lira:. I. Vet. res. anim. Sri.

So Paulo, v. 32. n. 3. p. 155-159, 1995

SHEEP ERYTHROCYTE SENSITISATION ANI) RESTRICTION TO LYSIS

BY THE COMPLEMENT SYSTEM FROM EQUINE AND SHEEP SERUM
SE N SIB IL IZ A O D E H EM C I A S DF. C A R N E IR O E R E STR I O L ISE PELO
SISTE M A C O M P LE M EN TO DO SO R O EQ IN O F D F C A R N E IR O
(iislen e 1KRKIRA1: Yarn Maria tu c isa n o V A I.IM 2: Ana Klisa C aleiro Seixas AZZOl.INl-'; Ana Isabel <le Assis lA NIK K'l II4

SUM M ARY
The effe c t o f th e d e g re e o f sh ee p red c ell sen sitisa tio n by a n tib o d y in the re sista n c e to lysis by sheep and h o rse co m p le m en t w as analysed.
T h e resu lts sh o w e d th a t, as ih e level o f sen sitisa tio n in c re a se d , so did the h o m o lo g o u s co m p le m en t a ctiv ity , e v en tu ally lead in g to o n e hundred
p ercen t o f lysis; the sam e w a s o b serv ed fo r h o rse seru m , g e n erally c o n sid e re d as not e ffic ie n t at lysing these cells. T h e re fo re, the h o m o lo g o u s
anil h e te ro lo g o u s restrictio n c an be o v e rrid d e n by th is treatm en t. T h is m ay be o f in terest for the c o m p re h e n sio n o f the m ec h an ism s involved
in the m o d u latio n o f th e re stric tio n p h en o m en o n in h ealth an in d ise a se s in v o lv in g c o m p le m en t in d o m e stic a n im a ls and in hum an, as w ell as
in lab o rato ry d ia g n o sis w h ere h e m o ly tic a ssa y s are u sed for m e a su re m e n t o f c o m p le m en t a ctivity and ev alu atio n o f p resen ce o f a n tib o d ie s in
serum sam ples.

UNITKRMS: L y sis: E ry th ro c y te s; A n tib o d ie s: H em o ly sis

INTRODUCTION
C o m p lem en t is not efficien t at lysing ery th ro cy tes from
the same species. M em bran e proteins regulate the C3 eonvertase71- and the m em brane attack com p lex ( M A C ) 1719,
restricting c o m p le m e n t activity. E ry th ro c y tes from in d i
viduals affected by paroxysm al nocturnal h em oglob inuria
are d efficient in D ecay A c c e le ra tin g F a c to r ( D A F ) 1-1-14,
H o m o lo g o u s R e stric tio n F a c to r ( H F R ) 518, an d C D 5 9 8,
a n d a r e s u s c e p t i b l e to h o m o l o g o u s c o m p l e m e n t .
Resistance to com plem en t action is also observed in het
erologous systems, dep ending on the cell and o f species o f
com plem ent. As an exam ple, horse co m p lem en t is ineffi
cient to lyse sheep red ce lls - - 1 i-12, and control o f the M AC
is in v o lv e d 11. H ow ever, this restriction appears to be te m
porary. S to ra g e in v itro in c r e a s e s the s u s c e p ti b ilit y o f
sheep red cells to horse c o m p le m e n t1, and hum an cells to
human c o m p le m e n t10. This m ay partially explain o p p o s
ing results in the literature9-1-', and iluslrates the im p o r
tance o f studies analysing possible factors involved in this
phenom enon.

