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Haemolysis of human erythrocytes heavily sensitized with sheep amboceptor by sheep complement chelated with EGTA or Mg2+-EGTA
Veterinary Immunology and Immunopathology, 2 ( 1 9 8 1 ) 2 5 3 - - 2 6 4
253
Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in Belgium
HAEMOLYSIS OF HUMAN ERYTHR~CYTESHEAVILY SENSITIZED WITH SHEEP AMBOCEPTORBY SHEEP COMPLEMENTCHELATED WITH EGTA OR Mg2+-EGTA M. STANKIEWICZ1, and W. JONAS2 IDepartment of Immunology and I n s t i t u t e of Zoology, Warsaw U n i v e r s i t y , 00927/1 Warszawa, Krakowskie Przemiescie 26/28 Poland 2Wallaceville Animal Research Centre, Research D i v i s i o n , M i n i s t r y of A g r i c u l t u r e and Fisheries, Private Bag, Upper Hutt, New Zealand (Accepted 24 March 1981) ABSTRACT Stankiewicz, M. and Jonas, W. 1981. Haemolysis of human erythrocytes heavily sensitized with sheep amboceptor by sheep complement chelated with EGTA or Mg2+-EGTA. Vet. Immunol. Immunopatho]., 2: 253-264. Human erythrocytes sensitized with an a g g l u t i n a t i n g dose of e i t h e r sheep anti-human erythrocyte antiserum, IgM, slow or fast y - g l o b u l i n antibody are haemolysed by foetal lamb or sheep serum complement (C) in the presence of e t h y l e n e g l y c o l t e t r a a c e t i c acid (EGTA) or Mg2+-EGTA but not ethylenediaminetetraacetic acid (EDTA) or triethylenetetraminehexaacetic acid (TTHA). Dialysis of the C source aqainst phosphate buffered saline rendered i t i n a c t i v e , but addition of Mg2+ or Mg2~-EGTA restored haemolytic a c t i v i t y . Haemolysis of sensitized erythrocyte~ took place more r a p i d l y in buffer containing Ca2+ and Mg2+ than in EGTA or Mg2T-EGTA. Heating the C source at 50 °C for 30 min or 56 °C for 3 min abolished haemolytic a c t i v i t y . In the presence of EGTA or Mg2+-EGTA, haemolytic a c t i v i t y was restored to 50 °C-heated serum by a CM-cellulose f r a c t i o n that contained sheep f a c t o r B. These results show that sheep C, in the presence of EGTA or Mg2+-EGTA w i l l haemolyse human erythrocytes heavily sensitized with sheep amboceptor. The a l t e r n a t i v e pathway of sheep C is thought to be involved. INTRODUCTION Lysis of sensitized erythrocytes has been a basic test for i n v e s t i g a t i n g complement (C) a c t i v i t y .
In the standard haemolytic assay system, sheep erythro-
cytes and r a b b i t anti-sheep erythrocyte antibody are used.
This system has a
l i m i t a t i o n in that i t cannot be used to demonstrate C a c t i v i t y in serum of some species of animals including sheep (Rice and Crowson, 1950).
However, sheep C
haemolytic a c t i v i t y can be r e a d i l y demonstrated when sheep antibodies are used to sensitize e i t h e r human erythrocytes (Feinstein and Hobart, 1969; Rhee et al. 1970) or r a b b i t erythrocytes (Barta et a l . ,
1975).
0165-2427/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishmg Company
254
Two major pathways have been described fo r the a c t i v a t i o n of C.
The classical
pathway is antibody dependent and involves the C components CI, C4 and C2. requires Caz+ and Mg2+ f o r a c t i v i t y ,
It
The a l t e r n a t i v e pathway may be activated
without antibody, is Mg2+ dependent and has a 50 °C l a b i l e component, factor B. These two a c t i v a t i o n pathways have been thoroughly investigated f o r guinea-pig and human C (MUller-Eberhard, 1975) but l i t t l e
is known f o r other animals.
Although i t is generally accepted that the classical pathway is involved in immune haemolysis, the f o l l o w i n g points indicated to us that sheep C immune haemolysis may not always use only the classical pathway.
Haemolysis of rabbit
erythrocytes sensitized with sheep amboceptor has a high Mg2+ requirement (Barta et a l . , 1975).
Sheep C is e i t h e r poorly or non-haemolytic f o r sheep erythrocytes
sensitized with r a b b i t amboceptor (Rice and Crowson, 1950; Barta et a l . , 1975), an amboceptor that is very e f f e c t i v e in a c t i v a t i n g the classical pathway. Classical pathway components C4 and C2 have not been detected (Barta and Hubert, 1978) or found in low t i t r e only (Borsos and Rapp, 1965) in sheep serum. The purpose of this paper is to show that sheep C haemolysis of human erythrocytes heavily sensitized by sheep amboceptor takes place in the presence of e t h y l e n e g l y c o l t e t r a a c e t i c acid (EGTA) or Mg2+-EGTA. This and other data indicates that this haemolytic a c t i v i t y is probably dependent on the a l t e r n a t i v e pathway of sheep C. MATERIALS AND METHODS Buffers and chelatinq aqent§ Phosphate buffered saline (PBS, 0.15 M, pH 7.2) w@s used for some procedures. Veronal buffered saline containing Ca2+ and Mg2+ (VBS2+) (Mayer, 1961) was used as the d i l u e n t in the haemolytic assays.
Stock solutions (0.1 M, pH 7.6) of
ethylenediaminetetraacetic acid (EDTA) and e t h y l e n e g l y c o l t e t r a a c e t i c acid (EGTA) were prepared as described by Fine et al.
(1972).
Stock solutions of Mg2+-EGTA
(stock 0.1M EGTA containing 10, 20 or 50 mM Mg2+) were prepared with MgCl2 and the pH adjusted to pH 7.6.
Stock solutions (pH 7.6) of MgCl2 (10, 20 or 50 mM
Mg2+) were prepared in 0.15 M saline. Ca2+) was prepared in saline.
A stock solution of CaCI2.2H20 (0.2 M
Triethylenetetraminehexaacetic acid (TTHA) was
prepared as described by Nagaki et al. (1974).
Complement ~iCJ Blood collected from near-term foetal lambs, adult sheep or guinea-pigs was allowed to c l o t at room temperature (2 h).
After standing overnight at 4 °C,
serum was separated and frozen in aliquots at -20 °C.
255
Some sheep serum was absorbed w i t h human e r y t h r o c y t e s by t r e a t i n g
1 ml o f
serum c o n t a i n i n g 0 . 1 M EDTA w i t h 0.5 ml o f packed e r y t h r o c y t e s f o r 1 h at 0 - i °C on f o u r occasions.
Some absorbed serum was d i a l y s e d o v e r n i g h t at 0-1 °C
a g a i n s t VBS2+ and some a g a i n s t PBS. Some serum samples were heated at e i t h e r 50 °C f o r 30 min, 56 °C f o r 3 min or 56 °C f o r 30 min. The f o e t a l
lamb serum (FLS) used was h a e m o l y t i c a l l y i n a c t i v e when incubated
u n d i l u t e d w i t h u n s e n s i t i z e d human e r y t h r o c y t e s and was negative f o r immunog l o b u l i n when checked by immunoelectrophoresis and an a n t i g l o b u l i n
inhibition
t e s t (Jonas, 1969). CM-cellulose f r a c t i o n The method f o r i s o l a t i n g was used w i t h FLS.
f a c t o r B from human serum (Lachmann e t a l . ,
The f r a c t i o n
1973)
used (11-13 mmhos, 0.49 mg/ml), by g r a d i e n t
p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s contained 5 p r o t e i n bands (515,330,140,107 and 84,000 d a l t o n s ) .
I t has a haemolytic t i t r e
w i t h sheep antibody s e n s i t i z e d
human e r y t h r o c y t e s of 1:32 in 1:4 50 °C sheep serum c o n t a i n i n g Mg2+-EGTA. A f t e r s e p a r a t i o n on Sephadex G-200, the haemolytic a c t i v i t y 107,000 d a l t o n p r o t e i n .
was associated w i t h the
Guinea-pig a n t i s e r a to the f r a c t i o n p r e c i p i t a t e d from
e l e c t r o p h o r e s e d sheep serum a s i n g l e band in the f a s t B - g l o b u l i n r e g i o n . Erythrocytes Group 0 R h - p o s i t i v e human e r y t h r o c y t e s or sheep e r y t h r o c y t e s , c o l l e c t e d i n t o A l s e v e r ' s s o l u t i o n were obtained weekly.
The e r y t h r o c y t e s were washed three
times in PBS before use. Antisera Sheep antiserum to human e r y t h r o c y t e s Six Romney ewes were each i n j e c t e d i n t r a m u s c u l a r l y (I/M) w i t h 4 ml o f a 30% suspension o f human e r y t h r o c y t e s e m u l s i f i e d w i t h an equal volume o f Freund's complete adjuvant (FCA). weeks. of IgM.
