- Email: [email protected]
Studies of tolerance in schistosomiasis
CELLULAR
IMMUNOLOGY
99,4
I l-42 1 ( 1986)
Studies of Tolerance in Schistosomiasis’ T.L.McCURLEY,T.
ABE,' C.E.CARTER, ANDD.G.COLLEY~
Departments of Pathology, Microbiology, and Biology, Vanderbilt University School of Medicine and the VeteransAdministration Medical Center, Nashville, Tennessee37203 Received October 11, 1985; acceptedJanuary 9, 1985 We have studied the induction of immune tolerance by exogenous exposure to schistosomal egg antigen (SEA). We compared routes of administration, exposure to different forms of SEA (eggs,normal SEA, reultracentrifuged SEA), and treatment schedules.Tolerance was evaluated in comparison to SEA sensitization, after challenge with SEA or eggs.Anti-SEA responseswere measured by ELISA assays,and cell-mediated responsivenessby 24-hr skin tests and pulmonary gmnuloma formation 8 and 16 days after intravenous egginjection. Oral administration of either SEA in PBSor NaHCOs, or eggsin PBSdid not induce toleranceor sensitization.Reuhracentrifuged SEA was similar to SEA in inducing cell-mediated immune response. Intrathymic exposure to SEA either had no effect or sensitized the animal for subsequent exposure to SEA. Two intraperitoneal injections of0.5 to 1.Omg of SEA lead to reduced cell-mediated responses,but resulted in high levels of anti-SEA antibody, suggesting a state of split tolerance or immune deviation. Our studies suggestthat with complex, poorly defined antigens such as SEA, many of the classic methods of eliciting tolerance fail. The biologic significance of the form of tolerance inducible by high dosesof SEA remains to be defined. 0 1986 Academic press, hc.
INTRODUCTION Mice infected with the human helminth Schistosomamansoni develop, and then progressively immunoregulate, cell-mediated responses(delayed type hypersensitivity and granuloma formation) to soluble schistosomal eggantigens (SEA) (l-3). In human schistosomiasis, patients may well have had exposure to SEA prior to their initial infections. This could have occurred systemically in utero, or orally through breast milk (4, 5). Upon infection, there may be continuous systematic exposure to high doses of SEA antigens that have been “biofiltered” through the portal system. The effects,if any, of such early or uncommon chronic exposure patterns to these antigens on subsequent responsesto SEA remain unclear. Similarly to Hang et al. (6) we have studied the induction of immune tolerance by exogenous exposure to SEA. We compared routes of administration, exposure to dif’ This work was supported by Grant AI-l 1289 from the National Institutes of Health and a grant from the Edna McConnell Clark Foundation, and the Veterans Administration. * Present address:Department of Parasitology, Miyazaki Medical College, Kiyotake, Miyazaki-gun, Miyazuki Pref., 889-16, Japan. 3To whom correspondence should be addressed:F323 Veterans Administration Medical Center, 1310 24th Avenue South, Nashville, Tenn. 37203. 411 0008-8749/86 $3.00 Copyrieht 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
412
MC CURLEY ET AL.
ferent forms of SEA (eggs,normal SEA, reultracentrifuged SEA), and treatment schedules. Tolerance was evaluated in comparison to SEA sensitization, after challenge with SEA or eggs.Anti-SEA humoral responseswere measured by ELISA assays,and cellmediated responsivenesswas assayedby 24-hr skin tests and pulmonary granuloma formation 8 and 16 days after intravenous egg injections. MATERIALS
AND METHODS
Animals Either adult (8-week-old) male CFl mice (Harlan Laboratories, Indianapolis, Ind.) or adult male CBA/J mice supplied by the National Cancer Institute Supply Contract (IGA VlOl (134) P-77014) were used in all experiments.
Preparation of SchistosomalEggs and SchistosomalEgg Antigen Eggsfor tail vein injection and oral feeding were harvested from the livers of mice 7-9 weeks after infection with cercariae of a Puerto Rican strain of S. mansoni. Eggs were kept in chilled hypertonic saline until immediately prior to injection. Three thousand to 5000 eggs in PBS were used for tail vein injection. Schistosomal egg antigenic preparation (SEA) was prepared by homogenization of similarly obtained eggsand ultracentrifugation of the resulting extract by methods previously described (7, 8). Protein determinations were performed by the Lowry method (9).
Serum Antibody by ELISA Mice were bled and serum was harvested at the time of sacrifice and stored at -70°C. Anti-SEA antibody was measured by an enzyme-linked immunosorbent assay (ELISA) at serum dilutions ranging from 1:100 to 1:400using a /3-galactosidasemethod on microtiter plates as previously described (10).
Lung Granuloma Measurement Eight or 16 days after a tail vein injection of schistosome eggs,the lungs of animals were perfused with 10% buffered Formalin. Tissues were processedand stained with hematoxylin and eosin for histopathologic analysis. Granulomas with central schistosome eggswere located sequentially and diameters were measured across opposing axes using an ocular micrometer. Based upon the generally spherical nature of the schistosome egg granuloma, the mean diameter and volume of each granuloma were calculated. Twenty-five granulomas from each mouse were measured to yield the mean granuloma volume for each mouse. Group means were determined on these values and compared statistically by Student’s t test (1 I).