In studies employing sheep serum, inconsistent results were

observed using different lots o f the same monoclonal anti
body to sensitise sheep red cells'. T he level of cell sensitisa
tion is known to interfere in the degree o f lysis, but its corre
lation to the hom ologous (and heterologous) restriction to
complement has not been focalized. Here sheep erythrocytes

were sensitised with variable amounts o f antibody and sub

jected to lysis by sheep (homologous) and horse (heterolo
gous) serum; guinea pig and hum an serum w ere used for
comparison. The results are discussed in terms o f degree of
restriction presented by these sensitised cell to complement
action. T he im portance of studies on this subject is rein
forced considering situations of occurrence of high levels of
antibodies and cell sensitisation in Wvo^, (he cytolitic reac
tions by homologous com plem ent4, and its role in immune
surveillance and disease.

MATERIAL AND METHOD

Complement Fixation Diluent (CFD) containing Ca-+. M g 2+
and 0.1% gelatin; and phosphate-buffered saline (PBS) were
prepared as described6. Tw o aliquots o f blood were collected
under sterile conditions from each adult healthy sheep: in 4%
sodium citrate for the cells, and without anticoagulant for the
serum. Human, horse and guinea pig sera were prepared from
blood of adult healthy individuals, pooled and stored at 70C.
When necessary, sera were absorbed at 0C with sheep red
cells. Rat monoclonal ( Ig M ) anti-sheep red cell antibody was
s u p p l i e d by t h e M R C C e n t r e , C a m b r i d g e , E n g l a n d .
Polyclonal (mainly IgG) anti-sheep red cell antibody was
prepared by injecting rabbits with cell stroma, collecting
serum, and inactivating it at 56C for 30 minutes. Hemolytic

1 - Ps-graduanda cm Bioqumica - Faculdade de Cincias Farmacuticas du USP - Campus dc Ribeiro Preto - SP.
2 - Professora Associada - Faculdadc dc Cincias Farmacuticas da USP - Campus de Ribeiro Preto - SP.
3 Farmacutica - Faculdade de Cincias Farmacuticas da USP - Campus dc Ribeiro Preto - SP.
4 - Professora Assistente - Faculdade de Cincias Farmacuticas da USP - Campus de Ribeiro Preto - SP.

155

P I-.R K 1R A . < V A I . I M . V M l . . ; A / / / . O L I N I . A . L C . S . IA M M X H I , A .I . A . S lic e p cr yllm K -yii* s e n s i i i s a l i o n a n d r c s lr ic lio n to l y s i s h y th e i m p l e m e n t s y s t e m rtn n e q u in e a n il s h e e p scrum.

H r a z . J . v c t . K o s . a n i m . S e i . . S o P a u In. \ .3 2 . 11. \ p . 15 5 - 15 9 . IW 5 .

assays were performed incubating 40 pi o f EA with scrum

diluted 1:2 in CFD to 1.0 ml. After 30 minutes of incubation
at 37"C. and ce n trifu gation, lysis was determ in e d by the
absorption o f the supernatant at 412 nm. Percentages o f lysis
were calculated considering the absorption o f EA lysed in
distilled water as 100%. Dilutions o f monoclonal antibody
used to EA preparation offered approximately 8.4 x 10! - 5.4
x 10 s molecules o f antibody/cell.

35%). O ur results com plem ent these observations showing

that, one hu ndred percent of h o m olog ous lysis occurs in
vitro since cells are heavily sensitised. Thus, the level of
sensitising antibody affects the restriction presented by cells
to c om p lem ent attack and may have an important role in
defense and disease mechanisms. High sensitisation ol cells
may occur in vivo in some cases leading to severe hemoly
sis or cell death. T h is is ilustralcd by the o ccurrence of
cytolytic reactions by h o m o lo g o u s c o m p le m e n t, such as