Two f u r t h e r
I/M i n j e c t i o n s were given at f o u r and ten
Serum o b t a i n e d ten days a f t e r the f i r s t
i n j e c t i o n was used as a source
Serum o b t a i n e d f o u r weeks a f t e r the t h i r d
i n j e c t i o n was used as a
source o f anti-human e r y t h r o c y t e antiserum and the source of slow and f a s t y-globulin.
The antiserum was pooled, heated at 56 °C f o r 30 min and t r e a t e d
w i t h s a t u r a t e d ammonium sulphate to a f i n a l
c o n c e n t r a t i o n of 40%. The
p r e c i p i t a t e d g l o b u l i n was washed, r e c o n s t i t u t e d to the o r i g i n a l d i a l y s e d a g a i n s t PBS and s t o r e d in a l i q u o t s at -20 °C.
serum volume,
256
IgM, slow and f a s t T - g l o b u l i n were prepared as described by Jonas (1969). The p r o t e i n c o n c e n t r a t i o n of the immunoglobulins ( I g ) was assessed at 280 nm a g a i n s t a standard curve prepared w i t h bovine T - g l o b u l i n .
The p u r i t y o f the
f r a c t i o n s was checked by immunoelectrophoresis. Rabbit antiserum to sheep er%_thro!yte § Haemolysin was purchased from the Commonwealth Serum L a b o r a t o r i e s , Melbourne, Australia.
I t was heated and p r e c i p i t a t e d as described above.
The h a e m a g g l u t i n a t i n g t i t r e s
of the amboceptors were determined using a 1~]
suspension of the a p p r o p r i a t e e r y t h r o c y t e s . Guinea-pj 9 antiserum to the CM-cellulose f r a c t i o n Five g u i n e a - p i g s were used.
One ml of PBS c o n t a i n i n g the CM-cellulose
f r a c t i o n was e m u l s i f i e d w i t h i ml of FCA. The immunization schedule was: day 1, 0.2 ml o f emulsion i n t o each hind f o o t p a d ; day 10 and 20, 0.2 ml o f emulsion I/M i n t o each hind l e g ; day 30, bled out. Guinea-pig antiserum to sheep serum Guinea-pigs were immunized (as above) but w i t h a 1:10 d i l u t i o n
of sheep
serum. Haemolytic assays Serum (the C source) was s e r i a l l y
d i l u t e d t w o - f o l d in 0.4 ml volumes of VBS2+.
A 0.5 ml volume o f VBS2+ or the reagent under t e s t was added and mixed.
To a 5%
suspension o f human or sheep e r y t h r o c y t e s was added an equal volume o f the sensitizing
sheep antiserum (or Ig f r a c t i o n )
respectively.
or r a b b i t antibody (haemolysin)
A f t e r at l e a s t 20 min at room t e m p e r a t u r e , 0.05 ml of s e n s i t i z e d
e r y t h r o c y t e s was added to the serum d i l u t i o n s ,
mixed and incubated f o r 45 min at
39 °C ( f o r sheep C) or 37 °C (foY g u i n e a - p i g C).
The tubes were c e n t r i f u g e d and
haemolysis read a g a i n s t a 50% haemolytic standard. In some e x p e r i m e n t s , u n s e n s i t i z e d human e r y t h r o c y t e s were used. experiments w i t h the CM-cellulose f r a c t i o n , 0.2 ml o f d i l u t e d FLS or sheep serum. was used as d i l u e n t throughout. C dilutions
In the
0.2 ml o f f r a c t i o n was added to
In the d i a l y s e d serum e x p e r i m e n t s , PBS
For the k i n e t i c e x p e r i m e n t s , a l i q u o t s of the
were mixed w i t h the reagents (VBS2+, EGTA or Mg2+-EGTA) and held in
an ice bath u n t i l
the s e n s i t i z e d e r y t h r o c y t e s were added.
The tubes were then
incubated at 39 °C and at the s p e c i f i e d time (up to 60 min) the a p p r o p r i a t e tubes were t r e a t e d w i t h 0.1 ml of s a t u r a t e d EDTA s o l u t i o n , centrifuged.
c h i l l e d and then
For the k i n e t i c experiments, the amount of haemolysis was determined
s p e c t r o p h o t o m e t r i c a l l y and the percentage read from a standard curve.
257
Immunoelectrophoresis (IEP) Scheidegger's (1955) method o f micro-IEP was used. RESULTS Haemolytic a c t i v i t y titre
o f sheep C.
E f f e c t o f EGTA and Mg2+-EGTA on haemolytic
o f sheep anti-human e r y t h r o c y t e a n t i b o d y and Ig f r a c t i o n s
Using FLS o r absorbed sheep serhm, VBSe+-treated serum lysed human e r y t h r o cytes to a h i g h e r t i t r e
o f the sheep anti-human e r y t h r o c y t e antibody than d i d
serum t r e a t e d w i t h EGTA (Table I ) . titre
Using 1 or 2 mM Mge+-EGTA, the haemolytic
was s i m i l a r to VBS2+-treated serum.
Five mM Mge+-EGTA was s l i g h t l y
inhibitory. TABLE I Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h various d i l u t i o n s o f sheep antibody. E f f e c t of EGTA and Mge+-EGTA on haemolytic a c t i v i t y o f f o e t a l lamb serum o r sheep serum t h a t had been absorbed w i t h human e r y t h r o c y t e s
Complement
Treatment
D i l u t i o n o f anti-human e r y t h r o c y t e antibody 1:512 1:1024 1:2048 1:4096
Undiluted f o e t a l lamb serum
VBS2+ EGTA, 0 . 0 1 M 1 mM Mg2+-EGTA 2 " 5 "
lOOt 50 100 100 100
50 0 50 50 0
0 0 0 0 0
0 0 0 0 0
Absorbed sheep serum 1:10
VBS2+ EGTA, 0 . 0 1 M 1 mM Mg2+-EGTA 2 " 5 "
100 100 100 100 100
100 100 100 100 100
100 100 100 100 50
50 0 50 50 0
* Haemagglutination t i t r e , f Percent haemolysis
1:4096
With the FLS and sheep serum used (Table I ) , dilutions
haemolysis occurred a t a l l
o f anti-human e r y t h r o c y t e a n t i b o d y to 1:512.
1:20 or 1:200 d i l u t i o n
In subsequent t e s t s ,
of antibody was used to ensure t h a t the C a c t i v i t y
was
not l i m i t e d by a n t i b o d y c o n c e n t r a t i o n . Human e r y t h r o c y t e s s e n s i t i z e d w i t h 1:20 or 1:200 antibody and washed t w i c e were j u s t as s u s c e p t i b l e to C a c t i o n as were unwashed s e n s i t i z e d human erythrocytes. IgM and the slow and f a s t y - g l o b u l i n f r a c t i o n s from sheep anti-human
a
258
e r y t h r o c y t e antiserum a l l s e n s i t i z e d the e r y t h r o c y t e s f o r haemolysis by FLS in the presence of VBS2+, EGTA or Mg2+-EGTA, but not EDTA (Table I I ) . Fast y - g l o b u l i n reacted w i t h FLS t o a higher t i t r e
than d i d IgM whereas slow
y - g l o b u l i n was i n t e r m e d i a t e . TABLE I I Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h d i l u t i o n s o f Ig f r a c t i o n s . E f f e c t of EGTA, Mg2+-EGTA and EDTA when used w i t h d i l u t i o n s of FLS FLS d i l u t i o n
VBS2+
EGTA
512t 64 0
512 32 0
5 10 20 40
64 16 8 0
32 16 8 0
5
1024
512
i0 20 40
512 64 8
256 32 4
1:5 10 20
IgM (1:16384)* 1 mM Mg2+-EGTA 2 mM Mg2+-EGTA 1024 128 0
EDTA
1024 128 0
0 0 0
64 16 8 0
0 0 0 0
1024
1024
0
512 64 8
512 64 8
0 0 0
Slow y - g l o b u l i n 64 32 8 0
(1:2048)*
Fast y - g l o b u l i n
(1:2048)*
* Haemagglutinating t i t r e of f r a c t i o n . t D i l u t i o n o f f r a c t i o n used to s e n s i t i z e e r y t h r o c y t e s t h a t gave 50% haemolysis w i t h the d i l u t i o n o f FLS shown. With 1:5 FLS (Table I I ) , dilutions
the haemolytic t i t r e
less than the h a e m a g g l u t i n a t i n g t i t r e ;
less whereas w i t h slow y - g l o b u l i n , was used at a standard d i l u t i o n
i t was 5-6.
