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Hemolytic complement measurement in eleven species of nonhuman primates
Veterinary Immunology and Immunopathology, 5 (1983/1984) 141--149 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
HEMOLYTIC COMPLEMENTMEASUREMENTIN ELEVENSPECIESOF NONHUMANPRIMATES L.R. ELLINGSWORTHl, C.A. HOLMBERG 2 and B.I. OSBURN 3 IDepartment of Experimental Immunology and Pathology, California Primate Research Center, University of California, Davis, CA, U.S.A. 2Veterinary Medicine Teaching and Research Center, 3981 South K, Tulare, CA, U.S.A. 3Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA, U.S.A.
(Accepted 4 May 1983)
Supported by National Institutes of Health Research Program Project Grant RROO169
ABSTRACT Ellingsworth, L.R., Holmberg, C.A. and Osburn, B.I., 1983. Hemolytic complement measurement in eleven species of nonhuman primates. Vet. Immunol. Immunopathol., 5: 141-149. A microtiter system was used to measure hemolytic complement levels in serum from eleven nonhuman primate species. The species studied were Macaca mulatta (rhesus macaque), Macaca radiata (bonnet macaque), Macaca nemestrina ~iled macaque), Maca-cac'a--f~aris (crab-eating macaque), Macaca (stumptailed macaq-'ETq-ue~,---P-apio cynocephalus (yellow b a b o o n ) , ~ live baboon), Cercopithecus aethiops (African green monkey), oAot-us t--r-TvT~atus (owl monkey), Ateles'"fusc~ robustus (spider m o n k e y ) ~ ~ c r a s s i c a u d a t u s panganTensis (-t'h-TEk-tai~g--gaTago). The optimal hemolytic complement t i t e r of the various nonhuman primate species was found to vary with different species sources of erythrocytes and anti-erythrocyte reagents used in the assay. No single erythrocyte and antierythrocyte test reagent produced optimal titers for all of the primate species examined. Sera from several species was found to have high spontaneous lytic activity towards non-sensitized sheep erythrocytes which for six species (M. mulatta, M. radiata, M. speciosa, P. cynocephalus, P. anubis and A. t r i ~ i r ~ w a s eq'u-al ~o the ~ Y - o r antibody Sensitized erythrocytes. Evidence of alternate pathway complement activation as a possible reason for the high t i t e r of lytic activity towards unsensitized erythrocytes could not be demonstrated for any nonhuman primate species. In one species, M. mulatta, the sensitizing activity of normal serum for sheep erythrocytes ~ a s ~ - t o be in the IgM containing fraction obtained with gel f i l t r a t i o n and to be absorbed by boiled sheep erythrocyte stroma which contains Forssman antigen.
~%
0165-2427/83/$03.00
© 1983 Elsevier Science Publishers B.V.
141
142
INTRODUCTION Normal host
inflammatory
and
immune responses
activation of the serum complement system.
are involved in chemotaxis of inflammatory c e l l s , inflammatory
response,
are
dependent
upon
Activated complement components
enhanced phagocytosis
of
induction of vascular foreign
substances
by
inflammatory c e l l s , neutralization and l y s i s of microorganisms, and l y s i s of c e l l s bearing foreign antigenic determinants (Muller, Kabat and Meyer, 1967; Fudenberg et a l . , 1978). the sequential complement cascade,
1975; Ruddy, 1974;
In addition, activation of
by either the classical or alternate
pathways, results in the f i n a l sequence capable of erythrocyte l y s i s , which provides the basis for quantitation of t o t a l complement a c t i v i t y by hemolytic assays (Kabat and Meyer, 1967). When hemolytic complement was measured, the source of erythrocytes and anti-erythrocyte antibody (hemolysin) was shown to vary between d i f f e r e n t species (Grant, 1977).
The widely used test system of sheep erythrocytes
sensitized with rabbit antibody against sheep red blood c e l l s , for instance, is not optimal for quantitation of complement for such species as the horse, cow, sheep, deer, c a t
r a b b i t , mouse, or chicken (Grant, 1977).