RESULTS ANI) DISCUSSION

destruction of cat cells infected with feline leukemia virus

by c a t a n t i - t u m o r IgG an d cat c o m p l e m e n t 1: or severe

Sheep erythrocytes from one animal were washed in PBS

and s e n sitis e d w ith v a r io u s c o n c e n t r a tio n s o f a n tib o d y
(Erythrocyte-Antibody complex. EA) according to H A R R I
SON: L A C H M A N N 6 (1986), washed 3 times in CFD, and
resuspended to a concentration of 1.7 x 10* cells/ml. The EA
were incubated at 37C with sheep serum from the same ani
mal or horse serum from a pool, and lysis was m easured
a fter 30 m inutes. T he results o f a re p re se n ta tiv e from 4
assays arc shown in Fig. 1. At adequate antibody concentra
tion. the sh e ep serum ly sed on e h u n d re d p erc en t o f the
autologous cells, and the sam e occurred for horse serum.
Fresh cells were used to abrogate age-related variations in
susceptibility to c o m p lem en t1. Therefore, hom ologous and
heterologous restriction to lysis by com plem ent can be over
ridden by this treatment. Similarly, reactive lysis by hom olo
gous com plem ent was observed when the num ber o f C'5b67
sites was sufficiently increased, com pensating the lysis inhi
bitio n 17. The effect was similar with both monoclonal and
polyclonal antibodies, indicating that it is not specific to a
p a r tic u la r a n tib o d y or, for the p o ly c lo n a l a n tis e ru m , to
blockage o f restricting factors on the cell m embrane. The
use o f one serum dilution throughout the experiment, and
the absence o f lysis by inactivated sera (not shown) elimi
nate the possibility o f interference o f factors other than com
plement in the results.

h em o ly sis asso cia te d with p atholo gical p rocesses where

high levels o f antibo dies and cell sensitisation occurs in
vivo, as in cold agglutinin sy n d ro m e in h u m a n s '6. Apart
from this, they may have interest also for the routine use of
sensitised sheep erythrocytes in hemolytic assays for labo
ratory diagnosis involving determination of antibody titres
or c o m p le m e n t levels; particu la rly c o n s id e rin g here the
large domesticated animals, whose sera show low lytic effi
ciency against these cells-1, and the possibility of improve
ment o f assay sensitivity by increasing the level o f cell sen
sitisation.

The mechanism by which high levels o f antibody sensitisa

tion overcom e homologous restriction is not clear. It is pos
sible that e n h a n c e d rates o f c o m p le m e n t activation may
allow M A C formation to occur sufficiently rapid to avoid
the action o f the restriction proteins. Further investigation ol
the mechanism is therefore in progress.

CONCLUSIONS
O ur results show that the level of cell sensitisation affect'h o m o lo g o u s (and h ete ro lo g o u s) restric tio n p resented b\

Data in Tab. I (representative from 4 assays) there is a c o m

parison of lysis of EA by sheep, horse, human and guinea
pig c o m p le m e n t, clearly sh ow ing the high effic ie n c y o f
human and guinea pig serum in this system. However, sim i
larly to data in Fig. 1. sheep and horse serum diluted 1 in 2
lysed the cells at the appropriate antibody concentration. In
this assay, the use o f antibody diluted only 1 in 32 or 1 in 64
would lead conversely to the conclusion that the activity of
r e s p e c t i v e l y s h e e p o r s h e e p an d h o r s e c o m p l e m e n t is
absolutely restricted by sheep red cells.

cells to com plem ent attack. This may help the com prehen
sion of the m echanism s underlying the modulation of (he
restriction pheno m eno n as well as its role in determining
com plem ent action in defense and diseases. In addition, or
using hemolytic assays for research and laboratorial diagno
sis, on e has to be a w a re that a n t ib o d y titre s may v a n
depending on the com plem ent source used in the titratior
assays, and that in some cases lest sensitivity for measuring
com plem ent levels can be improved by increasing cell sensi
tisation. T h is may be im portant in v eterinary pathology
since, as previously stated, serum from most com mon large

Lysis by homologous com plem ent in vitro was observed to

human red cells sensitised by a n tib o d y 10, how ever it peaked
at levels o f 40-60%, with routinely lower values (less than
156

dom estic anim als do not Ivse sheep cells, and there is. it
consequence, little information on their complement le\el;
in health and in disease*.