Antibody Characterization by WesternBlot Analysis SDS gel electrophoretically separated SEA components were transferred to nitrocellulose paper and incubated with sera from animals that had been infected with S. mansoni for 16 weeks, or animals given PBS or various dosesof SEA intraperitonally (ip). The sites of antibody binding were detected with goat anti-mouse IgG coupled with horseradish peroxidase followed by incubation with diaminobenzidine (12).
TOLERANCE IN SCHISTOSOMIASIS
413
Experimental Protocols Oral exposure to whole S. mansoni eggs. Twenty thousand intact S. mansoni eggs were given by intragastric intubation to 8-week male CBA/J mice in 0.5 cc PBS 2 weeks (Day - 14) prior to tail vein injection of eggs(Day 0). Animals were sacrificed at Day 8 and Day 16 after egg injection. Sera were harvested for measurements of anti-SEA antibody and lungs were taken for granuloma measurement. Oral exposure to schistosomal egg antigens (SEA). CFl mice were given (a) 0.5 cc PBS; (b) 0.5 cc of 150 pg SEA/cc in PBS; (c) 0.5 cc of 1500 pg SEA/cc in PBS; (d) 0.5 cc 1.3%NaHC03; (e) 0.5 of 15OpgSEA/cc in 1.3%NaHCO,; or (f) 0.5 cc of 1500 pg SEA/cc in 1.3% NaHC03, by intragastric intubation, on Day -28 and Day - 14. On Day 0 eggswere injected intravenously. Lungs and sera were harvested on Day 8 and Day 16. Reultracentrifuged SEA CFl mice were given single ip injections of SEA or reultracentrifuged SEA 1 week (Day -7) prior to tail vein injection of eggs(Day 0). Reultracentrifuged SEA consisted of SEA taken from the upper third of a tube after centrifugation at 100,OOOgfor 60 min immediately before use. Animals were sacrificed on Day 8 and lungs and sera were obtained for measurement of mean granuloma volumes and anti-SEA antibody. Intrathymic Injection of Bovine Serum Albumin (BSA) or SEA CBA/J mice were either injected intrathymically ( 10 &lobe) with saline, or a total of 10, 50, or 250 Kg of BSA, or the mediastinal area surgically exposed in parallel but left uninjected (sham control). Three weeks later each mouse was immunized at the base of the tail (13) with a 50 ~1 volume of 100 pg BSA emulsified in Complete Freund’s adjuvant. Eight days later the mice were exposed to 500 pg BSA by footpad, rested 6 days and skin test challenged (14) with 30 ~1 of a 2% suspension of heataggregatedBSA (13). Ear thickness was measured with an engineer’s micrometer at 5 and 24 hr after challenge. Sera were collected after the 24-hr skin test readings. The effectsof intrathymic exposure to SEA were studied similarly. Thymic injections ranged from 25 to 80 pg of SEA and sensitizations at the base of the tail were with only one exposure to 25 pg in CFA. Ear injection challenges were with 15 pg/injection versus saline injections in the contralateral ear. Sera were collected after the 24-hr measurements. High Dose and Low Dose SEA Mean lung granuloma volumes and anti-SEA antibody were measured 8 days after tail vein egginjection (Day 0). PBS or 500 pg SEA were given ip on Day -7 as negative and positive sensitization controls, respectively. Animals given two injections of SEA received 100, 500, or 1000 rg of SEA on Day -28 and Day -14. Animals were sacrificed and lungs and sem harvested on Day 8. RESULTS Oral Exposure to Schistosome Eggs Mean lung granuloma volumes in mm3 X 1O-3 f SEM and serum antibody by ELISA (optical density) +- SEM were compared on Day -8 and Day -16 following
414
MC CURLEY ET AL.
intravenous tail vein injection of eggs(Day -0) in animals previously given eggsorally on Day - 14. Mean lung granuloma volumes and antibody levels by ELISA did not differ statistically between experimental groups and controls given PBS whether the assayswere 8 or 16 days after challenge (Fig. 1). This oral regimen of eggs neither sensitized nor tolerized mice to SEA.
Oral Exposure to SEA Mean lung granuloma volumes and serum antibody levels were compared in animals given SEA intragastrically on Day -28 and Day -14. There were no statistically significant differences in granuloma volumes or anti-SEA antibody levels on Day 8 or Day 16 with either low or high dose SEA. The results were similar using either PBS or WaHC03 as the vehicle (Fig. 2A and B).
ReultracentrijugedSEA SEA and reultracentrifuged preparations of SEA produced similar responses as measured by lung granuloma volumes levels. Both materials caused sensitization. Preexposure to 50 E.cg doses of reultracentrifuged SEA did occasionally lead to lower antibody responsesas measured by ELISA (Fig. 3). However, these differences were not observed consistently.
Intrathymic Exposure to BSA or SEA Intrathymic injection of lo-250 pg of BSA induced effective tolerance as seen by anti-BSA serum antibody levels and DTH reactions (Fig. 4). Sham thymic exposure or intrathymic saline injection had no effect on BSA sensitization. In three experiments (Fig. 5), it was seenthat intrathymic exposure to SEA either had no effect or sensitized for subsequent parenteral exposure to SEA. This was true of both serum antibody and DTH responsiveness.