Another
possible source of v a r i a t i o n , in hemolytic assays, is the contribution of non-antibody dependent alternate pathway activation of complement observed when bovine complement is measured with human erythrocytes (Pang and Aston, 1977). The present
study u t i l i z e d
determine
50% hemolytic
the
a sensitive spectrophotometric method to endpoint
(CH50) and compares additional
erythrocyte test reagents. Rabbit, sheep, guinea pig, and rhesus monkey sera served as sources of sensitizing antibody for bovine and sheep erythrocytes. Alternate pathway activation as a possible factor contributing to the high hemolytic a c t i v i t y of primate serum to non-sensitized erythrocytes was also examined. MATERIAL AND METHODS Serum Whole blood obtained by venipuncture was allowed to clot at room temperature for 2 hours in s t e r i l e glass tubes. The serum was c l a r i f i e d by centrifugation and frozen at -80°C. a c t i v i t y immediately after thawing.
Samples were assayed for complement All animals were c l i n i c a l l y normal.
143
Hemoiyticcomplement assay Serum t i t r a t i o n s were made using O.l ml amounts of twofold
serial
dilutions in round bottom microtiter plates (Microbiological Associates, Walkersville, Md.). An equal volume of optimally sensitized or control (nonsensitized) erythrocytes (I.5 x lO8 cells/ml) were added to each well
This
was incubated at 37°C for l hour and overnight at 4°C. Using a micropipette, O.l ml of supernatant was withdrawn from the well and diluted tenfold.
The
optical density was determined at 414 ~m and the complement t i t e r calculated from a standard curve (Kabat and Meyer, 1967). The t i t e r s were expressed in CH50 units which is the reciprocal of the serum dilution producing 50% hemolysis. Preparation of erythrocytes Sheep and bovine erythrocytes were collected in Alsever's solution and used within l week. The cells were washed and optimally sensitized with a non-agglutinating dilution of anti-erythrocyte antibody.
Bovine red blood
cell antiserum was produced in guinea pigs, sheep, and rabbits by immunizing at two week intervals with washed erythrocytes. Rabbit anti-sheep hemolysin was obtained commercially (Cappel Laboratories,
Inc., Cochranville, Pa.).
Rhesus anti-bovine and sheep erythrocyte antibody consisted of pooled whole rhesus serum from non-immunized animals (natural antibody).
Both sensitized
and non-sensitized erythrocytes were suspended to 1.5 x lO8 cells/ml in 0.02 M phosphate buffer containing l mMMgCI2 and 0.15 mM CaCl at pH 7.2. Activatio n of alternate pathway All complement dependent l y t i c a c t i v i t y was inhibited by chelation with 0.02 M phosphate buffered saline containing lO mMethylene diaminetetracetic acid (PBS/EDTA).
Conditions for selective activation of the alternate
pathway was performed in 0.02 M phosphate buffered saline containing lO mM ethyleneglycol-bis-(B-amino ethyl ether) N,N-tetraacetic acid and 4 mMMgCl2 (PBS/EGTA). classical
The EGTA selectively chelates the calcium necessary for the pathway, permitting
the
calcium independent, but
magnesium
u t i l i z i n g alternation pathway, to remain active (Pang and Aston, 1977). Characterization of natural ant!body .in rhesus serum Whole rhesus serum was separated into IgM and IgG containing fractions by gel f i l t r a t i o n (Sephadex G-200, Pharmacia, Piscataway, N.J.) in O.l M Tris-
144
HCL, 0.5 M NaCl, and 0.4% sodium azide (pH 8.0) and concentrated to the original serum volume (Diaflo Ultrafiltration Systems, Amicon Corp.).