P K R F .IR A . ( .. V A I IM . Y \ 1 I ..: A Z Z O I .I N I . A .I :.C S . P A N D O C H I . A I A

S h e e p e r y t h r o c y t e x e n M ii> iiiio n ;jn<J r e s tr ic tio n t o ly s i s b y d ie c o m p le m e n t sV M em fr o m e q u in e a n d s h e e p s e r u m .

B r a / . .1. \ e t . R e s . a n i m . S e i . . S o P a u lo . \ .^ 2 . n..V p. 1 5 5 1 5 9 . 1 9 9 5 .

TABI.F. 1
P e rc e n ta g e o f lysis o f sh ee p F^A (m o n o c lo n a l a n tib o d y ) by sh eep , horse
h u m a n o r g u in e a pig seru m * . R ib eiro P reto - S P , 1993.
R ecip ro cals
o f an tib o d y
dilu tio n s

*>

l(>

.2

64

C o m p le m en t
S ource *
hum an

KKI.O

KKI.O

100.0

100.0

KKI.O

lOO.O

g u in e a pig

100.0

100.0

100.0

KKI.O

KKI.O

KKI.O

equine

100.0

100.0

100.0

9 7 .6

26. 1

0.0

sheep

100.0

88.4

52.9

14.9

(1.0

0 .0

* T he hem olytic assay w as preform ed as in Fig. 1.

** C o m p le m en t w as d ilu ted 1:2 in CT'I) (see m ethods for diluent com posi
lion).

ACKNOWLKDGKMKNT
Wc acknow ledge Prof. P.J. Lachmann for his helpful 1 dis
cussion, the help of R. Okiroyd in supplying the monoclonal
antibody, Maria Regina P. Raphaloski and S. C'oppendale
for her help with the manuscript, and Sergio A. Uyemura
and [)r. Carlos Curti for the illustration.

FK il'Rl-: I
Litfeci o f th e d e g re e o f cell se n s itis a tio n b \ a n tib o d y in the ly sis o f sh ee p
F.A b \ sh ee p (O ) an d h o rse (O ) sera.
F.A w ere in c u b a ted at 37C lo r 3 0 m in u te s w ith d ilu te d seru m . A fte r c e n
trifu g a tio n . I\s is w as d e te rm in e d by the a b so rp tio n o f th e s u p e rn a le n t at
412 m m , c o n s id e rin g th e a b s o r p tio n o f KA ly s e d in d is tille d w a te r as

100#.

157

P P R I .I K A . ( i . Y A 1 .I M . Y M .I ..I A Z Z O L I N I . A l C . S . P A N D O C H I . A .I .A . S l u r p e r y llim c y t c s c n s i l i s a l u m a n d r e s t r ic t io n l o l y s i s b y lh e e o m p le in c n l s y s io m Ir o m e q u in e atui s h e e p scrum,

l i r a / . J . v e t. Kes. a n iin . Sei.. S o P a u lo , v 32. n. V p . 15 5 - 159. 1995.

RK SUM O

Neste trabalho foi analisado o eleito do grau de sensibilizao de hetrueias de carneiro por anticorpo, na resistncia lise pelo
complemento do soro eqino e de carneiro. Os resultados mostraram que, medida que houve 11111 aumento do nvel de sensi
bilizao. ocorreu tambm 11111 aumento da atividade do complemento homlogo, levando a 1009; de lise: o mesmo foi obser
vado para o complemento do soro eqino, geralmente considerado ineficiente na lise destas clulas. A restrio lise pode.
assim, ser sobrepujada por este tratamento. listes dados podem ser de interesse para a compreenso dos mecanismos envolvi
dos na modulao do fenmeno de restrio, na sade e etn doenas envolvendo o sistema complemento em animais domsti
cos c 110 homem, bem como em diagnstico laboratorial, onde so utilizados ensaios hemolticos para medida de atividade do
complemento e pesquisa de anticorpos em amostras de soro.
U M T K R M O S : 1.ise:

F.ritrcitos: Anticorpos: Hemlise

REFERENCES
1-ASSIS. A.I..; P E R E IR A . G.; A Z Z O L IN I. A.E.C.S.:
BARBOSA, J.L. I ' fleet o f the in vitro ageing on the restric
tio n o f s h e e p e r y t h r o c y t e l y s i s by h o r s e s e r u m .
S c a n d i n a v i a n J o u r n a l o f I m m u n o l o g y , v.33. p.579-84.
1991.

8-1IO L G U IN . N.H.: F R E D R IC K . L.R.: BERNSHAW,

N.J.; W IL C O X , I..A. PA R K E R . C.J. Isolation and charac
terization o f a membrane protein from normal human ery
throcytes that inhibits reactive lysis o f the erythrocytes of
paroxysmal nocturnal hemoglobinuria. J o u r n a l o f Clinical
Investigation, v.84. p.717. 1989.

2 -B A R T A. ().: B A R I A . V .; W I L L I A M S . L .L . A
m e t h o d fo r t i t r a t i n g e q u i n e h a e m o l y t i e c o m p l e m e n t .
Z eitsch rift f u r I m m u n it a ts f o r s c h u n g Bd.. v. 146. p. 114-22.
1973.
3-G R A N T . C.K. C o m p lem en t "sp ecificity " and inter
changeability: measurement o f hemolytic com plem ent levels
and use of the complement fixation test with sera from c o m
mon d o m e s tic a te d a n im a ls. A m e r i c a n J o u r n a l of
V e te rin a ry R e sea rch , v . 3 8 . 11.10. p. 1611-7. 1977.

9-H O U L E . J.J.: H O FF M A N N . E.M. The use o f a passive

hemolysis system to evaluate the com plem ent activities of
six m a m m a lia n s p e c ie s. V e t e r i n a r y I m m u n o l o g y and
Im m u n o p a th o lo g y . \ . 16 . p.259-70. 1987.
1 0 -H O U L E . J.J.: H O F F M A N N . E .M .. E S S E R . A.F.
Restriction of cell lysis by homologous complement. I. An
analysis of m em brane attack com plex formation on target
membranes. Blood, v.7 l. n.2. p.280-6. 1988.

4-G R A N T . C.K.; D eB O L R . D.J.: ESS E X . M.: W O R

LEY, M B.: H IG G IN S , J. A n tib o d ie s from h ea lthy cats
exposed to feline leukemia virus lyse feline lymphom a cells
show lysis with cat complement. J o u r n a l o f Im m u n o lo g y ,
v. I 19. n.2. p.401-6. 1977.
5-IIA N SCH . G.M .; S C H O N E R M A R K . S.: RO ELCK E.
I). Paroxysmal nocturnal hemoglobinuria type III: lack o f an
erythrocyte membrane protein restricting the lysis by C5b-9.
J o u r n a l o f Clinical Investigation. v.XO. p.7-12. I9S7.

6 - H ARR ISON. R.A.; L A C IIM A N N . P.J. C om plem en t

T e c h n o l o g y . In: W E I R . D .M . e d . H a n d b o o k o f
E x p e r i m e n t a l I m i m i n o c h e m i s t r y . O x f o r d . B l a c k w e ll
Scientific Publications, v .l, p.39.1-39.49, 1986.
7 -H O F F M A N N . K.M. Inhibition o f c o m p le m e n t by a
substance isolated from hum an erythrocytes. I. Extraction
from hum an erythrocyte stroma. I m im i n o c h e m is tr y . v. 6 .
p.3 9 1-403. 1969.
158

11- L A C II M A N N . P.J.: B O W Y E R . D.E.; N IC O L, P.;

D A W SO N , R.M.C.; Ml INN. II. A. Studies on the terminal
stages o f com plem ent lysis. Im m u n o lo g y , v.24. 11. 1, p. 13545. 1973.
12-NIGHOLSON W E L L E R . A.: BURGE. J.: AUSTEN.
K.F. Purification from guinea pig erythrocyte stroma o f a
decay accelerating factor for the classical C3 convertase.
C 4 b 2 a . J o u r n a l o f I m m u n o l o g y . \ .127. n.5. p . 2035-9.
1981.
I 3 - N I C II O L S O N - W E L L E R .