High Dose SEA A single exposure to 500 pg of SEA induced sensitization for secondary granuloma formation at 8 days (Fig. 6), but did not lead to antibody formation (Fig. 7). In contrast,
m GRANULOMA VOLUME IN MM% d%SEM 20 DAY BPBS
?&
DAY 16PBS
w--+
30 I
’
DAYl6CkWEGGS~ I I I I 0.3 0 0.1 0.2 SERUM ANTlBODY BY ELISA (Optical Densityk SEM Fsssl FIG. 1. Mice were given either PBS or 20,000 intact S. mansoni eggsin PBS orally 2 weeks (Day -14) prior to tail vein injection of eggs(Day 0). Mean lung granuloma volumes and anti-SEA antibody by ELISA (serum diluted 1:lOO)were compared. Animals were sacrificed at Day 8 and Day 16 after egg injections. Neither sensitization nor tolerance induction was observed following this oral regimen of eggs.
TOLERANCE IN SCHISTOSOMIASIS GRANULOMA
VOLUME
415
IN MM’xl&SEM
75~19 SEA IN PBS m 75~~9 SEA m IN NaHCOam
1 0 A
I 0.1
I 0.2
E L ISA
(Optical
GRANULOMA
NoHCO
I 0.3
I 0.4
Demity)
VOLUME
I 0.5
t SEM
IN MM’% lb
C-SEM
‘-
75~4 SEA m IN PBS w 75~ SEA w IN Nnt-KO,~ 750~~3 SEA w IN P0S m 750~ SEAIN NoHC03~ I 0 B
I 0.1 ELISA
I 0.2
I 0.3
1 0.4
I 0.5
(0pticalOensity)~SEM
FIG. 2. Varying dosesof SEA in PBS and NaHC03 were given orally on Day -28 and Day - 14 prior to iv tail vein injection of whole schistosomeeggs.Mean lung granuloma volumes in mm3 X lo-’ f SEM and antibody optical density + SEM were not statistically different from controls on Day 8 (A) or Day 16 (B) postinjection.
two exposures to 100 pg of SEA, while inducing some degree of sensitization for granuloma formation (Fig. 6) lead to high levels of anti-SEA antibody (Fig. 7). Multiple prior exposures to 31000 pg SEA (either as 2X 500 pg or 2X 1000 pg) induced significantly decreasedgranuloma formation and significantly elevated anti-SEA antibody levels compared to the positive SEA controls given a single injection of 500 pg SEA. Antibody levels in the highest dose SEA (1000 pg X2) group were significantly decreased
416
MC CURLEY ET AL.
m GRANULOMA 0 I
IO I
VOLUME
20 I
30 I
I I I .20 .I0 .30 ANTIBODY BY ELISA m
I 0 SERUM
INhdxlb33+SEM 40 I
50 I
60 I
I I 40 .50 (Opt~alDensity)
J .60 +SEM
70 I
FIG. 3. Single ip injections of SEA and reultracentrifuged SEA (from the upper f of the tube after lOO,OOOg/ 60 min) were given 1 week (Day -7) prior to tail vein egg administration (Day 0). Mean lung granuloma volumes and anti-SEA antibody by ELISA (serum diluted I: 100) were measured on Day 8.
relative to the 100 pg X2 SEA group. The granuloma reactivity of the 1000 pg X2 group is significantly decreasedrelative to all other SEA-exposed groups. Immunoelectrophoretic transfer blot analysis of the reactivity of pooled group sera against SEA antigenic components electrophoretically separatedby SDS gels revealed that fewer bands were recognized by the sera of all animals treated with SEA relative to 16-week chronically infected mice. Exposure to 1000 pg or more of SEA appeared to lead to production of antibodies against a major low-molecular-weight antigenic fraction of approximately 36,000 Da (Fig. 8). The antibody levels against most other components of SEA were low following high-dose exposure. DISCUSSION Immunologic tolerance to antigens has been classically induced by manipulation of the dose of the antigen, form of the antigen, and its route of administration. Multiple
MEAN 5
BSA IO
SPECIFIC
DTH
I5
20
IN Kj3CMkSEM 25
30
35
ELISA O.D. 0.53
I
0.640 0.312 0. I98 0.154
FIG. 4. Antibody levels by ELISA and ear thicknesspostchallengewith BSA in animals by shamthymectomy or with intrathymic saline or BSA.
417
TOLERANCE IN SCHISTOSOMIASIS MEAN 5 I
SEA SPECIFIC IO I
I5 ,
DTH 20 !
IN
10-3CM 5 SEM 25
30
3! 1
A
ELISA 0.D. 0.07 I 0. loo 0.208 0.223
0.275
FIG. 5. Antibody levels by ELBA and ear thickness postchallenge with SEA in animals treated by sham thymectomy or with intrathymic saline or SEA concentrations.
parenteral exposures to antigen at both low and high doseshave led to immunologic tolerance upon subsequent challenge ( 15, 16). Low dosesof antigen appear to induce tolerance by different mechanisms from that induced by high dosesof the sameantigen (17). Similarly, the use of monomeric forms of antigen as prepared by ultracentrifugation has been shown to facilitate the induction of tolerance ( 18, 19). Deliberate exposure to antigen via the gastrointestinal route is likewise an established means of producing systemic hyporesponsiveness to the same antigen when later administered in an immunogenic form (20, 21). Direct intrathymic injection of antigen has successfully induced tolerance in both rats (22-24) and mice (25).