The
activity of natural antibody in rhesus serum was determined by comparing the sensitizing activity of whole rhesus serum, the IgG and IgM containing fractions both before and after absorption with boiled (100°C) sheep erythrocyte stroma which contains intact Forssman antigen (Campbell et al., 1970; Boyd, 1966). Guinea pig serum was used as a complement source in these sensitization assays. RESULTS Hemolytic complement titers The hemolytic complement t i t e r (CH50) of II species of nonhuman primates using bovine and sheep erythrocytes sensitized with various sources of hemolysin is summarized in Table I and II. Whenthe results were examined for the erythrocyte-hemolysin combination, which gave maximal CH50 values for most species, the rhesus antibody sensitized sheep erythrocytes resulted in maximal CH50values for seven species (M. mulatta, M. radiata, M. fascicularis, M. spec!osa, P. ~nocephalus, P. anubis, andA. fusceps, robustus). Of the four species that did not have maximal CH50values with rhesus sensitized sheep erythrocytes, three species (M. nemestrina, C. aethiops, and A. trivirgatus ) were found to have maximal CH50 values with rabbit antibody sensitized bovine erythrocytes while one species (G. crassicaudatus panganiensis) did not have significant levels of hemolytic complement as measured by any of these systems. Results with the test reagent most commonly used for measurement of human serum complement (rabbit antibody sensitized sheep erythrocytes), resulted in CH50 values comparable to the optimal system for only 3 of the primate species (M. speciosa, P. cynocephalus, and A. trivirgatus). There was one species serum (A. trivir~atus) for which the CH50 for unsensitized erythrocytes was clearly higher than with the sensitized erythrocyte (rhesus sensitized sheep erythrocyte). This suggests M. mulatta antibody not only did not activate A. trivirBatus complement but that i t blocked 'natural' antibody in the complement source from recognizing erythrocyte antigens. The CH50 of all primate species sera for unsensitized erythrocytes (spontaneous lysis) was noted to be higher for sheep erythrocytes than for bovine erythrocytes. When the CH50 for sensitized and unsensitized
145 TABLE I Comparison of hemolytic complement t i t e r s * in nonhuman primate sera using bovine erythrocytes. Bovine Erythrocytes
Species Macaca mulatta Macaca radiata Macaca nemestrina Macaca fascicularis Macaca seeciosa Papio cynocephalus Papio anubis Cercopithecus aethiops Aotus trivirgatus Ateles fusceps robustus Galago crassicaudatus panganiensis
No. of samples I0
I0 2 3 6 8 3 l 3
1 I0
Unsensitized
Sheep antibovine
Guinea Rabbit pig anti- antibovine bovine
21+19 (0:62) 36+7 (Ig 43) 21+7 (20-21)
75+31 (40-144) 68+16 (40-85) 111+44 (80~143)
213+81 162+34 (90:330) (89-202) 252+105 99+32 (79:328) (70-154) 143 116+53 (79Z154)
19+8 (I~-26) 48+4 (40 50) 52+52 (IT-77) 46+9 (30-56) 24
67+29 (3~-91) 93+4 (90-100) 91+8 (7~-97) 89+5 (8~-92) 362
60+35 (20 82) 160+4 (15~ 163) 132+81 (76:160) 157+8 (14~-163) 160
56+28 (3~-88) 9
88+5 (8~ 92) 78
NDt
ND
237+106 (167-359) 148
I0
17+I (1~-18)
21+8 (16-34)
16+4 (10-22)
I15+68 (37=155) 177+23 (15~-199) 192+51
(131-288) 174+28 (141-190) 379
*Complement t i t e r is expressed in CH50 units, equal to the reciprocal of the serum dilution resulting in 50% red blood cell lysis. tNot determined. erythrocytes are equal, i t is not clear whether the assay is quantitating the hemolytic complement or the amount of naturally occurring anti-erythrocyte antibody present in the serum sample being titrated. The results in Table II show only two species (M. speciosa and [ . c~nocephalus) which had comparable t i t e r s with both the sensitized and unsensitized sheep erythrocytes. For
146
these two species, numerically comparable CH50 values were observed with rabbit sensitized bovine erythrocytes while the unsensitized bovine erythrocytes t i t e r s were appreciably less indicating that hemolytic complement was the limiting factor for hemolysis.
TABLE II Comparison of hemolytic complement t i t e r s * in nonhuman primate sera using sheep erythrocytes. Sheep Erythrocytes
Unsensitized
Rabbit anti-sheep
Pooled rhesus serum
I0
205+105 (69Z416)
190+80 (62Z301)
273+83 (13~-365)
I0
108+48 (45Z190) 39+0.9 (3g-46) 36+17 (21-47)
125+80 (73z333)
300+116 (145-575)
40
40
164+41 (87;197 176+62 (98:310 137+60 (76:195 88
68+21 (45-75) 180+70 (91:307) 202+91 (93;372) 109+53 (70-170) 148
174+21 (153-195) 172+18 (14~-199) 189+82 (79;300) 198+162 (13~-320) 198
144+52 (93~156 36
229+144 (135-395) 53
63+20 (40-78) 141
14+3 (11-19)
18+2
I0
Species
No. of samples
Macaca mulatta Macaca radiata Macaca nemestrina Macaca fascicularis Macaca speciosa Papio cynocephalus Papio anubis Cercopithecus aethiops Aotus trivirgatus Ateles fusceps robustus Galago crassicaudatus panganiensis
2 3 6 8 3 1 3 1
I0
(15-22)
*Complement t i t e r is expressed in CH50 units, equal to the reciprocal of the serum dilution resulting in 50% red blood cell lysis. CH50 C standard deviation with range
Mean
147
Alternate pathway activation To determine i f there was alternate pathway activation of complement on mixing the serum samples with unsensitized erythrocytes, aliquots from a l l serum samples studied (Table I , I I )
were also simultaneously incubated with
unsensitized bovine and sheep erythrocytes in the presence of the PBS/EDTA buffer and the PBS/EGTA buffer.