of f a s t y - g l o b u l i n was 1-2 w i t h IgM i t was 4-5 d i l u t i o n s In subsequent experiments, IgM
o f 1:32, slow y - g l o b u l i n at 1:4 and f a s t
y - g l o b u l i n a t 1:64. When used u n d i l u t e d , n e i t h e r these f r a c t i o n s nor the FLS produced any haemolysis of u n s e n s i t i z e d human e r y t h r o c y t e s . E f f e c t o f h e a t i n g , c h e l a t o r s , the CM-cellulose f r a c t i o n , activity
Ca2+ and Mg2+ on C
f o r human e r y t h r o c y t e s s e n s i t i z e d w i t h sheep antiserum or Ig f r a c t i o n ~
FLS Haemolytic a c t i v i t y
was abolished by heating FLS at 50 °C or 56 °C or by
0 . 0 1 M EDTA or TTHA but not 0 . 0 1 M EGTA or Mg2+-EGTA (Table I l l ) . activity
Haemolytic
of FLS heated a t 50 °C f o r 30 min was r e s t o r e d by the CM-cellulose
f r a c t i o n or the f r a c t i o n
plus EGTA or Mg2+-EGTA. Haemolytic a c t i v i t y
was not
259
r e s t o r e d by the f r a c t i o n a f t e r heating at 50 °C or the unheated f r a c t i o n plus EDTA o r TTHA. TABLE I I I Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h a standard d i l u t i o n o f sheep antiserum or Ig f r a c t i o n . E f f e c t o f h e a t i n g , c h e l a t o r s and the CM-cellulose f r a c t i o n on C a c t i v i t y o f pooled f o e t a l lamb serum Human e r y t h r o c y t e s s e n s i t i z e d w i t h Sheep y-globulin Antiserum IgM Slow Fast 1:200 1:32 1:4 1:64
Foetal lamb serum t r e a t m e n t VBS2+ 50 °C 30 min 56 °C 3 min 56 °C 30 min EDTA, 0 . 0 1 M TTHA, " EGTA, " 1,2 or 5 mM Mg2+-EGTA
16" 0 0 0 0 0 16 16
16 0 0 0 0 0 8 16
16 0 0 0 0 0 8 16
16 0 0 0 0 0 16 16
16 0 0 0 16 16
16 0 0 0 8 16
16 0 0 0 8 16
16 0 0 0 16 16
Heated 50 °C 30 min plus CM-cellulose . . . . . . . . . . . . . . . * Titre
fraction . . . . .
(reciprocal)
(FLS) heated 50 °C 30 min + EDTA, 0 . 0 1 M + TTHA, " + EGTA, " + I mM Mg2+-EGTA
o f FLS g i v i n g 50% haemolysis
Control experiments using sheep e r y t h r o c y t e s s e n s i t i z e d w i t h r a b b i t a n t i b o d y (haemagglutination titre,
1:320; haemolytic t i t r e ,
guinea-pig C (haemolytic titre,
1:~000-9000) showed t h a t
1:512) was i n h i b i t e d by 0 . 0 1 M EGTA, EDTA and
TTHA (even w i t h u n d i l u t e d g u i n e a - p i g serum) but not by heating at 50 °C f o r 30 min. Individual
FLS samples had haemolytic t i t r e s
human e r y t h r o c y t e s .
of 1:8-1:32 w i t h s e n s i t i z e d
For a l l heated (50 °C f o r 30 min) FLS samples t e s t e d ,
haemolytic a c t i v i t y
was r e s t o r e d to the o r i g i n a l
cellulose fraction,
even in the presence of EGTA or Mg2+-EGTAo
titre
by a d d i t i o n of the CM-
Sheep serum When human e r y t h r o c y t e s s e n s i t i z e d w i t h sheep antiserum o r IgM, slow or f a s t y - g l o b u l i n a n t i b o d i e s were added to d i l u t i o n s occurred to a t i t r e
of 1:32-128.
of sheep serum, haemolysis
This serum haemolytic a c t i v i t y
was abolished
by heating a t 50 °C or 56 °C or t r e a t m e n t w i t h 0 . 0 1 M EDTA or TTHA. Haemolysis occurred in the presence of 0 . 0 1 M EGTA or 1 mM Mg2+-EGTA and w i t h heated serum
260
(50 °C f o r 30 min) plus the C M - c e l l u l o s e f r a c t i o n ,
even in the presence o f EGTA
or Mg2+-EGTA.
Absorbed sheep serum The h a e m o l y t i c t i t r e
o f sheep serum absorbed w i t h human e r y t h r o c y t e s dropped
from 1:16 t o g i v e o n l y a t r a c e o f haemolysis when used u n d i l u t e d w i t h unsensitized
human e r y t h r o c y t e s .
This absorbed serum (as a source o f C) gave s i m i l a r
r e s u l t s w i t h s e n s i t i z e d human e r y t h r o c y t e s or a f t e r
in the presence o f VBS2+, c h e l a t o r s
h e a t - t r e a t m e n t as did unabsorbed sheep serum.
D i a l y s e d sheep serum Absorbed serum t h a t was d i a l y s e d a g a i n s t PBS had no h a e m o l y t i c a c t i v i t y the presence or absence o f EGTA (Table I V ) . r e s t o r e d the h a e m o l y t i c a c t i v i t y
in
A d d i t i o n o f Mg2+ or Mg2+-EGTA
to t h a t o f u n t r e a t e d serum or serum d i a l y s e d
a g a i n s t VBS2+.
TABLE IV Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h a standard d i l u t i o n o f sheep a n t i s e r u m o r Ig f r a c t i o n . E f f e c t o f Mg2+ on a c t i v i t y o f d i a l y s e d serum Treatment o f sheep serum after absorption with human e r y t h r o c y t e s
Human e r y t h r o c y t e s s e n s i t i z e d w i t h Sheep IgM y-globulin Antiserum Slow Fast 1:200 1:32 1:4 1:64
None D i a l y s e d PBS o n l y . . . . + EGTA, 0 . 0 1 M . . . . + 1 mM Mg2+ . . . . + 2 o r 5 mM Mg2+ . . . . + 1, 2 o r 5 mM Mg2+-EGTA . . . . + VBS2+ *Titre
(reciprocal)
32* 0 0 32 32 32 32
16 0 0 8 16 16 16
16 0 0 8 16 16 16
16 0 0 8 16 16 16
o f serum g i v i n g 50% haemolysis.
A d d i t i o n o f Ca2+ ( f i n a l
c o n c e n t r a t i o n s from 0 . 5 - 2 0 mM) to d i a l y s e d serum t h a t
had no or v e r y low h a e m o l y t i c C a c t i v i t y
had no e f f e c t .
I n f l u e n c e o f EGTA and Mg2+-EGTA on the k i n e t i c s
o f sheep C h a e m o l y t i c a c t i v i t y
Sheep a n t i s e r u m and sheep C The r a t e o f haemolysis by sheep serum c o n t a i n i n g 0 . 0 1 M EGTA or Mg2+-EGTA was s l o w e r than f o r serum c o n t a i n i n g VBS2+ (Table V).
261
TABLE V
K i n e t i c s of C l y s i s of human erythrocytes s e n s i t i z e d with sheep antibody. E f f e c t of EGTA and Mg2+-EGTA on haemolytic a c t i v i t y of sheep serum Sheep serum* treatment
i0
Percent l y s i s at (min)t 20 30 45
VBS2+ EGTA, 0.01M i mM Mg2+-EGTA 2 ...... 5 ......
67 I 1 1 2
95 3 6 6 4
99 15 41 48 43
60
100 81 94 97 88
I00 100 100 100 98
tWith human e r y t h r o c y t e s s e n s i t i z e d with a 1:200 d i l u t i o n of sheep antibody. * T i t r e of 1:128 with s e n s i t i z e d human e r y t h r o c y t e s . Used at 1:100. Sheep Ig f r a c t i o n s and FLS C With Ig s e n s i t i z e d e r y t h r o c y t e s , haemolysis proceeded f a s t e r in the presence of 5 mM Mg2+-EGTA than with EGTA, but not as f a s t as with VBSe+.
Rates
intermediate between the values obtained f o r VBS2+ and 5 mM Mg2+-EGTA were obtained in the presence of I and 2 mM Mge+-EGTA ( F i g . l ) . c----o Fast~ 1:6L lo07
~ _ _ _ o
80
..
~ ffl
~
1:32
o..... ~ S l o w ~ 1:4 c _4 ,~''O"~ o
.... o
/P"
/
...'"
//
VBS2÷
~1 /:: /
,
o ...... °
//
20
o-9
...'"
iI
.""
50~/ 40
o--olgM
.:
?
=': 0.01M EGTA
i
i
i
0
E lO0 ci
.o ......... -o
ZE
/
....... ""
B050!
f
40-
.....'""
,.
.... .:
,
:.:
! .d 20- r..." o~ IO
2raM Mg2+-EGTA
2'o 3~
4'5 Time
&
10
20
30
/-5
60
(rain)
Fig 1. FLS complement haemolysis of human erythrocytes s e n s i t i z e d with Ig fractions. E f f e c t of EGTA and Mge+-EGTA on the r a t e of haemolysis.