MEAN 0 I
SEA 5cQqx
LUNG IO I
GRANULOMA 20 30 I I
VOLUME 40 I
IN MM3 x Id3 _+ SEM 50 60 70 I 1 I
00 I
I
FIG. 6. High dose tolerance. Mean lung granuloma volumes 8 days after tail vein egg injection (Day 0). SEA 500 pg X 1 is a positive sensitization control with SEA given at Day -7. Animals given 2 injections of SEA received 100,500, or 1000 pg SEA on Day -28 and Day - 14. *P < 0.05for starred groups compared to the SEA (500 pg X 1) control.
418
MC CURLEY ET AL.
0
I
ANTI 0.1
I
SEA ANTIBODY
0.2 -I
SEA
0.3 I
BY ELISA
0.4 I
@ticolDmsity)?
0.5 I
SEM
0.6 I
0.7 I
0.8 I
*
loopq12
FIG. 7. High dose tolerance. Anti-SEA antibody in the serum (1:500 dilution) detected by ELISA in the same groups as Fig. 6. *P i .05 for starred groups compared to SEA (500 pg Xl) control.
Many of these tolerogenic patternsof antigen presentation to the host could occur in chronic schistosomiasis.In human schistosomiasis,patients may well have exposure to SEA prior to their initial infections. Circulating schistosomal antigens are known to be present in schistosomiasis. They are found in serum, urine, and milk, and cross biological membranes in the kidney and lacteals (4, 5, 26). Some uninfected children born to mothers with chronic schistosomiasis have heightened skin test reactivity and demonstrable macrophage migration inhibition with schistosomal antigens relative to children born of uninfected mothers (27, 28). The effect of early exposure to antigen upon later infection is unknown though Gelfand has noted that the Katayama syn-
ABCDEF FIG. 8. Western blot showing reactivity of group sera with electrophoretically separated SEA antigenic components. Sera used to develop the separated SEA were obtained from infected mice or those injected with the materials stated below. (A) 16-weekchronically infected mouse, (B) PBS-injected control, (C) 500 pg Xl, (D) 100 pg SEA X2, (E) 500 pg SEA X2, (F) 1000 pg SEA X2. The positions of molecular weight standards are shown on the right.
TOLERANCE IN SCHISTOSOMIASIS
419
drome, a hypersensitivity reaction, occurs in schistosomeinfected Europeans in Africa but not in the indigenous African population (29). In mice, at least one study suggests that offspring of infected mothers have reduced delayed type hypersensitivity reactions to injected eggs compared to offspring of uninfected controls (30). Similarly, once infected, the host may be exposed to circulating schistosomal egg antigen which is biofiltered by liver (15). Furthermore, it is feasible that chronic intravascular antigen exposure could lead to a breach of the thymic/blood barrier. Whether these exposure patterns contribute to the establishment of the immunoregulation of cell-mediated responses (DTH and granuloma formation) to SEA seen in mice with 5’. mansoni (l-3) is unknown. Several studies have shown that oral feeding of antigen may lead to diminished humoral and cell-mediated responses to that antigen on subsequent challenge (20, 21). Many of these studies have been performed with relatively simple antigens such as TNCB or ovalbumin. Perrotto showed that in neonatal CFl mice, oral feeding of schistosome eggslead to augmented delayed type hypersensitivity responses(granuloma formation) on subsequent challenge with SEA (3 1). With adult mice, Perrotto found augmented DTH (granuloma formation on Day -8) responsesin animals given schistosome eggsin NaHC03, but not in animals given SEA or eggsin PBS. Recently, Weinstock has shown that direct injection of 25,000 eggsinto the cecal pouch of mice infected for 4 weeks leads to modulation of hepatic granuloma formation which may be adoptively transferred to infected animals (32). With intragastrically administered SEA in either PBS and NaHC03 vehicles or eggsgiven orally in PBS, we were unable to seeeither increased or diminished responses(DTH and humoral) to later challenge with parenterally administered eggs.No differences were detected at either Day -8 or Day - 16 following challenge. In the 1960sDresser demonstrated that intraperitoneal injection of small quantities of deaggregatedbovine -y-globulin produced specific inhibition of antibody production in adult CBA mice on subsequent challenge ( 18). This unresponsiveness was specific and was observed only with the ultracentrifuged fraction of the antigen. We wondered if ultracentrifuged preparations of SEA would produce tolerance to subsequent challenge with immunogenic forms of SEA. We studied the effect of multiple exposures to ultracentrifuged SEA at a variety of doses.Subsequent challenge with intravenously injected eggsdemonstrated similar humoral and DTH responsesin all groups of animals. Preexposure to deaggregatedpreparations of SEA did not influence later responsiveness. Direct intrathymic injection of antigen is a relatively unique method to induce specific tolerance (22-25). However, it was considered that long-term, intravascular exposure to eggsand SEA might lead to crossing of the thymic/blood barrier during chronic intravascular infection. Our studies with BSA provide a positive control for this intrathymic form of induction of humoral and cell-mediated tolerance. However, parallel regimens using SEA failed to develop tolerance to SEA. It is possible that the antigenic heterogeneity of crude SEA is such that critical concentrations of any given component are not achieved in the amounts available. Hang et al., demonstrated hyporesponsiveness to granuloma formation in both adult and neonatal mice treated with large doses of SEA (6). Similar treatment with lower doses resulted in sensitization to granuloma formation in both groups. In our studies high dosesof SEA (2 1000 pg) also resulted in a significant reduction in DTH as measured by lung granuloma diameters. Lower dosesproduced consistent sensiti-
420
MC CURLEY ET AL.
