27 Immune Reactivities of Antibodies against Glycolipids Downloaded via TUFTS UNIV on July 12, 2018 at 21:22:04 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Natural Antibodies ROBERTA L. RICHARDS and CARL R. ALVING Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, DC 20012 Naturally-occurring antibodies against simple glycolipids have been described only in scattered reports in the literature. About 75% of a l l normal humans have complement-activating anti-Forssman activity (1), and a monoclonal Waldenstrom macroglobulin IgM antibody (McG) having specificity for Forssman glycolipid was derived from the plasma of a patient (2,3). Some normal, or abnormal, human sera have anti-monogalactosyl (4) or anti-digalactosyl diglyceride antibodies (5). Recently we reported the occurrence of "natural" antibodies, apparently autoantibodies, with specificity against d i - and trihexosyl ceramide haptens (CDH and CTH), in normal rabbit sera (6). We also found natural anti-ganglioside G antibodies in normal human, guinea pig, and rabbit sera (7). The major purpose of the present study was to describe, and to quantify, the widespread occurrence of natural complement-fixing autoantibodies against numerous simple glycolipids. We show that every individual rabbit and human serum tested had complement-fixing autoantibodies against glycolipids that are widely distributed in circulating blood cells and other tissues. Numerous studies of specificities of anti-glycolipid antibodies produced by immunizing rabbits have been reported (8,9), and some discrepancies have been noted between laboratories (9). The widespread occurrence of natural antibodies might cause confusion in analyzing specificities produced by experimental immunization. Because of this we have purified both immune and natural anti-glycolipid antibodies from rabbit sera, and we have compared their specifities against the same, and different, glycolipids. M1
Methods In a l l the experiments reported here, antibody activities were determined by antibody-mediated complement-dependent release of trapped marker from liposomes (3,10). The liposomes contained dimyristoylphosphatidylcholine (except where indicated), choles-
0-8412-0556-6/80/ 47-128-461 $5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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t e r o l , and d i c e t y l phosphate i n molar r a t i o s of 2:1.5:0.22, and the phospholipid was 10 mM i n the f i n a l aqueous s w e l l i n g volume. G l y c o l i p i d antigens were incorporated i n t o the liposomes where i n d i c a t e d , g e n e r a l l y a t the l e v e l of 150 ug per ymole of phos p h o l i p i d . P u r i f i e d g a n g l i o s i d e s and a s i a l o - G ^ were k i n d l y pro vided by Drs. Roscoe 0. Brady and Peter H. Fishman. Galactosyl ceramide (CMH) from bovine b r a i n and s y n t h e t i c l a c t o s y l ceramide (CDH) were from M i l e s L a b o r a t o r i e s , Inc., E l k h a r t , Indiana. Cer amide t r i h e x o s i d e (CTH) and globoside from human erythrocytes and Forssman g l y c o l i p i d from sheep erythrocytes were i s o l a t e d as pre v i o u s l y described (3). The sources of s y n t h e t i c and n a t u r a l phos p h o l i p i d s , c h o l e s t e r o l , d i c e t y l phosphate and d i g a l a c t o s y l d i g l y c e r i d e have been given elsewhere (11). Antibody-dependent complement damage to the liposomes was detected by r e l e a s e of trapped glucose, using a spectrophotometr i c assay as described p r e v i o u s l y (10). A l l sera tested f o r the presence of a n t i b o d i e s were i n a c t i v a t e d a t 56° f o r 30 min., and f r e s h (unheated) guinea pig serum was used as the complement source. The assays f o r n a t u r a l a n t i b o d i e s contained 5 y l of l i p osomes, 500 μΐ of glucose assay reagent, e i t h e r 30 p i of r a b b i t serum or 50 y l of human serum, 120 y l of guinea pig serum, and s u f f i c i e n t 0.15 M NaCl to b r i n g the f i n a l volume to 1 ml. Glu cose release was measured a f t e r 30 min at room temperature (ca 22°) (_3). Glucose r e l e a s e of more than 5% was considered a pos i t i v e antibody r e a c t i o n . Results A n a l y s i s of n a t u r a l a n t i b o d i e s against g a l a c t o s y l ceramide (CMH) and l a c t o s y l ceramide (CDH) i n sera from s e v e r a l normal humans and from s e v e r a l normal r a b b i t s r e s u l t e d i n the data shown i n Figure 1. None of the humans, and very few r a b b i t s , had a c t i v i t y a g a i n s t liposomes when no g l y c o l i p i d was present. All the data shown f o r n a t u r a l a n t i - g l y c o l i p i d a n t i b o d i e s were c o r rected f o r any a c t i v i t y observed with liposomes l a c k i n g g l y c o lipid. Few of the human sera had even a s l i g h t l e v e l of a c t i v i t y a g a i n s t CMH, but about h a l f of the i n d i v i d u a l humans d i d have anti-CDH a c t i v i t y . The r a b b i t s were more r e a c t i v e a g a i n s t both CMH and CDH. About 40% of the r a b b i t s had a c t i v i t y a g a i n s t CMH, while only 2% lacked anti-CDH a c t i v i t y . The d i f f e r e n c e s i n the r e a c t i v i t y of r a b b i t sera with CMH and CDH suggests that there i s l i t t l e cross r e a c t i v i t y between these two a c t i v i t i e s i n unimmunized animals - a conclusion p r e v i o u s l y reported by Rapport and colleagues ( 8 ) . These same human sera and some of the r a b b i t sera a l s o were tested against ceramide t r i h e x o s i d e (CTH) and d i g a l a c t o s y l d i g l y c e r i d e (Table I ) . Several of the human sera reacted with d i g a l a c t o s y l d i g l y c e r i d e , i n confirmation of the r e s u l t s of H i r s c h and Parks (_5). Although only a few human sera reacted with CTH, a l l of the r a b b i t sera tested showed r e a c t i v i t y .
Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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A L V I N G
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Glycolipids
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100
GLYCOLIPID ANTIGEN NONE
:
80 60 40 20 uJ ω
υ
0-5
100
6-10
11-15
I6 20 L
2'-30
31-40 4 h50
>50
GALACTOSYL CERAMIDE
Λ 80 < ν
Q
> 60 40 Η 20 h 0 60 40 20
U M
0^5
J IHÏÏS
J
J
16^20 2^30 31-40 41-50 >50
LACTOSYL CERAMIDE
La
. . . . . . . . Figure 1. Natural antibodies in normal human and rabbit sera against liposomes containing no glycolipid, galactosylceramide, or lactosylceramide. Glucose release % OF TRAPPED measured from liposomes containing DMPC, GLUCOSE CHOL, RELEASED DCP, and, where indicated, galactosylceramide (150 pg/pjnol PC) or lactosylceramide (150 μg/μmol PC). Closed bars, humans; open bars, rabbits.
Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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TABLE I . HUMAN AND RABBIT NATURAL ANTIBODIES AGAINST DIGALACTOSYL DIGLYCERIDE AND CERAMIDE TRIHEXOSIDE HUMAN % OF TRAPPED GLUCOSE % RELEASED REACTING
GLYCOLIPID
Digalactosyl diglyceride Ceramide trihexoside
3
6.1±1.7 (17)
76
2.3±3.6 (17)
12
RABBIT % OF TRAPPED GLUCOSE % RELEASED REACTING 3
9.8±
8.1(42)
69
0
46.7110.8(12) 100
0
a
E x p r e s s e d a s : mean±standard d e v i a t i o n (number of sera t e s t e d ) . ^Present i n the liposomes a t 150 yg per ymole of p h o s p h o l i p i d . P r e s e n t i n the liposomes a t 150 nmoles per ymole of phospholipid. c
Natural a n t i b o d i e s were p u r i f i e d from r a b b i t serum by a f f i n i t y b i n d i n g to liposomes (6,12). B r i e f l y , t h i s i n v o l v e d adsorb ing the a n t i b o d i e s from the serum onto liposomes containing the a p p r o p r i a t e g l y c o l i p i d , washing the liposome-antibody complexes f r e e of unreacted serum, then e l u t i n g the a n t i b o d i e s from the liposomes i n 1M Nal. Both anti-CDH and anti-CTH were i s o l a t e d from the same batch of normal r a b b i t serum and were compared for s p e c i f i c i t y . As shown i n Figure 2, the anti-CDH d i d not react with CTH-containing liposomes. In c o n t r a s t , the anti-CTH d i d r e a c t with CDH liposomes (Figure 3 ) , though t o a l e s s e r extent than d i d the anti-CDH. Since no anti-CMH a c t i v i t y was observed i n the whole serum, the p u r i f i e d a n t i b o d i e s were not tested a gainst this antigen. A somewhat d i f f e r e n t pattern o f r e a c t i v i t y was observed with p u r i f i e d a n t i b o d i e s obtained from r a b b i t s immunized with CDH or CTH. As Figure 4 shows, immune anti-CDH a n t i b o d i e s d i d c r o s s - r e a c t with CMH. This observation i s i n contrast to the l a c k of c r o s s - r e a c t i v i t y observed with the n a t u r a l antibody (see above), but i s i n agreement with the r e s u l t s of Arnon et^ a l . (13) ob tained with r a b b i t s that were immunized with l a c t o s y l s p h i n g o s i n e conjugated to a polypeptide. The immune anti-CTH studied here showed l i t t l e or no r e a c t i v i t y with CMH. The normal human sera shown i n Figure 1 a l s o were tested a g a i n s t four g l y c o l i p i d s (globoside, Forssman, a s i a l o - G ^ * °M2) having terminal N-acetylgalactosamine residues (Table I I ) . The f i n d i n g that a l l the i n d i v i d u a l human sera had a c t i v i t y against globoside (Table II) was s u r p r i s i n g , since globoside i s the major g l y c o l i p i d of human e r y t h r o c y t e s , and only i n d i v i d u a l s of the r a r e ρ and P^ blood types l a c k globoside (14). Several, but not a l l , of the i n d i v i d u a l s tested a l s o had a c t i v i t y a g a i n s t Forssman g l y c o l i p i d , as reported p r e v i o u s l y (1,15). This l a c k of c o r r e l a -
Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Immunochemistry Figure 2. Reactivities of purified natural anti-CDH and anti-CTH antibodies against CTH-containing liposomes. Glucose release measured from liposomes con taining DMPC, CHOL, DCP, and CTH (50 nmol/'μίηοΐ PC) (6).
ELUTED ANTIBODIES (μ\) Immunochemistry Figure 3. Reactivities of purified natural anti-CDH and anti-CTH antibodies against CDH-containing liposomes. Glucose release measured from liposomes con taining DMPC, CHOL, DCP, and CDH (154 nmol/^mol PC) (6).
Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Lu CO