262
P u r i t y of Ig f r a c t i o n s The p r o t e i n c o n c e n t r a t i o n s o f the Ig f r a c t i o n s were: IgM, 4-6 mg/m!; slow y-globulin,
6.25 mg/ml; f a s t y - g l o b u l i n ,
was pure, and on d i l u t i o n
18.0 mg/ml.
By IEP, each Ig f r a c t i o n
IgM p r e c i p i t a t e d to a d i l u t i o n
of 1:16, slow
y - g l o b u l i n to 1:128 and f a s t y - g l o b u l i n to 1:256. DISCUSSION The importance o f Mg2+ in haemolysis of sheep a n t i b o d y s e n s i t i z e d human e r y t h r o c y t e s by sheep C has been demonstrated by use o f c h e l a t o r s and d i a l y s e d serum.
Human e r y t h r o c y t e s s e n s i t i z e d w i t h antiserum or the Ig f r a c t i o n s were
lysed by sheep C in the presence o f EGTA or MgZ+-EGTA (Tables I-V and Fig. 1) but not EDTA or TTHA (Tables I - I l l ) .
(EGTA has a 10 O00-fold preference f o r
Ca2+ over Mgz+ (Bryant and Jenkins, 1968) whereas EDTA and TTHA have high a s s o c i a t i o n constants f o r both Ca2+ and Mg2+ (Bryant and Jenkins, 1968; Nagaki et al.,
1974).)
The EGTA d i d , however, have an e f f e c t on the assays. the t i t r e
e r y t h r o c y t e s f o r l y s i s by a standard d i l u t i o n standard d i l u t i o n Ill).
There was, r e l a t i v e
to
in VBS2+, an increased requirement f o r antibody to s e n s i t i z e the of Ig, the C t i t r e
of C in EGTA (Table I ) .
was o f t e n s l i g h t l y
With a
lower w i t h EGTA (Table
There was also a reduced r a t e of haemolysis in EGTA compared w i t h VBS2+
(Table V and F i g . l ) .
R e l a t i v e to EGTA, MgZ+-EGTA decreased the amount of
antibody r e q u i r e d to s e n s i t i z e the e r y t h r o c y t e s the C t i t r e VBS2+.
(Table I l l ) ,
giving titres
(Tables I and I I )
and increased
t h a t were s i m i l a r to those obtained w i t h
The r a t e of haemolysis was also increased but the VBS2+ r a t e was not
reached (Table V and F i g . l ) . Mg2+ and Mg2+-EGTA were able to r e s t o r e haemolytic a c t i v i t y
to sheep serum
t h a t had been absorbed w i t h human e r y t h r o c y t e s and then d i a l y s e d a g a i n s t PBS (Table I V ) , but Ca2+ had no e f f e c t .
As s i m i l a r antibody and C t i t r e s
obtained in the presence of Mg2+-EGTA and VBSz+ (Tables I - I l l ) Mg2+ and Mg2+-EGTA were able to f u l l y (Table IV), t h i s
restore C activity
and as 2 and 5 mM
to d i a l y s e d serum
i n d i c a t e s t h a t t h i s sheep C haemolytic a c t i v i t y
the absence of Ca2+.
As C a c t i v i t y
were
can proceed in
in EGTA or Mg2+-EGTA chelated serum from
various animals has been considered to i n d i c a t e a l t e r n a t i v e
pathway a c t i v i t y
(Sandberg and O s l e r , 1971; Forsgren and Quie, 1974; P l a t t s - M i l l s 1974; Fine, 1977), the sheep C haemolytic a c t i v i t y
and I s h i z a k a ,
in EGTA and Mg2+-EGTA
described in t h i s paper probably also i n v o l v e s the a l t e r n a t i v e pathway. Heating C at 50 °C f o r 30 min (Goodkofsky and Lepow, 1971) or 56 °C f o r 3 min (Pang and Aston, 1978) i n a c t i v a t e s a l t e r n a t i v e haemolytic a c t i v i t y
pathway f a c t o r B.
The sheep
mediated by sheep antiserum and Ig f r a c t i o n s was also l a b i l e
263
to this heat treatment (Table I I I ) .
The FLS CM-cellulose f r a c t i o n that restored
haemolytic a c t i v i t y to 50 °C-treated sheep serum (Table I I I ) protein that was responsible f o r t h i s a c t i v i t y .
had a 107 000 dalton
This protein is considered to
be sheep factor B as the mol~cular weight is s i m i l a r to that reported f o r bovine factor B (Pang and Aston, 1978).
Good evidence f o r a l t e r n a t i v e pathway
involvement is that the factor B containing f r a c t i o n , in the presence o~ EGTA or Mg2+-EGTA could restore haemolytic a c t i v i t y to 50 °C-treated serum. The r e l a t i o n s h i p between the haemagglutination and the haemolytic t i t r e of the sheep anti-human erythrocyte antibody is of i n t e r e s t .
With the standard
sheep-erythrocyte rabbit-antibody i n d i c a t o r system, the rabbit antiserum used had a haemagglutination t i t r e of 1:320 and a haemolytic t i t r e of 1:7000-9000. By contrast, the sheep anti-human erythrocyte antiserum had a haemagglutination t i t r e of 1:4096 and a haemolytic t i t r e of 1:512-4096. haemagglutination t i t r e ,
IgM had the highest
but i t s haemolytic t i t r e was much lower and s i m i l a r to
that of fast y - g l o b u l i n .
The slow and fast v-globulins had s i m i l a r
haemagglutination t i t r e s ,
but there was a marked difference in haemolytic t i t r e s
(Table I I ) .
No explanation is a v a i l a b l e f o r the difference between the
haemagglutination and haemolytic t i t r e of the sheep amboceptors r e l a t i v e to the rabbit amboceptor. Lachmann and Nicol (1973) have suggested that the a l t e r n a t i v e pathway requires a much higher concentration of C fo r a c t i v i t y than does the classical pathway. In our experiments, FLS had a maximum haemolytic t i t r e of 1:32 and sheep serum, a maximum of 1:128.
These t i t r e s were lower than the 1:512 r e g u l a r l y obtained
with guinea-pig C.
Thus, r e l a t i v e to the classical pathway a c t i v i t y of guinea-
pig C, this sheep haemolytic system required a high concentration of both amboceptor and C. Although the data presented indicate that the a l t e r n a t i v e pathway is involved in sheep C haemolytic a c t i v i t y in EGTA and Mg2+-EGTA, the mechanism of C a c t i v a t i o n is not known. The sheep amboceptor is apparently required on the erythrocyte surface as washed sensitized c e l l s were j u s t as susceptible as unwashed sensitized c e l l s to C. agglutinated by the
Even though the erythrocytes were heavily
doses of sheep amboceptor used, provided the tubes were
shaken r e g u l a r l y , complete haemolysis occurred. ACKNOWLEDGEMENTS We would l i k e to thank Mr Hugh Pulford for technical assistance. M. Stankiewicz g r a t e f u l l y acknowledges a New Zealand Government National Research Advisory Council Post-Doctoral Fellowship.