zation similar to that of positive controls. Paradoxically, these reduced cell-mediated reactions following high doses of SEA were seen in animals with significantly higher levels of antibody relative to the positive control. Western Blot analysis of this altered antibody response showed that antibody was directed predominantly against lowermolecular-weight components of SEA with a pattern similar to, but more intense than that of positive controls. We attempted to elicit tolerance to schistosomal antigens in mice by patterns of SEA administration paralleling potential natural exposures. We were unsuccessful in inducing tolerance with oral or intrathymic introduction of SEA, or with parenteral deaggregatedforms of the antigen. However, tolerance in terms of delayed-type hypersensitivity was successfully induced with high dosesof SEA. Interestingly, antibody levels were high in these animals suggesting a divergence of anti-SEA humoral and DTH responses. This observation generally reflects the situation observed during chronic murine infection (3). Our studies suggestthat with complex, poorly defined antigens such as SEA, many of the classic methods of eliciting immunologic tolerance fail. This inability may not accurately reflect the situations that occur during chronic infections in endemic populations. Certainly our ability to reproduce the conditions, duration, amount and presentation of SEA exposure may not parallel those of chronically infected mice or patient populations. These studies indicate that certain approaches to the study of SEA-induced tolerance will be experimentally difficult. This may well rest on the current complexity of the highly heterogeneousmaterials (8) termed SEA. Later studies using purified antigens or antigens produced by recombinant DNA technology may alleviate these problems. As previously reported (6), upon use of extraordinary amounts of SEA, immunomanipulation can yield exogenously induced granuloma modulation or tolerance/immunoregulation. The biologic significance of the form of tolerance inducible by high doses of SEA remains to be defined. REFERENCES 1. Andrade, Z. A., and Warren, K. S., Trans. Roy. Sot. Trop. Med. Hyg. S&53, 1964. 2. Domingo, E. O., and Warren, K. S., Amer. J. Pathol. 52, 369, 1968. 3. Colley, D. G., J. Zmmunol. 115, 150, 1975. 4. Deedler, A. M., KIappc, H. T. M., and Van Den Aardweg, G. J. M. J., Exp. Parasitol. 40,435, 1977. 5. Santoro, F., Carher, Y., Borojevic, R., Bout, D., Tachon, P., and Chapron, A., Ann. Trop. Med. Parasitol. 71, 121, 1977. 6. Hang, L. M., Boros, D. L., and Warren, K. S., J. Infect. Dis. 130, 515, 1974. 7. Bores, D. L., and Warren, K. S., J. Exp. Med. 132,488, 1970. 8. Carter, C. E., and Colley, D. G., J. Parasitol. 64,385, 1978. 9. Lowry, 0. H., Rosebrough, N. J., Farr, L., and Randall, R. J., J. Biol. Chem. 193,265, 1951. 10. Little, J., Carter, C. E., and Colley, D. G., J. Parasitol. 68, 5 19, 1982. 11. Colley, D. G., J. Exp. Med. 43, 696, 1976. 12. Tsang, V. C. W., Peralta, J. M., and Simons, A. R., In “Methods in Enzymology” (J. J. Langone and H. Van Vunakis, Eds.), Vol. 92, p. 377. Academic Press,Orlando, FIa., 1983. 13. Titus, R. G., and Chiller, J. M., J. Zmmunol. Methods 45, 65, 1981. 14. Colley, D. G., Proc. Sac. Exp. Biol. Med. 140,772, 1972. 15. Battisto, J. R., and Miller, J., Proc. Sot. Exp. Biol. Med. 111, 111, 1962. 16. Dixon, F. J., and Maurer, P. H., .Z.Exp. Med. 101,245, 1955. 17. Green, D. R., Hood, P. M., and Gershon, R. K., In “Annual Review of Immunology” (Paul, W. E., Fatbman, C. G., and Metzger, H. Eds.), pp. 439-464, Annual Reviews, Inc., Palo Alto, 1983. 18. Dresser, D. W., Zmmunology5,378, 1962. 19. Gamble, C. N., Znt. Arch. Allergy 30, 446, 1966.
TOLERANCE IN SCHISTOSOMIASIS 20. 21. 22. 23. 24. 25. 26.