The PBS/EDTA buffer prevented l y s i s (CH50
<10)
as would
in
all
samples which
is
be expected
if
the
lysis
of
unsensitized erythrocytes was mediated by classical complement pathway which depends on both calcium and magnesium ions.
The PBS/EGTA buffer
also
prevented l y s i s (CH50
Guinea pig serum complement was used
to t i t r a t e the sensitizing antibody (Table I I I ) .
In these experiments, the
amount of IgM and IgG fractions used to sensitize erythrocytes represent equal amounts of the original serum pool and was comparable to the amount used for
"whole rhesus
serum" sensitization.
Most of
the sensitizing
a c t i v i t y of normal rhesus serum was in the IgM containing f r a c t i o n .
The
sensitizing a c t i v i t y of the whole serum, IgM fraction and IgG f r a c t i o n were readily
absorbed
by
prior
incubation
with
boiled
sheep erythrocytes
containing Forssman antigen. DISCUSSION The hemolytic complement present in the serum of
II
nonhuman primate
species was assayed with several d i f f e r e n t species sources of erythrocyte and anti-erythrocyte
antibody
(hemolysin).
No
single
erythrocyte/anti-
erythrocyte combination gave maximal CH50 values for a l l species tested. I t was found that by using 2 d i f f e r e n t test reagent combinations (rhesus serum sensitized
sheep erythrocytes
and
rabbit
serum sensitized
bovine
erythrocytes) a maximal CH50 value could be obtained f o r a l l but one of the primate species assayed.
I t was found that rabbit serum sensitized sheep
erythrocytes, the most widely used test system, resulted in maximal CH50 t i t e r s for only 3 species.
Only one species was examined, the prosimian G.
148
crassicaudatus panganiensis, for which no system was capable of demonstrating the hemolytic complement. There was no evidence of alternate pathway complement activation in any of the test systems investigated as demonstrated by use of EGTA and EDTA containing buffers; which selectively chelate divalent cations (Ca and Mg). In at least one species, Macaca mulatta, the sensitizing a c t i v i t y of whole serum for sheep erythrocytes was shown to be present in the IgM containing fraction with all of the sensitizing a c t i v i t y readily absorbed by a Forssman antigen containing erythrocyte preparation.
TABLE I l l Sensitizing a c t i v i t y of normal rhesus serum for sheep erythrocytes after gel f i l t r a t i o n and Forssman antigen absorption.*
Sensitizing Antibody t Whole rhesus serum Forssman antigen absorbed whole rhesus serum IgM f r a c t i o n
CH50 of Guinea Pig Serum 275 18 320
Forssman antigen absorbed IgM f r a c t i o n
I0
IgG f r a c t i o n
43
Forssman antigen absorbed IgG f r a c t i o n
I0
*TheoForssman antigen (washed sheep erythrocyte stroma after heating at lO0 C fOro30 minutes) absorption of the sensitizing antibody was carried out at 37 C for l hour. tSerum and serum fractions were heat inactivated at 56°C for 30 minutes before sensitization. REFERENCES Boyd, W.C., 1966. Fundamentals of Immunology. John Wiley Co., New York, 773 PP. Campbell, D., Garvey, F., Cremer, N. and Susdorf, D.H., 1970. Methods in Immunology: A Laboratory Text for Instruction and Research. W. Benjamin, Inc., London, 454 pp. Fudenberg, H., Stites, D., Caldwell, J. and Wells, J., 1978. Basic and Clinical Immunology. Lange Medical Publications, Los Altos, California, 758 pp. Grant, C.K., 1977. Complement ' s p e c i f i c i t y ' and interchangeability: Measurement of hemolytic levels and use of the complement fixation test with sera from common domesticated animals. American Journal of Veterinary Research 38: 1611-1617.
149
Kabat, E. and Mayer, M., 1967. Experimental Immunochemistry. C. Thomas Publishing Co., Springfield, I11inois, 905 pp. Muller-Berhard, H.J., 1975. Complement. Annual Review of Biochemistry 44: 697-724. Pang, A. and Aston, W., 1977. Alternative complement pathway in bovine serum: Lysis of human erythrocytes. American Journal of Veterinary Research 38: 355-359. Ruddy, S., 1974. Chemistry and biological activity of the complement system. Transplantation Proceedings 6: I-7.