264
REFERENCES Barta, 0., Barta, V., S h i r l e y , R.A. and McMurry, J.D., 1975. Haemolytic assay of sheep complement. Zentbl. VetMed., 22:254. Barta, O. and Hubbert, N.L., 1978. Testing of haemolytic complement components in domestic animals. Am. J. Vet. Res., 39:1303. Borsos, T. and Rapp, H.J., 1965. Estimation of molecular size of complement components by Sephadex chromatography. J. Immunol., 94:510. Bryant, R.E. and Jenkins, D.E., 1968. Calcium requirements for complement dependent haemolytic reactions. J. Immunol. 101:664. Feinstein, A. and Hobart, M.J., 1969. Structural r e l a t i o n s h i p and complement f i x i n g a c t i v i t y of sheep and other ruminant immunoglobulin G subclasses. Nature (Lond.), 223:950. Fine, D.P., 1977. Comparison of e t h y l e n e g l y c o l t e t r a a c e t i c acid and i t s magnesium s a l t as reagents for studying a l t e r n a t i v e complement pathway function. Inf. Immun., 16:124. Fine, D.P., Marney, S.R., Colley, D.G., Sergent, J.A. and Des Prez, R.M., 1972. C3 shunt a c t i v a t i o n in human serum chelated with EGTA. J. Immunol., 109:807. Forsgren, A. and Quie, P.G., 1974. Opsonic a c t i v i t y in human serum chelated with ethylene g l y c o l t e t r a - a c e t i c acid. Immunology, 26:1251. Goodkofsky, I. and Lepow, I . H . , 1971. Functional r e l a t i o n s h i p of factor B in the properdin system to C3 proactivator of human serum. J. Immunol., 107:1200. Jonas, W.E., 1969. Determination of sheep anti-Salmonella typhi~urium immunog l o b u l i n class by a n t i g l o b u l i n t e s t i n g . Res. Vet. S c i . , 19:311. Lachmann, P.J., Hobart, M.J. and Aston, W.P., 1973. Complement technology. In: Handbook of Experimental Immunology. Ed by D.M. Weir, 2nd edn., p.5.1. Blackwell S c i e n t i f i c Publications, Oxford. Lachmann, P.J. and Nicol, P., 1973. Reaction mechanisms of the a l t e r n a t i v e pathway of complement f i x a t i o n . The Lancet, 1:465. Mayer, M.M., 1961. In: Kabat & Mayer's Experimental Immunochemistry, 2nd edn.: Charles C. Thomas, Publisher, S p r i n g f i e l d , I I I . , p.149. M~ller-Eberhard, H.J., 1975. Complement. A. Rev. Biochem., 44:697. Ngaki, K., l i d a , K. and I n a i , S., 1974. A new method for the preparations of EACI4 cell with human or guinea-pig serum. J. Immunol. Meth., 5:307. Pang, A.S.D. and Aston, W.P., 1978. The a l t e r n a t i v e complement pathway in bovine serum: the i s o l a t i o n of a serum protein with f a c t o r B a c t i v i t y . Immunochem., 15:529. P l a t t s - M i l l s , T.A.E. and Ishizaka, K., 1974. A c t i v a t i o n of the alternate pathway of human complement by rabbit c e l l s . J. Immunol., 113:348. Rhee, Y.O., Broad, S. and Jonas, W.E., 1970. A g g l u t i n a t i n g and complement-mediated a c t i v i t i e s of sheep antisera, IgM slow and fast y - g l o b u l i n antibodies. Res. Vet. S c i . , 11:123. Rice, C.E. and Crowson, C.N., 1950. The i n t e r c h a n g e a b i l i t y of the complement components of d i f f e r e n t animal species. I I . In the hemolysis of sheep erythrocytes sensitized with rabbit amboceptor. J. Immunol., 65:201. Sandberg, A.L. and Osler, A.G., 1971. Dual pathways of complement i n t e r a c t i o n with guinea pig immunoglobulins. J. Immunol., 197:1268. Scheidegger, J . J . , 1955. Une micro-m~thode de l'immuno~lectrophor~se. Int. Archs. A l l e r g y Appl. Immun., 7:103.
253
Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in Belgium
HAEMOLYSIS OF HUMAN ERYTHR~CYTESHEAVILY SENSITIZED WITH SHEEP AMBOCEPTORBY SHEEP COMPLEMENTCHELATED WITH EGTA OR Mg2+-EGTA M. STANKIEWICZ1, and W. JONAS2 IDepartment of Immunology and I n s t i t u t e of Zoology, Warsaw U n i v e r s i t y , 00927/1 Warszawa, Krakowskie Przemiescie 26/28 Poland 2Wallaceville Animal Research Centre, Research D i v i s i o n , M i n i s t r y of A g r i c u l t u r e and Fisheries, Private Bag, Upper Hutt, New Zealand (Accepted 24 March 1981) ABSTRACT Stankiewicz, M. and Jonas, W. 1981. Haemolysis of human erythrocytes heavily sensitized with sheep amboceptor by sheep complement chelated with EGTA or Mg2+-EGTA. Vet. Immunol. Immunopatho]., 2: 253-264. Human erythrocytes sensitized with an a g g l u t i n a t i n g dose of e i t h e r sheep anti-human erythrocyte antiserum, IgM, slow or fast y - g l o b u l i n antibody are haemolysed by foetal lamb or sheep serum complement (C) in the presence of e t h y l e n e g l y c o l t e t r a a c e t i c acid (EGTA) or Mg2+-EGTA but not ethylenediaminetetraacetic acid (EDTA) or triethylenetetraminehexaacetic acid (TTHA). Dialysis of the C source aqainst phosphate buffered saline rendered i t i n a c t i v e , but addition of Mg2+ or Mg2~-EGTA restored haemolytic a c t i v i t y . Haemolysis of sensitized erythrocyte~ took place more r a p i d l y in buffer containing Ca2+ and Mg2+ than in EGTA or Mg2T-EGTA. Heating the C source at 50 °C for 30 min or 56 °C for 3 min abolished haemolytic a c t i v i t y . In the presence of EGTA or Mg2+-EGTA, haemolytic a c t i v i t y was restored to 50 °C-heated serum by a CM-cellulose f r a c t i o n that contained sheep f a c t o r B. These results show that sheep C, in the presence of EGTA or Mg2+-EGTA w i l l haemolyse human erythrocytes heavily sensitized with sheep amboceptor. The a l t e r n a t i v e pathway of sheep C is thought to be involved. INTRODUCTION Lysis of sensitized erythrocytes has been a basic test for i n v e s t i g a t i n g complement (C) a c t i v i t y .
In the standard haemolytic assay system, sheep erythro-
cytes and r a b b i t anti-sheep erythrocyte antibody are used.
This system has a
l i m i t a t i o n in that i t cannot be used to demonstrate C a c t i v i t y in serum of some species of animals including sheep (Rice and Crowson, 1950).
However, sheep C
haemolytic a c t i v i t y can be r e a d i l y demonstrated when sheep antibodies are used to sensitize e i t h e r human erythrocytes (Feinstein and Hobart, 1969; Rhee et al. 1970) or r a b b i t erythrocytes (Barta et a l . ,
1975).
0165-2427/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishmg Company
254
Two major pathways have been described fo r the a c t i v a t i o n of C.
The classical
pathway is antibody dependent and involves the C components CI, C4 and C2. requires Caz+ and Mg2+ f o r a c t i v i t y ,
It
The a l t e r n a t i v e pathway may be activated
without antibody, is Mg2+ dependent and has a 50 °C l a b i l e component, factor B. These two a c t i v a t i o n pathways have been thoroughly investigated f o r guinea-pig and human C (MUller-Eberhard, 1975) but l i t t l e
is known f o r other animals.
Although i t is generally accepted that the classical pathway is involved in immune haemolysis, the f o l l o w i n g points indicated to us that sheep C immune haemolysis may not always use only the classical pathway.
Haemolysis of rabbit
erythrocytes sensitized with sheep amboceptor has a high Mg2+ requirement (Barta et a l . , 1975).
Sheep C is e i t h e r poorly or non-haemolytic f o r sheep erythrocytes
sensitized with r a b b i t amboceptor (Rice and Crowson, 1950; Barta et a l . , 1975), an amboceptor that is very e f f e c t i v e in a c t i v a t i n g the classical pathway. Classical pathway components C4 and C2 have not been detected (Barta and Hubert, 1978) or found in low t i t r e only (Borsos and Rapp, 1965) in sheep serum. The purpose of this paper is to show that sheep C haemolysis of human erythrocytes heavily sensitized by sheep amboceptor takes place in the presence of e t h y l e n e g l y c o l t e t r a a c e t i c acid (EGTA) or Mg2+-EGTA. This and other data indicates that this haemolytic a c t i v i t y is probably dependent on the a l t e r n a t i v e pathway of sheep C. MATERIALS AND METHODS Buffers and chelatinq aqent§ Phosphate buffered saline (PBS, 0.15 M, pH 7.2) w@s used for some procedures. Veronal buffered saline containing Ca2+ and Mg2+ (VBS2+) (Mayer, 1961) was used as the d i l u e n t in the haemolytic assays.
Stock solutions (0.1 M, pH 7.6) of
ethylenediaminetetraacetic acid (EDTA) and e t h y l e n e g l y c o l t e t r a a c e t i c acid (EGTA) were prepared as described by Fine et al.
(1972).
Stock solutions of Mg2+-EGTA
(stock 0.1M EGTA containing 10, 20 or 50 mM Mg2+) were prepared with MgCl2 and the pH adjusted to pH 7.6.
Stock solutions (pH 7.6) of MgCl2 (10, 20 or 50 mM
Mg2+) were prepared in 0.15 M saline. Ca2+) was prepared in saline.
A stock solution of CaCI2.2H20 (0.2 M
Triethylenetetraminehexaacetic acid (TTHA) was
prepared as described by Nagaki et al. (1974).
Complement ~iCJ Blood collected from near-term foetal lambs, adult sheep or guinea-pigs was allowed to c l o t at room temperature (2 h).
After standing overnight at 4 °C,
serum was separated and frozen in aliquots at -20 °C.
255
Some sheep serum was absorbed w i t h human e r y t h r o c y t e s by t r e a t i n g
1 ml o f
serum c o n t a i n i n g 0 . 1 M EDTA w i t h 0.5 ml o f packed e r y t h r o c y t e s f o r 1 h at 0 - i °C on f o u r occasions.