A .:

M A R C H . J .P .:

R O S E N F E L D . S.I.: A U ST E N . K.F. Affected erythrocytes

o f patients with paroxysmal nocturnal hem oglobinuria are
deficient in the com plem ent regulatory protein decay-accel
erating factor. P r o c e e d in g s o f th e N a tio n a l A c a d e m y of
Sciences o f th e U n ite d S ta te s o f A m e r ic a , v.80, p.506670.1983.

P E R E I R A . G .. V A L I M . Y .M .L .; A Z / O I .I N I . A .E .C . S .; P A N D O C H I , A .I .A . S h e e p c r y t h r o e y t c s e n s it is a t io n a n d r e s tr ic tio n t o l y s i s b y t h e c o m p le m e n t s y s t e m fr o m e q u in e a n d s h e e p s e r u m .
B r a /.. J . v e t . R e s . a n i m . S c i . , S o P a u lo , v . 3 2 , n .3 , p . 1 5 5 1 5 9 . 1 9 9 5 .

14-PANGBURN, M.K.; S C H RE IBER, R.D.; M U LLER EBERHARD. H J . Deficiency o f an erythrocyte membrane

protein with com plem ent regulatory activity in paroxysmal
nocturnal h em oglobin uria. P r o c e e d i n g s o f t h e N a ti o n a l
A c a d e m y o f S cie n ce s o f t h e U n ite d S ta te s o f A m e r ic a ,
v.80, p.5430-4, 1983.
15-REIS. K.J. An hemolytic assay for the measurement
of equine co m p lem ent. V e te r in a r y Im m u n o lo g y a n d
Im m u n o p a th o lo g y , v.23, p. 129-37, 1989.
1 6 -R O S S E , W .F .: A D A M S , J.P . T h e v a r ia b ilit y o f
hemolysis in the Cold Agglutinin syndrome. Blood, v.56,
p.409-16, 1980.
17 - S C H O N E R M A R K , S .; R A U T E R B E R G . E .W .;
SHIN. M.L.. L O R E , S.; R O E L C H E . D.: H A N S C H . Ci.M.
Homologous species restriction in lysis o f hum an erythro

cytes: a membrane-derived protein with C 8-binding capacity

f u n c tio n s as an inhibitor. J o u r n a l o f I m m u n o l o g y a n d
Im m u n o p a th o lo g y . v. 136, n.5. p. 1772-6, 1986.
I 8 - Z A L M A N , L .S .; W O O D . L .M .; F R A N K , M .M .;
M U L L E R -E B E R H A R D , H.J. Deficiency o f the homologous
restriction factor in paroxysmal nocturnal hemoglobinuria.
J o u r n a l o f E x p e r im e n ta l M edicine, v.165, p.527-7, 1987.
19-Z A L M A N . L.S.; W O O D , L.M .; M U L L E R -E B E R
H A R D , H.J. Isolation o f a hum an erythrocyte m em brane
protein c a p a b le o f in h ibiting ex p re ssio n o f h o m o lo g o u s
com plem ent transm cm branc channels. P ro c e e d in g s o f the
N a ti o n a l A c a d e m y o f S c ie n c e s o f th e U n ite d S t a t e s o f
A m e ric a , v.83, p.6975-9. 1986.
R e ce b ido p a r a puhlica^'ao em 11/02/94
A p r o v a d o p a r a publicai^ao em 13/12/94