HEMOLYTIC COMPLEMENTMEASUREMENTIN ELEVENSPECIESOF NONHUMANPRIMATES L.R. ELLINGSWORTHl, C.A. HOLMBERG 2 and B.I. OSBURN 3 IDepartment of Experimental Immunology and Pathology, California Primate Research Center, University of California, Davis, CA, U.S.A. 2Veterinary Medicine Teaching and Research Center, 3981 South K, Tulare, CA, U.S.A. 3Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, CA, U.S.A.
(Accepted 4 May 1983)
Supported by National Institutes of Health Research Program Project Grant RROO169
ABSTRACT Ellingsworth, L.R., Holmberg, C.A. and Osburn, B.I., 1983. Hemolytic complement measurement in eleven species of nonhuman primates. Vet. Immunol. Immunopathol., 5: 141-149. A microtiter system was used to measure hemolytic complement levels in serum from eleven nonhuman primate species. The species studied were Macaca mulatta (rhesus macaque), Macaca radiata (bonnet macaque), Macaca nemestrina ~iled macaque), Maca-cac'a--f~aris (crab-eating macaque), Macaca (stumptailed macaq-'ETq-ue~,---P-apio cynocephalus (yellow b a b o o n ) , ~ live baboon), Cercopithecus aethiops (African green monkey), oAot-us t--r-TvT~atus (owl monkey), Ateles'"fusc~ robustus (spider m o n k e y ) ~ ~ c r a s s i c a u d a t u s panganTensis (-t'h-TEk-tai~g--gaTago). The optimal hemolytic complement t i t e r of the various nonhuman primate species was found to vary with different species sources of erythrocytes and anti-erythrocyte reagents used in the assay. No single erythrocyte and antierythrocyte test reagent produced optimal titers for all of the primate species examined. Sera from several species was found to have high spontaneous lytic activity towards non-sensitized sheep erythrocytes which for six species (M. mulatta, M. radiata, M. speciosa, P. cynocephalus, P. anubis and A. t r i ~ i r ~ w a s eq'u-al ~o the ~ Y - o r antibody Sensitized erythrocytes. Evidence of alternate pathway complement activation as a possible reason for the high t i t e r of lytic activity towards unsensitized erythrocytes could not be demonstrated for any nonhuman primate species. In one species, M. mulatta, the sensitizing activity of normal serum for sheep erythrocytes ~ a s ~ - t o be in the IgM containing fraction obtained with gel f i l t r a t i o n and to be absorbed by boiled sheep erythrocyte stroma which contains Forssman antigen.
~%
0165-2427/83/$03.00
© 1983 Elsevier Science Publishers B.V.
141
142
INTRODUCTION Normal host
inflammatory
and
immune responses
activation of the serum complement system.
are involved in chemotaxis of inflammatory c e l l s , inflammatory
response,
are
dependent
upon
Activated complement components
enhanced phagocytosis
of
induction of vascular foreign
substances
by
inflammatory c e l l s , neutralization and l y s i s of microorganisms, and l y s i s of c e l l s bearing foreign antigenic determinants (Muller, Kabat and Meyer, 1967; Fudenberg et a l . , 1978). the sequential complement cascade,
1975; Ruddy, 1974;
In addition, activation of
by either the classical or alternate
pathways, results in the f i n a l sequence capable of erythrocyte l y s i s , which provides the basis for quantitation of t o t a l complement a c t i v i t y by hemolytic assays (Kabat and Meyer, 1967). When hemolytic complement was measured, the source of erythrocytes and anti-erythrocyte antibody (hemolysin) was shown to vary between d i f f e r e n t species (Grant, 1977).
The widely used test system of sheep erythrocytes
sensitized with rabbit antibody against sheep red blood c e l l s , for instance, is not optimal for quantitation of complement for such species as the horse, cow, sheep, deer, c a t
r a b b i t , mouse, or chicken (Grant, 1977).
Another
possible source of v a r i a t i o n , in hemolytic assays, is the contribution of non-antibody dependent alternate pathway activation of complement observed when bovine complement is measured with human erythrocytes (Pang and Aston, 1977). The present
study u t i l i z e d
determine
50% hemolytic
the
a sensitive spectrophotometric method to endpoint
(CH50) and compares additional
erythrocyte test reagents. Rabbit, sheep, guinea pig, and rhesus monkey sera served as sources of sensitizing antibody for bovine and sheep erythrocytes. Alternate pathway activation as a possible factor contributing to the high hemolytic a c t i v i t y of primate serum to non-sensitized erythrocytes was also examined. MATERIAL AND METHODS Serum Whole blood obtained by venipuncture was allowed to clot at room temperature for 2 hours in s t e r i l e glass tubes. The serum was c l a r i f i e d by centrifugation and frozen at -80°C. a c t i v i t y immediately after thawing.