Some absorbed serum was d i a l y s e d o v e r n i g h t at 0-1 °C
a g a i n s t VBS2+ and some a g a i n s t PBS. Some serum samples were heated at e i t h e r 50 °C f o r 30 min, 56 °C f o r 3 min or 56 °C f o r 30 min. The f o e t a l
lamb serum (FLS) used was h a e m o l y t i c a l l y i n a c t i v e when incubated
u n d i l u t e d w i t h u n s e n s i t i z e d human e r y t h r o c y t e s and was negative f o r immunog l o b u l i n when checked by immunoelectrophoresis and an a n t i g l o b u l i n
inhibition
t e s t (Jonas, 1969). CM-cellulose f r a c t i o n The method f o r i s o l a t i n g was used w i t h FLS.
f a c t o r B from human serum (Lachmann e t a l . ,
The f r a c t i o n
1973)
used (11-13 mmhos, 0.49 mg/ml), by g r a d i e n t
p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s contained 5 p r o t e i n bands (515,330,140,107 and 84,000 d a l t o n s ) .
I t has a haemolytic t i t r e
w i t h sheep antibody s e n s i t i z e d
human e r y t h r o c y t e s of 1:32 in 1:4 50 °C sheep serum c o n t a i n i n g Mg2+-EGTA. A f t e r s e p a r a t i o n on Sephadex G-200, the haemolytic a c t i v i t y 107,000 d a l t o n p r o t e i n .
was associated w i t h the
Guinea-pig a n t i s e r a to the f r a c t i o n p r e c i p i t a t e d from
e l e c t r o p h o r e s e d sheep serum a s i n g l e band in the f a s t B - g l o b u l i n r e g i o n . Erythrocytes Group 0 R h - p o s i t i v e human e r y t h r o c y t e s or sheep e r y t h r o c y t e s , c o l l e c t e d i n t o A l s e v e r ' s s o l u t i o n were obtained weekly.
The e r y t h r o c y t e s were washed three
times in PBS before use. Antisera Sheep antiserum to human e r y t h r o c y t e s Six Romney ewes were each i n j e c t e d i n t r a m u s c u l a r l y (I/M) w i t h 4 ml o f a 30% suspension o f human e r y t h r o c y t e s e m u l s i f i e d w i t h an equal volume o f Freund's complete adjuvant (FCA). weeks. of IgM.
Two f u r t h e r
I/M i n j e c t i o n s were given at f o u r and ten
Serum o b t a i n e d ten days a f t e r the f i r s t
i n j e c t i o n was used as a source
Serum o b t a i n e d f o u r weeks a f t e r the t h i r d
i n j e c t i o n was used as a
source o f anti-human e r y t h r o c y t e antiserum and the source of slow and f a s t y-globulin.
The antiserum was pooled, heated at 56 °C f o r 30 min and t r e a t e d
w i t h s a t u r a t e d ammonium sulphate to a f i n a l
c o n c e n t r a t i o n of 40%. The
p r e c i p i t a t e d g l o b u l i n was washed, r e c o n s t i t u t e d to the o r i g i n a l d i a l y s e d a g a i n s t PBS and s t o r e d in a l i q u o t s at -20 °C.
serum volume,
256
IgM, slow and f a s t T - g l o b u l i n were prepared as described by Jonas (1969). The p r o t e i n c o n c e n t r a t i o n of the immunoglobulins ( I g ) was assessed at 280 nm a g a i n s t a standard curve prepared w i t h bovine T - g l o b u l i n .
The p u r i t y o f the
f r a c t i o n s was checked by immunoelectrophoresis. Rabbit antiserum to sheep er%_thro!yte § Haemolysin was purchased from the Commonwealth Serum L a b o r a t o r i e s , Melbourne, Australia.
I t was heated and p r e c i p i t a t e d as described above.
The h a e m a g g l u t i n a t i n g t i t r e s
of the amboceptors were determined using a 1~]
suspension of the a p p r o p r i a t e e r y t h r o c y t e s . Guinea-pj 9 antiserum to the CM-cellulose f r a c t i o n Five g u i n e a - p i g s were used.
One ml of PBS c o n t a i n i n g the CM-cellulose
f r a c t i o n was e m u l s i f i e d w i t h i ml of FCA. The immunization schedule was: day 1, 0.2 ml o f emulsion i n t o each hind f o o t p a d ; day 10 and 20, 0.2 ml o f emulsion I/M i n t o each hind l e g ; day 30, bled out. Guinea-pig antiserum to sheep serum Guinea-pigs were immunized (as above) but w i t h a 1:10 d i l u t i o n
of sheep
serum. Haemolytic assays Serum (the C source) was s e r i a l l y
d i l u t e d t w o - f o l d in 0.4 ml volumes of VBS2+.
A 0.5 ml volume o f VBS2+ or the reagent under t e s t was added and mixed.
To a 5%
suspension o f human or sheep e r y t h r o c y t e s was added an equal volume o f the sensitizing
sheep antiserum (or Ig f r a c t i o n )
respectively.
or r a b b i t antibody (haemolysin)
A f t e r at l e a s t 20 min at room t e m p e r a t u r e , 0.05 ml of s e n s i t i z e d
e r y t h r o c y t e s was added to the serum d i l u t i o n s ,
mixed and incubated f o r 45 min at
39 °C ( f o r sheep C) or 37 °C (foY g u i n e a - p i g C).
The tubes were c e n t r i f u g e d and
haemolysis read a g a i n s t a 50% haemolytic standard. In some e x p e r i m e n t s , u n s e n s i t i z e d human e r y t h r o c y t e s were used. experiments w i t h the CM-cellulose f r a c t i o n , 0.2 ml o f d i l u t e d FLS or sheep serum. was used as d i l u e n t throughout. C dilutions
In the
0.2 ml o f f r a c t i o n was added to
In the d i a l y s e d serum e x p e r i m e n t s , PBS
For the k i n e t i c e x p e r i m e n t s , a l i q u o t s of the
were mixed w i t h the reagents (VBS2+, EGTA or Mg2+-EGTA) and held in
an ice bath u n t i l
the s e n s i t i z e d e r y t h r o c y t e s were added.
The tubes were then
incubated at 39 °C and at the s p e c i f i e d time (up to 60 min) the a p p r o p r i a t e tubes were t r e a t e d w i t h 0.1 ml of s a t u r a t e d EDTA s o l u t i o n , centrifuged.
c h i l l e d and then
For the k i n e t i c experiments, the amount of haemolysis was determined
s p e c t r o p h o t o m e t r i c a l l y and the percentage read from a standard curve.
257
Immunoelectrophoresis (IEP) Scheidegger's (1955) method o f micro-IEP was used. RESULTS Haemolytic a c t i v i t y titre
o f sheep C.
E f f e c t o f EGTA and Mg2+-EGTA on haemolytic
o f sheep anti-human e r y t h r o c y t e a n t i b o d y and Ig f r a c t i o n s
Using FLS o r absorbed sheep serhm, VBSe+-treated serum lysed human e r y t h r o cytes to a h i g h e r t i t r e
o f the sheep anti-human e r y t h r o c y t e antibody than d i d
serum t r e a t e d w i t h EGTA (Table I ) . titre
Using 1 or 2 mM Mge+-EGTA, the haemolytic
was s i m i l a r to VBS2+-treated serum.
Five mM Mge+-EGTA was s l i g h t l y
inhibitory. TABLE I Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h various d i l u t i o n s o f sheep antibody. E f f e c t of EGTA and Mge+-EGTA on haemolytic a c t i v i t y o f f o e t a l lamb serum o r sheep serum t h a t had been absorbed w i t h human e r y t h r o c y t e s
Complement
Treatment
D i l u t i o n o f anti-human e r y t h r o c y t e antibody 1:512 1:1024 1:2048 1:4096
Undiluted f o e t a l lamb serum
VBS2+ EGTA, 0 . 0 1 M 1 mM Mg2+-EGTA 2 " 5 "
lOOt 50 100 100 100
50 0 50 50 0
0 0 0 0 0
0 0 0 0 0
Absorbed sheep serum 1:10
VBS2+ EGTA, 0 . 0 1 M 1 mM Mg2+-EGTA 2 " 5 "
100 100 100 100 100
100 100 100 100 100
100 100 100 100 50
50 0 50 50 0
* Haemagglutination t i t r e , f Percent haemolysis
1:4096
With the FLS and sheep serum used (Table I ) , dilutions
haemolysis occurred a t a l l
o f anti-human e r y t h r o c y t e a n t i b o d y to 1:512.
