmer, R.M., Andrews, T., Foxwell, N.A. and Moncada,
S. (1992) Glucocorticoids do not affect the induction
of a novel calcium-dependent nitric oxide synthase
in rabbit chondrocytes. Biochem. Biophys. Res.
Commun. 188, 209-215.
Palmer, R.M., Hickery, M.S., Charles, I.G., Moncada, S.
and Bayliss, M.T. (1993) Induction of nitric oxide
synthase in human chondrocytes. Biochem. Biophys.
Res. Commun. 193, 398-405.
Pelletier, J.P., Mineau, F., Ranger, P., Tardif G. and
Martel-Pelletier, J. (1996) The increased synthesis of
inducible nitric oxide inhibits IL-1Ra synthesis by
human articular chondrocytes; possible role in
osteoarthritis cartilage degradation. Osteoarthritis
Cartilage 4, 77-84.
Pelletier, J.P., Lascau-Coman, V., Jovanovich, D.
Fernandes, J.C., Manning, P., Connor, J.R., Currie,
M.G. and Martel-Pelletier, J. (1999) Selective
inhibition of inducible nitric oxide synthase in
experimental osteoarthritis is associated with
reduction in tissue levels of catabolic factors.
J. Rheumato. 26, 2002-2014.
101
LIST OF PARTICIPANTS
DOROTHY AINSWORTH
United States
DOUG ANTCZAK
United States
CORMAC BREATHNAC
United States
JOHN CARON
United States
LAURENT COUTIL
United States
BENJAMIN DARIEN
United States
JULIA FLAMINIO
United States
STEEVE GIGURE
United States
GITTAN GRNDAHL
Sweden
DUNCAN HANNANT
United Kingdom
STEVE HINES
United States
MARK HOLMES
United Kingdom
DAVID HOROHOV
United States
TOM KLEI
United States
JULIA KYDD
United Kingdom
JEAN-PIERRE LAVOIE
Canada
DR PAUL LUNN
United States
JAMIE MACLEOD
United States
BRUCE MCGORUM
United Kingdom
TRAVIS MCGUIRE
United States
WAYNE MCILWRAITH
United States
JAMES MOORE
United States
RUSTIN MOORE
United States
LESLEY NICOLSON
United Kingdom
GENE PRANZO
United States
SHARANNE RAIDAL
Australia
ED ROBINSON
United States
BONNIE RUSH
United States
ELIZABETH SANTSCHI
United States
JOSH SLATER
United Kingdom
GISELA SOBLL
United States
JEFF STOTT
United States
JOHN TIMONEY
United States
LAURENT VIEL
Canada
JAN WADE
United Kingdom
BETTINA WAGNER
Germany
JOIE WATSON
United States
EVA WATTRANG
Sweden
DAVID WILSON
United States
Immunology in 2001
102
AUTHOR INDEX
AINSWORTH. D.M. et al., 84
ALLEN, G.P. see BREATHNACH,
C.C. et al.; KYDD, J.H. et al.
ANTCZAK, D.F., 29 and see
AINSWORTH. D.M. et al.
APPLETON, J.A. see
ARGYLE, D. see NICOLSON, L.
et al.
AVIZA, G.A. see AINSWORTH.
D.M. et al.
BEATTIE. C.W. see SANTSCHI,
E.M. et al.
BRAZIL, T. see MCGORUM, B. and
BRAZIL, T.
BREATHNACH, C.C. et al., 26
CARON, J.P. see TUNG, J.T.
et al.
COUTIL, L.L. et al., 86
DARIEN, B.J., 91
DAVIS, E.G. see RUSH, B.R. et al.;
FLAMINIO, M.J.B.F. et al.
EADES, S.C. see HOLM, A.S. et al.
FLAMINIO, M.J.B.F. et al., 69 and
see RUSH, B.R. et al.
FRASER, D.G. see McGUIRE, T.C.
et al.
FUXLER, L. see WATTRANG, E.
et al.
GIGU
`
ERE, S., 11; 59
GRNDAHL, G., 46
HANNANT, D., 19 and see
WATTRANG, E. et al.; KYDD,
J.H. et al.
HINES, M. see HINES, S. et al.
HINES, S. et al., 42
HOLLIMAN, A. see HOLMES,
M.A. et al.
HOLM, A.S. et al., 89
HOLMES, M.A. et al., 35
HOPKINS, C. see NICOLSON, L.
et al.
HOROHOV, D.W., 7; 54 and see
LAVOIE, J-P. and HOROHOV,
D.W.; SOBOLL, G. et al.
HUNT, M.A. see COUTIL, L.L.
et al.
JACKSON, K.A. see WATSON, J.L.
et al.
JESSETT, D.M. see WATTRANG,
E. et al.
KLEI, T.R., 17
KYDD, J.H. et al., 65
LAVOIE, J-P. and HOROHOV, D.W.,
82
LEIB, S.R. see McGUIRE, T.C.
et al.
LUNN, D.P., 41 and also SOBOLL,
G. et al.
MACLEOD, J., 97
MCGUIRE, T.C. et al., 3 and see
HINES, S. et al.
MCGORUM, B. and BRAZIL, T., 79
MCILWRAITH, C.W., 94
MCMONAGLE, L. see NICOLSON,
L. et al.
MARTI, E., 33
MAY, P.D.F. see HOLMES, M.A.
et al.
MEALEY, R.H. see McGUIRE, T.C.
et al.
MOORE, J.N., 87
MOORE, R.M. see HOLM, A.S.
et al.
NICOLSON, L. et al., 57
NORTON, L. see HINES, S. et al.
OLIVER, J.L. see HOLM, A.S.
et al.
OLSEN, C.W. see SOBOLL, G. et
al.
ONEILL, T. see KYDD, J.H. et al.
ONIONS, D. see NICOLSON, L.
et al.
PRIEUR, D.J. see McGUIRE, T.C.
et al.
RAIDAL, S.L., 49
RIDGELY, S.L. see McGUIRE, T.C.
et al.
103
RINK, A. see SANTSCHI, E.M.
et al.
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Proceedings of a Workshop on

EQUINE IMMUNOLOGY IN 2001

, T. ONeill and D. Hannant

Gluck Equine Research Centre, Department of Veterinary Science,

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