Samples were assayed for complement All animals were c l i n i c a l l y normal.
143
Hemoiyticcomplement assay Serum t i t r a t i o n s were made using O.l ml amounts of twofold
serial
dilutions in round bottom microtiter plates (Microbiological Associates, Walkersville, Md.). An equal volume of optimally sensitized or control (nonsensitized) erythrocytes (I.5 x lO8 cells/ml) were added to each well
This
was incubated at 37°C for l hour and overnight at 4°C. Using a micropipette, O.l ml of supernatant was withdrawn from the well and diluted tenfold.
The
optical density was determined at 414 ~m and the complement t i t e r calculated from a standard curve (Kabat and Meyer, 1967). The t i t e r s were expressed in CH50 units which is the reciprocal of the serum dilution producing 50% hemolysis. Preparation of erythrocytes Sheep and bovine erythrocytes were collected in Alsever's solution and used within l week. The cells were washed and optimally sensitized with a non-agglutinating dilution of anti-erythrocyte antibody.
Bovine red blood
cell antiserum was produced in guinea pigs, sheep, and rabbits by immunizing at two week intervals with washed erythrocytes. Rabbit anti-sheep hemolysin was obtained commercially (Cappel Laboratories,
Inc., Cochranville, Pa.).
Rhesus anti-bovine and sheep erythrocyte antibody consisted of pooled whole rhesus serum from non-immunized animals (natural antibody).
Both sensitized
and non-sensitized erythrocytes were suspended to 1.5 x lO8 cells/ml in 0.02 M phosphate buffer containing l mMMgCI2 and 0.15 mM CaCl at pH 7.2. Activatio n of alternate pathway All complement dependent l y t i c a c t i v i t y was inhibited by chelation with 0.02 M phosphate buffered saline containing lO mMethylene diaminetetracetic acid (PBS/EDTA).
Conditions for selective activation of the alternate
pathway was performed in 0.02 M phosphate buffered saline containing lO mM ethyleneglycol-bis-(B-amino ethyl ether) N,N-tetraacetic acid and 4 mMMgCl2 (PBS/EGTA). classical
The EGTA selectively chelates the calcium necessary for the pathway, permitting
the
calcium independent, but
magnesium
u t i l i z i n g alternation pathway, to remain active (Pang and Aston, 1977). Characterization of natural ant!body .in rhesus serum Whole rhesus serum was separated into IgM and IgG containing fractions by gel f i l t r a t i o n (Sephadex G-200, Pharmacia, Piscataway, N.J.) in O.l M Tris-
144
HCL, 0.5 M NaCl, and 0.4% sodium azide (pH 8.0) and concentrated to the original serum volume (Diaflo Ultrafiltration Systems, Amicon Corp.).
The
activity of natural antibody in rhesus serum was determined by comparing the sensitizing activity of whole rhesus serum, the IgG and IgM containing fractions both before and after absorption with boiled (100°C) sheep erythrocyte stroma which contains intact Forssman antigen (Campbell et al., 1970; Boyd, 1966). Guinea pig serum was used as a complement source in these sensitization assays. RESULTS Hemolytic complement titers The hemolytic complement t i t e r (CH50) of II species of nonhuman primates using bovine and sheep erythrocytes sensitized with various sources of hemolysin is summarized in Table I and II. Whenthe results were examined for the erythrocyte-hemolysin combination, which gave maximal CH50 values for most species, the rhesus antibody sensitized sheep erythrocytes resulted in maximal CH50values for seven species (M. mulatta, M. radiata, M. fascicularis, M. spec!osa, P. ~nocephalus, P. anubis, andA. fusceps, robustus). Of the four species that did not have maximal CH50values with rhesus sensitized sheep erythrocytes, three species (M. nemestrina, C. aethiops, and A. trivirgatus ) were found to have maximal CH50 values with rabbit antibody sensitized bovine erythrocytes while one species (G. crassicaudatus panganiensis) did not have significant levels of hemolytic complement as measured by any of these systems. Results with the test reagent most commonly used for measurement of human