1:20 or 1:200 d i l u t i o n
In subsequent t e s t s ,
of antibody was used to ensure t h a t the C a c t i v i t y
was
not l i m i t e d by a n t i b o d y c o n c e n t r a t i o n . Human e r y t h r o c y t e s s e n s i t i z e d w i t h 1:20 or 1:200 antibody and washed t w i c e were j u s t as s u s c e p t i b l e to C a c t i o n as were unwashed s e n s i t i z e d human erythrocytes. IgM and the slow and f a s t y - g l o b u l i n f r a c t i o n s from sheep anti-human
a
258
e r y t h r o c y t e antiserum a l l s e n s i t i z e d the e r y t h r o c y t e s f o r haemolysis by FLS in the presence of VBS2+, EGTA or Mg2+-EGTA, but not EDTA (Table I I ) . Fast y - g l o b u l i n reacted w i t h FLS t o a higher t i t r e
than d i d IgM whereas slow
y - g l o b u l i n was i n t e r m e d i a t e . TABLE I I Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h d i l u t i o n s o f Ig f r a c t i o n s . E f f e c t of EGTA, Mg2+-EGTA and EDTA when used w i t h d i l u t i o n s of FLS FLS d i l u t i o n
VBS2+
EGTA
512t 64 0
512 32 0
5 10 20 40
64 16 8 0
32 16 8 0
5
1024
512
i0 20 40
512 64 8
256 32 4
1:5 10 20
IgM (1:16384)* 1 mM Mg2+-EGTA 2 mM Mg2+-EGTA 1024 128 0
EDTA
1024 128 0
0 0 0
64 16 8 0
0 0 0 0
1024
1024
0
512 64 8
512 64 8
0 0 0
Slow y - g l o b u l i n 64 32 8 0
(1:2048)*
Fast y - g l o b u l i n
(1:2048)*
* Haemagglutinating t i t r e of f r a c t i o n . t D i l u t i o n o f f r a c t i o n used to s e n s i t i z e e r y t h r o c y t e s t h a t gave 50% haemolysis w i t h the d i l u t i o n o f FLS shown. With 1:5 FLS (Table I I ) , dilutions
the haemolytic t i t r e
less than the h a e m a g g l u t i n a t i n g t i t r e ;
less whereas w i t h slow y - g l o b u l i n , was used at a standard d i l u t i o n
i t was 5-6.
of f a s t y - g l o b u l i n was 1-2 w i t h IgM i t was 4-5 d i l u t i o n s In subsequent experiments, IgM
o f 1:32, slow y - g l o b u l i n at 1:4 and f a s t
y - g l o b u l i n a t 1:64. When used u n d i l u t e d , n e i t h e r these f r a c t i o n s nor the FLS produced any haemolysis of u n s e n s i t i z e d human e r y t h r o c y t e s . E f f e c t o f h e a t i n g , c h e l a t o r s , the CM-cellulose f r a c t i o n , activity
Ca2+ and Mg2+ on C
f o r human e r y t h r o c y t e s s e n s i t i z e d w i t h sheep antiserum or Ig f r a c t i o n ~
FLS Haemolytic a c t i v i t y
was abolished by heating FLS at 50 °C or 56 °C or by
0 . 0 1 M EDTA or TTHA but not 0 . 0 1 M EGTA or Mg2+-EGTA (Table I l l ) . activity
Haemolytic
of FLS heated a t 50 °C f o r 30 min was r e s t o r e d by the CM-cellulose
f r a c t i o n or the f r a c t i o n
plus EGTA or Mg2+-EGTA. Haemolytic a c t i v i t y
was not
259
r e s t o r e d by the f r a c t i o n a f t e r heating at 50 °C or the unheated f r a c t i o n plus EDTA o r TTHA. TABLE I I I Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h a standard d i l u t i o n o f sheep antiserum or Ig f r a c t i o n . E f f e c t o f h e a t i n g , c h e l a t o r s and the CM-cellulose f r a c t i o n on C a c t i v i t y o f pooled f o e t a l lamb serum Human e r y t h r o c y t e s s e n s i t i z e d w i t h Sheep y-globulin Antiserum IgM Slow Fast 1:200 1:32 1:4 1:64
Foetal lamb serum t r e a t m e n t VBS2+ 50 °C 30 min 56 °C 3 min 56 °C 30 min EDTA, 0 . 0 1 M TTHA, " EGTA, " 1,2 or 5 mM Mg2+-EGTA
16" 0 0 0 0 0 16 16
16 0 0 0 0 0 8 16
16 0 0 0 0 0 8 16
16 0 0 0 0 0 16 16
16 0 0 0 16 16
16 0 0 0 8 16
16 0 0 0 8 16
16 0 0 0 16 16
Heated 50 °C 30 min plus CM-cellulose . . . . . . . . . . . . . . . * Titre
fraction . . . . .
(reciprocal)
(FLS) heated 50 °C 30 min + EDTA, 0 . 0 1 M + TTHA, " + EGTA, " + I mM Mg2+-EGTA
o f FLS g i v i n g 50% haemolysis
Control experiments using sheep e r y t h r o c y t e s s e n s i t i z e d w i t h r a b b i t a n t i b o d y (haemagglutination titre,
1:320; haemolytic t i t r e ,
guinea-pig C (haemolytic titre,
1:~000-9000) showed t h a t
1:512) was i n h i b i t e d by 0 . 0 1 M EGTA, EDTA and
TTHA (even w i t h u n d i l u t e d g u i n e a - p i g serum) but not by heating at 50 °C f o r 30 min. Individual
FLS samples had haemolytic t i t r e s
human e r y t h r o c y t e s .
of 1:8-1:32 w i t h s e n s i t i z e d
For a l l heated (50 °C f o r 30 min) FLS samples t e s t e d ,
haemolytic a c t i v i t y
was r e s t o r e d to the o r i g i n a l
cellulose fraction,
even in the presence of EGTA or Mg2+-EGTAo
titre
by a d d i t i o n of the CM-
Sheep serum When human e r y t h r o c y t e s s e n s i t i z e d w i t h sheep antiserum o r IgM, slow or f a s t y - g l o b u l i n a n t i b o d i e s were added to d i l u t i o n s occurred to a t i t r e
of 1:32-128.
of sheep serum, haemolysis
This serum haemolytic a c t i v i t y
was abolished
by heating a t 50 °C or 56 °C or t r e a t m e n t w i t h 0 . 0 1 M EDTA or TTHA. Haemolysis occurred in the presence of 0 . 0 1 M EGTA or 1 mM Mg2+-EGTA and w i t h heated serum
260
(50 °C f o r 30 min) plus the C M - c e l l u l o s e f r a c t i o n ,
even in the presence o f EGTA
or Mg2+-EGTA.
Absorbed sheep serum The h a e m o l y t i c t i t r e
o f sheep serum absorbed w i t h human e r y t h r o c y t e s dropped
from 1:16 t o g i v e o n l y a t r a c e o f haemolysis when used u n d i l u t e d w i t h unsensitized
human e r y t h r o c y t e s .
This absorbed serum (as a source o f C) gave s i m i l a r
r e s u l t s w i t h s e n s i t i z e d human e r y t h r o c y t e s or a f t e r
in the presence o f VBS2+, c h e l a t o r s
h e a t - t r e a t m e n t as did unabsorbed sheep serum.
D i a l y s e d sheep serum Absorbed serum t h a t was d i a l y s e d a g a i n s t PBS had no h a e m o l y t i c a c t i v i t y the presence or absence o f EGTA (Table I V ) . r e s t o r e d the h a e m o l y t i c a c t i v i t y
in
A d d i t i o n o f Mg2+ or Mg2+-EGTA
to t h a t o f u n t r e a t e d serum or serum d i a l y s e d
a g a i n s t VBS2+.
TABLE IV Haemolysis o f human e r y t h r o c y t e s s e n s i t i z e d w i t h a standard d i l u t i o n o f sheep a n t i s e r u m o r Ig f r a c t i o n . E f f e c t o f Mg2+ on a c t i v i t y o f d i a l y s e d serum Treatment o f sheep serum after absorption with human e r y t h r o c y t e s
Human e r y t h r o c y t e s s e n s i t i z e d w i t h Sheep IgM y-globulin Antiserum Slow Fast 1:200 1:32 1:4 1:64
None D i a l y s e d PBS o n l y . . . . + EGTA, 0 . 0 1 M . . . . + 1 mM Mg2+ . . . . + 2 o r 5 mM Mg2+ . . . . + 1, 2 o r 5 mM Mg2+-EGTA . . . . + VBS2+ *Titre
(reciprocal)
32* 0 0 32 32 32 32
16 0 0 8 16 16 16
16 0 0 8 16 16 16
16 0 0 8 16 16 16
o f serum g i v i n g 50% haemolysis.
A d d i t i o n o f Ca2+ ( f i n a l
c o n c e n t r a t i o n s from 0 . 5 - 2 0 mM) to d i a l y s e d serum t h a t
had no or v e r y low h a e m o l y t i c C a c t i v i t y
had no e f f e c t .
I n f l u e n c e o f EGTA and Mg2+-EGTA on the k i n e t i c s
o f sheep C h a e m o l y t i c a c t i v i t y
Sheep a n t i s e r u m and sheep C The r a t e o f haemolysis by sheep serum c o n t a i n i n g 0 . 0 1 M EGTA or Mg2+-EGTA was s l o w e r than f o r serum c o n t a i n i n g VBS2+ (Table V).
261
TABLE V
K i n e t i c s of C l y s i s of human erythrocytes s e n s i t i z e d with sheep antibody. E f f e c t of EGTA and Mg2+-EGTA on haemolytic a c t i v i t y of sheep serum Sheep serum* treatment
i0
Percent l y s i s at (min)t 20 30 45
VBS2+ EGTA, 0.01M i mM Mg2+-EGTA 2 ...... 5 ......