421
Tomasi, T. B., Transplantation 29, 353, 1980. Stokes, C. R., and Swarbick, E. T., Biochem. Sot. Trans. 5, 1573, 1977. Horiuchi, A., and Waksman, B. H., J. Immunol. 101, 1322, 1968. Horiuchi, A., and Waksman, B. H., J. Immunol. 100,914, 1968. Waksman, B. H., Clin. Exp. Immunol. 28, 363, 1977. Shim& M., Kimura, T., Kakinuma, M., and G’Hara, T., J. Immunol. Methods 31,41, 1979. Carlier, Y., Bout, D., Bina, J. C., Camus, D., Figueiredo, J. F. M., and Capron, A., Amer. J. Trop. Med. Hyg. 24, 949, 1975. 27. Camus, D., Carlier, Y., Bina, J. C., Borojevic, R., Prata, A., and Capron, A., J. Infect. Dis. 134, 405, 1976. 28. Tachon, P., and Borojevic, R., Trans. R. Sot. Trop. Med. Hyg. 72,605, 1978. 29. Gelfand, M., In “Bilharziasis” (F. K. Mostofi, Ed.), p. 104-I 15, Springer Verlag, New York, 1967. 30. Lewert, R. M., and Mandlowitz, S., Nature (London) 224, 1030, 1969. 31. Perrotto, J. L., Hang, L. M., Iselbacher, K. J., and Warren, K. S., J. Exp. Med. 140, 296, 1974. 32. Weinstock, J. V., Blum, A. M., and Kassab, J. T., J. Immunol. 135, 560, 1985.
IMMUNOLOGY
99,4
I l-42 1 ( 1986)
Studies of Tolerance in Schistosomiasis’ T.L.McCURLEY,T.
ABE,' C.E.CARTER, ANDD.G.COLLEY~
Departments of Pathology, Microbiology, and Biology, Vanderbilt University School of Medicine and the VeteransAdministration Medical Center, Nashville, Tennessee37203 Received October 11, 1985; acceptedJanuary 9, 1985 We have studied the induction of immune tolerance by exogenous exposure to schistosomal egg antigen (SEA). We compared routes of administration, exposure to different forms of SEA (eggs,normal SEA, reultracentrifuged SEA), and treatment schedules.Tolerance was evaluated in comparison to SEA sensitization, after challenge with SEA or eggs.Anti-SEA responseswere measured by ELISA assays,and cell-mediated responsivenessby 24-hr skin tests and pulmonary gmnuloma formation 8 and 16 days after intravenous egginjection. Oral administration of either SEA in PBSor NaHCOs, or eggsin PBSdid not induce toleranceor sensitization.Reuhracentrifuged SEA was similar to SEA in inducing cell-mediated immune response. Intrathymic exposure to SEA either had no effect or sensitized the animal for subsequent exposure to SEA. Two intraperitoneal injections of0.5 to 1.Omg of SEA lead to reduced cell-mediated responses,but resulted in high levels of anti-SEA antibody, suggesting a state of split tolerance or immune deviation. Our studies suggestthat with complex, poorly defined antigens such as SEA, many of the classic methods of eliciting tolerance fail. The biologic significance of the form of tolerance inducible by high dosesof SEA remains to be defined. 0 1986 Academic press, hc.
INTRODUCTION Mice infected with the human helminth Schistosomamansoni develop, and then progressively immunoregulate, cell-mediated responses(delayed type hypersensitivity and granuloma formation) to soluble schistosomal eggantigens (SEA) (l-3). In human schistosomiasis, patients may well have had exposure to SEA prior to their initial infections. This could have occurred systemically in utero, or orally through breast milk (4, 5). Upon infection, there may be continuous systematic exposure to high doses of SEA antigens that have been “biofiltered” through the portal system. The effects,if any, of such early or uncommon chronic exposure patterns to these antigens on subsequent responsesto SEA remain unclear. Similarly to Hang et al. (6) we have studied the induction of immune tolerance by exogenous exposure to SEA. We compared routes of administration, exposure to dif’ This work was supported by Grant AI-l 1289 from the National Institutes of Health and a grant from the Edna McConnell Clark Foundation, and the Veterans Administration. * Present address:Department of Parasitology, Miyazaki Medical College, Kiyotake, Miyazaki-gun, Miyazuki Pref., 889-16, Japan. 3To whom correspondence should be addressed:F323 Veterans Administration Medical Center, 1310 24th Avenue South, Nashville, Tenn. 37203. 411 0008-8749/86 $3.00 Copyrieht 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
412
MC CURLEY ET AL.
ferent forms of SEA (eggs,normal SEA, reultracentrifuged SEA), and treatment schedules. Tolerance was evaluated in comparison to SEA sensitization, after challenge with SEA or eggs.Anti-SEA humoral responseswere measured by ELISA assays,and cellmediated responsivenesswas assayedby 24-hr skin tests and pulmonary granuloma formation 8 and 16 days after intravenous egg injections. MATERIALS
AND METHODS
Animals Either adult (8-week-old) male CFl mice (Harlan Laboratories, Indianapolis, Ind.) or adult male CBA/J mice supplied by the National Cancer Institute Supply Contract (IGA VlOl (134) P-77014) were used in all experiments.