serum complement (rabbit antibody sensitized sheep erythrocytes), resulted in CH50 values comparable to the optimal system for only 3 of the primate species (M. speciosa, P. cynocephalus, and A. trivirgatus). There was one species serum (A. trivir~atus) for which the CH50 for unsensitized erythrocytes was clearly higher than with the sensitized erythrocyte (rhesus sensitized sheep erythrocyte). This suggests M. mulatta antibody not only did not activate A. trivirBatus complement but that i t blocked 'natural' antibody in the complement source from recognizing erythrocyte antigens. The CH50 of all primate species sera for unsensitized erythrocytes (spontaneous lysis) was noted to be higher for sheep erythrocytes than for bovine erythrocytes. When the CH50 for sensitized and unsensitized
145 TABLE I Comparison of hemolytic complement t i t e r s * in nonhuman primate sera using bovine erythrocytes. Bovine Erythrocytes
Species Macaca mulatta Macaca radiata Macaca nemestrina Macaca fascicularis Macaca seeciosa Papio cynocephalus Papio anubis Cercopithecus aethiops Aotus trivirgatus Ateles fusceps robustus Galago crassicaudatus panganiensis
No. of samples I0
I0 2 3 6 8 3 l 3
1 I0
Unsensitized
Sheep antibovine
Guinea Rabbit pig anti- antibovine bovine
21+19 (0:62) 36+7 (Ig 43) 21+7 (20-21)
75+31 (40-144) 68+16 (40-85) 111+44 (80~143)
213+81 162+34 (90:330) (89-202) 252+105 99+32 (79:328) (70-154) 143 116+53 (79Z154)
19+8 (I~-26) 48+4 (40 50) 52+52 (IT-77) 46+9 (30-56) 24
67+29 (3~-91) 93+4 (90-100) 91+8 (7~-97) 89+5 (8~-92) 362
60+35 (20 82) 160+4 (15~ 163) 132+81 (76:160) 157+8 (14~-163) 160
56+28 (3~-88) 9
88+5 (8~ 92) 78
NDt
ND
237+106 (167-359) 148
I0
17+I (1~-18)
21+8 (16-34)
16+4 (10-22)
I15+68 (37=155) 177+23 (15~-199) 192+51
(131-288) 174+28 (141-190) 379
*Complement t i t e r is expressed in CH50 units, equal to the reciprocal of the serum dilution resulting in 50% red blood cell lysis. tNot determined. erythrocytes are equal, i t is not clear whether the assay is quantitating the hemolytic complement or the amount of naturally occurring anti-erythrocyte antibody present in the serum sample being titrated. The results in Table II show only two species (M. speciosa and [ . c~nocephalus) which had comparable t i t e r s with both the sensitized and unsensitized sheep erythrocytes. For
146
these two species, numerically comparable CH50 values were observed with rabbit sensitized bovine erythrocytes while the unsensitized bovine erythrocytes t i t e r s were appreciably less indicating that hemolytic complement was the limiting factor for hemolysis.
TABLE II Comparison of hemolytic complement t i t e r s * in nonhuman primate sera using sheep erythrocytes. Sheep Erythrocytes
Unsensitized
Rabbit anti-sheep
Pooled rhesus serum
I0
205+105 (69Z416)
190+80 (62Z301)
273+83 (13~-365)
I0
108+48 (45Z190) 39+0.9 (3g-46) 36+17 (21-47)
125+80 (73z333)
300+116 (145-575)
40
40
164+41 (87;197 176+62 (98:310 137+60 (76:195 88
68+21 (45-75) 180+70 (91:307) 202+91 (93;372) 109+53 (70-170) 148
174+21 (153-195) 172+18 (14~-199) 189+82 (79;300) 198+162 (13~-320) 198
144+52 (93~156 36
229+144 (135-395) 53
63+20 (40-78) 141
14+3 (11-19)
18+2
I0
Species
No. of samples
Macaca mulatta Macaca radiata Macaca nemestrina Macaca fascicularis Macaca speciosa Papio cynocephalus Papio anubis Cercopithecus aethiops Aotus trivirgatus Ateles fusceps robustus Galago crassicaudatus panganiensis
2 3 6 8 3 1 3 1
I0
(15-22)
*Complement t i t e r is expressed in CH50 units, equal to the reciprocal of the serum dilution resulting in 50% red blood cell lysis. CH50 C standard deviation with range
Mean
147
Alternate pathway activation To determine i f there was alternate pathway activation of complement on mixing the serum samples with unsensitized erythrocytes, aliquots from a l l serum samples studied (Table I , I I )
were also simultaneously incubated with
unsensitized bovine and sheep erythrocytes in the presence of the PBS/EDTA buffer and the PBS/EGTA buffer.