67 I 1 1 2
95 3 6 6 4
99 15 41 48 43
60
100 81 94 97 88
I00 100 100 100 98
tWith human e r y t h r o c y t e s s e n s i t i z e d with a 1:200 d i l u t i o n of sheep antibody. * T i t r e of 1:128 with s e n s i t i z e d human e r y t h r o c y t e s . Used at 1:100. Sheep Ig f r a c t i o n s and FLS C With Ig s e n s i t i z e d e r y t h r o c y t e s , haemolysis proceeded f a s t e r in the presence of 5 mM Mg2+-EGTA than with EGTA, but not as f a s t as with VBSe+.
Rates
intermediate between the values obtained f o r VBS2+ and 5 mM Mg2+-EGTA were obtained in the presence of I and 2 mM Mge+-EGTA ( F i g . l ) . c----o Fast~ 1:6L lo07
~ _ _ _ o
80
..
~ ffl
~
1:32
o..... ~ S l o w ~ 1:4 c _4 ,~''O"~ o
.... o
/P"
/
...'"
//
VBS2÷
~1 /:: /
,
o ...... °
//
20
o-9
...'"
iI
.""
50~/ 40
o--olgM
.:
?
=': 0.01M EGTA
i
i
i
0
E lO0 ci
.o ......... -o
ZE
/
....... ""
B050!
f
40-
.....'""
,.
.... .:
,
:.:
! .d 20- r..." o~ IO
2raM Mg2+-EGTA
2'o 3~
4'5 Time
&
10
20
30
/-5
60
(rain)
Fig 1. FLS complement haemolysis of human erythrocytes s e n s i t i z e d with Ig fractions. E f f e c t of EGTA and Mge+-EGTA on the r a t e of haemolysis.
262
P u r i t y of Ig f r a c t i o n s The p r o t e i n c o n c e n t r a t i o n s o f the Ig f r a c t i o n s were: IgM, 4-6 mg/m!; slow y-globulin,
6.25 mg/ml; f a s t y - g l o b u l i n ,
was pure, and on d i l u t i o n
18.0 mg/ml.
By IEP, each Ig f r a c t i o n
IgM p r e c i p i t a t e d to a d i l u t i o n
of 1:16, slow
y - g l o b u l i n to 1:128 and f a s t y - g l o b u l i n to 1:256. DISCUSSION The importance o f Mg2+ in haemolysis of sheep a n t i b o d y s e n s i t i z e d human e r y t h r o c y t e s by sheep C has been demonstrated by use o f c h e l a t o r s and d i a l y s e d serum.
Human e r y t h r o c y t e s s e n s i t i z e d w i t h antiserum or the Ig f r a c t i o n s were
lysed by sheep C in the presence o f EGTA or MgZ+-EGTA (Tables I-V and Fig. 1) but not EDTA or TTHA (Tables I - I l l ) .
(EGTA has a 10 O00-fold preference f o r
Ca2+ over Mgz+ (Bryant and Jenkins, 1968) whereas EDTA and TTHA have high a s s o c i a t i o n constants f o r both Ca2+ and Mg2+ (Bryant and Jenkins, 1968; Nagaki et al.,
1974).)
The EGTA d i d , however, have an e f f e c t on the assays. the t i t r e
e r y t h r o c y t e s f o r l y s i s by a standard d i l u t i o n standard d i l u t i o n Ill).
There was, r e l a t i v e
to
in VBS2+, an increased requirement f o r antibody to s e n s i t i z e the of Ig, the C t i t r e
of C in EGTA (Table I ) .
was o f t e n s l i g h t l y
With a
lower w i t h EGTA (Table
There was also a reduced r a t e of haemolysis in EGTA compared w i t h VBS2+
(Table V and F i g . l ) .
R e l a t i v e to EGTA, MgZ+-EGTA decreased the amount of
antibody r e q u i r e d to s e n s i t i z e the e r y t h r o c y t e s the C t i t r e VBS2+.
(Table I l l ) ,
giving titres
(Tables I and I I )
and increased
t h a t were s i m i l a r to those obtained w i t h
The r a t e of haemolysis was also increased but the VBS2+ r a t e was not
reached (Table V and F i g . l ) . Mg2+ and Mg2+-EGTA were able to r e s t o r e haemolytic a c t i v i t y
to sheep serum
t h a t had been absorbed w i t h human e r y t h r o c y t e s and then d i a l y s e d a g a i n s t PBS (Table I V ) , but Ca2+ had no e f f e c t .
As s i m i l a r antibody and C t i t r e s
obtained in the presence of Mg2+-EGTA and VBSz+ (Tables I - I l l ) Mg2+ and Mg2+-EGTA were able to f u l l y (Table IV), t h i s
restore C activity
and as 2 and 5 mM
to d i a l y s e d serum
i n d i c a t e s t h a t t h i s sheep C haemolytic a c t i v i t y
the absence of Ca2+.
As C a c t i v i t y
were
can proceed in
in EGTA or Mg2+-EGTA chelated serum from
various animals has been considered to i n d i c a t e a l t e r n a t i v e
pathway a c t i v i t y
(Sandberg and O s l e r , 1971; Forsgren and Quie, 1974; P l a t t s - M i l l s 1974; Fine, 1977), the sheep C haemolytic a c t i v i t y
and I s h i z a k a ,
in EGTA and Mg2+-EGTA
described in t h i s paper probably also i n v o l v e s the a l t e r n a t i v e pathway. Heating C at 50 °C f o r 30 min (Goodkofsky and Lepow, 1971) or 56 °C f o r 3 min (Pang and Aston, 1978) i n a c t i v a t e s a l t e r n a t i v e haemolytic a c t i v i t y
pathway f a c t o r B.
The sheep
mediated by sheep antiserum and Ig f r a c t i o n s was also l a b i l e
263
to this heat treatment (Table I I I ) .
The FLS CM-cellulose f r a c t i o n that restored
haemolytic a c t i v i t y to 50 °C-treated sheep serum (Table I I I ) protein that was responsible f o r t h i s a c t i v i t y .
had a 107 000 dalton
This protein is considered to
be sheep factor B as the mol~cular weight is s i m i l a r to that reported f o r bovine factor B (Pang and Aston, 1978).
Good evidence f o r a l t e r n a t i v e pathway
involvement is that the factor B containing f r a c t i o n , in the presence o~ EGTA or Mg2+-EGTA could restore haemolytic a c t i v i t y to 50 °C-treated serum. The r e l a t i o n s h i p between the haemagglutination and the haemolytic t i t r e of the sheep anti-human erythrocyte antibody is of i n t e r e s t .
With the standard
sheep-erythrocyte rabbit-antibody i n d i c a t o r system, the rabbit antiserum used had a haemagglutination t i t r e of 1:320 and a haemolytic t i t r e of 1:7000-9000. By contrast, the sheep anti-human erythrocyte antiserum had a haemagglutination t i t r e of 1:4096 and a haemolytic t i t r e of 1:512-4096. haemagglutination t i t r e ,
IgM had the highest
but i t s haemolytic t i t r e was much lower and s i m i l a r to
that of fast y - g l o b u l i n .
The slow and fast v-globulins had s i m i l a r
haemagglutination t i t r e s ,
but there was a marked difference in haemolytic t i t r e s
(Table I I ) .
No explanation is a v a i l a b l e f o r the difference between the
haemagglutination and haemolytic t i t r e of the sheep amboceptors r e l a t i v e to the rabbit amboceptor. Lachmann and Nicol (1973) have suggested that the a l t e r n a t i v e pathway requires a much higher concentration of C fo r a c t i v i t y than does the classical pathway. In our experiments, FLS had a maximum haemolytic t i t r e of 1:32 and sheep serum, a maximum of 1:128.
These t i t r e s were lower than the 1:512 r e g u l a r l y obtained
with guinea-pig C.
Thus, r e l a t i v e to the classical pathway a c t i v i t y of guinea-
pig C, this sheep haemolytic system required a high concentration of both amboceptor and C. Although the data presented indicate that the a l t e r n a t i v e pathway is involved in sheep C haemolytic a c t i v i t y in EGTA and Mg2+-EGTA, the mechanism of C a c t i v a t i o n is not known. The sheep amboceptor is apparently required on the erythrocyte surface as washed sensitized c e l l s were j u s t as susceptible as unwashed sensitized c e l l s to C. agglutinated by the
Even though the erythrocytes were heavily
doses of sheep amboceptor used, provided the tubes were
shaken r e g u l a r l y , complete haemolysis occurred. ACKNOWLEDGEMENTS We would l i k e to thank Mr Hugh Pulford for technical assistance. M. Stankiewicz g r a t e f u l l y acknowledges a New Zealand Government National Research Advisory Council Post-Doctoral Fellowship.
264
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