Preparation of SchistosomalEggs and SchistosomalEgg Antigen Eggsfor tail vein injection and oral feeding were harvested from the livers of mice 7-9 weeks after infection with cercariae of a Puerto Rican strain of S. mansoni. Eggs were kept in chilled hypertonic saline until immediately prior to injection. Three thousand to 5000 eggs in PBS were used for tail vein injection. Schistosomal egg antigenic preparation (SEA) was prepared by homogenization of similarly obtained eggsand ultracentrifugation of the resulting extract by methods previously described (7, 8). Protein determinations were performed by the Lowry method (9).
Serum Antibody by ELISA Mice were bled and serum was harvested at the time of sacrifice and stored at -70°C. Anti-SEA antibody was measured by an enzyme-linked immunosorbent assay (ELISA) at serum dilutions ranging from 1:100 to 1:400using a /3-galactosidasemethod on microtiter plates as previously described (10).
Lung Granuloma Measurement Eight or 16 days after a tail vein injection of schistosome eggs,the lungs of animals were perfused with 10% buffered Formalin. Tissues were processedand stained with hematoxylin and eosin for histopathologic analysis. Granulomas with central schistosome eggswere located sequentially and diameters were measured across opposing axes using an ocular micrometer. Based upon the generally spherical nature of the schistosome egg granuloma, the mean diameter and volume of each granuloma were calculated. Twenty-five granulomas from each mouse were measured to yield the mean granuloma volume for each mouse. Group means were determined on these values and compared statistically by Student’s t test (1 I).
Antibody Characterization by WesternBlot Analysis SDS gel electrophoretically separated SEA components were transferred to nitrocellulose paper and incubated with sera from animals that had been infected with S. mansoni for 16 weeks, or animals given PBS or various dosesof SEA intraperitonally (ip). The sites of antibody binding were detected with goat anti-mouse IgG coupled with horseradish peroxidase followed by incubation with diaminobenzidine (12).
TOLERANCE IN SCHISTOSOMIASIS
413
Experimental Protocols Oral exposure to whole S. mansoni eggs. Twenty thousand intact S. mansoni eggs were given by intragastric intubation to 8-week male CBA/J mice in 0.5 cc PBS 2 weeks (Day - 14) prior to tail vein injection of eggs(Day 0). Animals were sacrificed at Day 8 and Day 16 after egg injection. Sera were harvested for measurements of anti-SEA antibody and lungs were taken for granuloma measurement. Oral exposure to schistosomal egg antigens (SEA). CFl mice were given (a) 0.5 cc PBS; (b) 0.5 cc of 150 pg SEA/cc in PBS; (c) 0.5 cc of 1500 pg SEA/cc in PBS; (d) 0.5 cc 1.3%NaHC03; (e) 0.5 of 15OpgSEA/cc in 1.3%NaHCO,; or (f) 0.5 cc of 1500 pg SEA/cc in 1.3% NaHC03, by intragastric intubation, on Day -28 and Day - 14. On Day 0 eggswere injected intravenously. Lungs and sera were harvested on Day 8 and Day 16. Reultracentrifuged SEA CFl mice were given single ip injections of SEA or reultracentrifuged SEA 1 week (Day -7) prior to tail vein injection of eggs(Day 0). Reultracentrifuged SEA consisted of SEA taken from the upper third of a tube after centrifugation at 100,OOOgfor 60 min immediately before use. Animals were sacrificed on Day 8 and lungs and sera were obtained for measurement of mean granuloma volumes and anti-SEA antibody. Intrathymic Injection of Bovine Serum Albumin (BSA) or SEA CBA/J mice were either injected intrathymically ( 10 &lobe) with saline, or a total of 10, 50, or 250 Kg of BSA, or the mediastinal area surgically exposed in parallel but left uninjected (sham control). Three weeks later each mouse was immunized at the base of the tail (13) with a 50 ~1 volume of 100 pg BSA emulsified in Complete Freund’s adjuvant. Eight days later the mice were exposed to 500 pg BSA by footpad, rested 6 days and skin test challenged (14) with 30 ~1 of a 2% suspension of heataggregatedBSA (13). Ear thickness was measured with an engineer’s micrometer at 5 and 24 hr after challenge. Sera were collected after the 24-hr skin test readings. The effectsof intrathymic exposure to SEA were studied similarly. Thymic injections ranged from 25 to 80 pg of SEA and sensitizations at the base of the tail were with only one exposure to 25 pg in CFA. Ear injection challenges were with 15 pg/injection versus saline injections in the contralateral ear. Sera were collected after the 24-hr measurements. High Dose and Low Dose SEA Mean lung granuloma volumes and anti-SEA antibody were measured 8 days after tail vein egginjection (Day 0). PBS or 500 pg SEA were given ip on Day -7 as negative and positive sensitization controls, respectively. Animals given two injections of SEA received 100, 500, or 1000 rg of SEA on Day -28 and Day -14. Animals were sacrificed and lungs and sem harvested on Day 8. RESULTS Oral Exposure to Schistosome Eggs Mean lung granuloma volumes in mm3 X 1O-3 f SEM and serum antibody by ELISA (optical density) +- SEM were compared on Day -8 and Day -16 following
414
MC CURLEY ET AL.
intravenous tail vein injection of eggs(Day -0) in animals previously given eggsorally on Day - 14. Mean lung granuloma volumes and antibody levels by ELISA did not differ statistically between experimental groups and controls given PBS whether the assayswere 8 or 16 days after challenge (Fig. 1). This oral regimen of eggs neither sensitized nor tolerized mice to SEA.