The PBS/EDTA buffer prevented l y s i s (CH50
<10)
as would
in
all
samples which
is
be expected
if
the
lysis
of
unsensitized erythrocytes was mediated by classical complement pathway which depends on both calcium and magnesium ions.
The PBS/EGTA buffer
also
prevented l y s i s (CH50
Guinea pig serum complement was used
to t i t r a t e the sensitizing antibody (Table I I I ) .
In these experiments, the
amount of IgM and IgG fractions used to sensitize erythrocytes represent equal amounts of the original serum pool and was comparable to the amount used for
"whole rhesus
serum" sensitization.
Most of
the sensitizing
a c t i v i t y of normal rhesus serum was in the IgM containing f r a c t i o n .
The
sensitizing a c t i v i t y of the whole serum, IgM fraction and IgG f r a c t i o n were readily
absorbed
by
prior
incubation
with
boiled
sheep erythrocytes
containing Forssman antigen. DISCUSSION The hemolytic complement present in the serum of
II
nonhuman primate
species was assayed with several d i f f e r e n t species sources of erythrocyte and anti-erythrocyte
antibody
(hemolysin).
No
single
erythrocyte/anti-
erythrocyte combination gave maximal CH50 values for a l l species tested. I t was found that by using 2 d i f f e r e n t test reagent combinations (rhesus serum sensitized
sheep erythrocytes
and
rabbit
serum sensitized
bovine
erythrocytes) a maximal CH50 value could be obtained f o r a l l but one of the primate species assayed.
I t was found that rabbit serum sensitized sheep
erythrocytes, the most widely used test system, resulted in maximal CH50 t i t e r s for only 3 species.
Only one species was examined, the prosimian G.
148
crassicaudatus panganiensis, for which no system was capable of demonstrating the hemolytic complement. There was no evidence of alternate pathway complement activation in any of the test systems investigated as demonstrated by use of EGTA and EDTA containing buffers; which selectively chelate divalent cations (Ca and Mg). In at least one species, Macaca mulatta, the sensitizing a c t i v i t y of whole serum for sheep erythrocytes was shown to be present in the IgM containing fraction with all of the sensitizing a c t i v i t y readily absorbed by a Forssman antigen containing erythrocyte preparation.
TABLE I l l Sensitizing a c t i v i t y of normal rhesus serum for sheep erythrocytes after gel f i l t r a t i o n and Forssman antigen absorption.*
Sensitizing Antibody t Whole rhesus serum Forssman antigen absorbed whole rhesus serum IgM f r a c t i o n
CH50 of Guinea Pig Serum 275 18 320
Forssman antigen absorbed IgM f r a c t i o n
I0
IgG f r a c t i o n
43
Forssman antigen absorbed IgG f r a c t i o n
I0
*TheoForssman antigen (washed sheep erythrocyte stroma after heating at lO0 C fOro30 minutes) absorption of the sensitizing antibody was carried out at 37 C for l hour. tSerum and serum fractions were heat inactivated at 56°C for 30 minutes before sensitization. REFERENCES Boyd, W.C., 1966. Fundamentals of Immunology. John Wiley Co., New York, 773 PP. Campbell, D., Garvey, F., Cremer, N. and Susdorf, D.H., 1970. Methods in Immunology: A Laboratory Text for Instruction and Research. W. Benjamin, Inc., London, 454 pp. Fudenberg, H., Stites, D., Caldwell, J. and Wells, J., 1978. Basic and Clinical Immunology. Lange Medical Publications, Los Altos, California, 758 pp. Grant, C.K., 1977. Complement ' s p e c i f i c i t y ' and interchangeability: Measurement of hemolytic levels and use of the complement fixation test with sera from common domesticated animals. American Journal of Veterinary Research 38: 1611-1617.
149
Kabat, E. and Mayer, M., 1967. Experimental Immunochemistry. C. Thomas Publishing Co., Springfield, I11inois, 905 pp. Muller-Berhard, H.J., 1975. Complement. Annual Review of Biochemistry 44: 697-724. Pang, A. and Aston, W., 1977. Alternative complement pathway in bovine serum: Lysis of human erythrocytes. American Journal of Veterinary Research 38: 355-359. Ruddy, S., 1974. Chemistry and biological activity of the complement system. Transplantation Proceedings 6: I-7.