Oral Exposure to SEA Mean lung granuloma volumes and serum antibody levels were compared in animals given SEA intragastrically on Day -28 and Day -14. There were no statistically significant differences in granuloma volumes or anti-SEA antibody levels on Day 8 or Day 16 with either low or high dose SEA. The results were similar using either PBS or WaHC03 as the vehicle (Fig. 2A and B).
ReultracentrijugedSEA SEA and reultracentrifuged preparations of SEA produced similar responses as measured by lung granuloma volumes levels. Both materials caused sensitization. Preexposure to 50 E.cg doses of reultracentrifuged SEA did occasionally lead to lower antibody responsesas measured by ELISA (Fig. 3). However, these differences were not observed consistently.
Intrathymic Exposure to BSA or SEA Intrathymic injection of lo-250 pg of BSA induced effective tolerance as seen by anti-BSA serum antibody levels and DTH reactions (Fig. 4). Sham thymic exposure or intrathymic saline injection had no effect on BSA sensitization. In three experiments (Fig. 5), it was seenthat intrathymic exposure to SEA either had no effect or sensitized for subsequent parenteral exposure to SEA. This was true of both serum antibody and DTH responsiveness.
High Dose SEA A single exposure to 500 pg of SEA induced sensitization for secondary granuloma formation at 8 days (Fig. 6), but did not lead to antibody formation (Fig. 7). In contrast,
m GRANULOMA VOLUME IN MM% d%SEM 20 DAY BPBS
?&
DAY 16PBS
w--+
30 I
’
DAYl6CkWEGGS~ I I I I 0.3 0 0.1 0.2 SERUM ANTlBODY BY ELISA (Optical Densityk SEM Fsssl FIG. 1. Mice were given either PBS or 20,000 intact S. mansoni eggsin PBS orally 2 weeks (Day -14) prior to tail vein injection of eggs(Day 0). Mean lung granuloma volumes and anti-SEA antibody by ELISA (serum diluted 1:lOO)were compared. Animals were sacrificed at Day 8 and Day 16 after egg injections. Neither sensitization nor tolerance induction was observed following this oral regimen of eggs.
TOLERANCE IN SCHISTOSOMIASIS GRANULOMA
VOLUME
415
IN MM’xl&SEM
75~19 SEA IN PBS m 75~~9 SEA m IN NaHCOam
1 0 A
I 0.1
I 0.2
E L ISA
(Optical
GRANULOMA
NoHCO
I 0.3
I 0.4
Demity)
VOLUME
I 0.5
t SEM
IN MM’% lb
C-SEM
‘-
75~4 SEA m IN PBS w 75~ SEA w IN Nnt-KO,~ 750~~3 SEA w IN P0S m 750~ SEAIN NoHC03~ I 0 B
I 0.1 ELISA
I 0.2
I 0.3
1 0.4
I 0.5
(0pticalOensity)~SEM
FIG. 2. Varying dosesof SEA in PBS and NaHC03 were given orally on Day -28 and Day - 14 prior to iv tail vein injection of whole schistosomeeggs.Mean lung granuloma volumes in mm3 X lo-’ f SEM and antibody optical density + SEM were not statistically different from controls on Day 8 (A) or Day 16 (B) postinjection.
two exposures to 100 pg of SEA, while inducing some degree of sensitization for granuloma formation (Fig. 6) lead to high levels of anti-SEA antibody (Fig. 7). Multiple prior exposures to 31000 pg SEA (either as 2X 500 pg or 2X 1000 pg) induced significantly decreasedgranuloma formation and significantly elevated anti-SEA antibody levels compared to the positive SEA controls given a single injection of 500 pg SEA. Antibody levels in the highest dose SEA (1000 pg X2) group were significantly decreased
416
MC CURLEY ET AL.
m GRANULOMA 0 I
IO I
VOLUME
20 I
30 I
I I I .20 .I0 .30 ANTIBODY BY ELISA m
I 0 SERUM
INhdxlb33+SEM 40 I
50 I
60 I
I I 40 .50 (Opt~alDensity)
J .60 +SEM
70 I
FIG. 3. Single ip injections of SEA and reultracentrifuged SEA (from the upper f of the tube after lOO,OOOg/ 60 min) were given 1 week (Day -7) prior to tail vein egg administration (Day 0). Mean lung granuloma volumes and anti-SEA antibody by ELISA (serum diluted I: 100) were measured on Day 8.
relative to the 100 pg X2 SEA group. The granuloma reactivity of the 1000 pg X2 group is significantly decreasedrelative to all other SEA-exposed groups. Immunoelectrophoretic transfer blot analysis of the reactivity of pooled group sera against SEA antigenic components electrophoretically separatedby SDS gels revealed that fewer bands were recognized by the sera of all animals treated with SEA relative to 16-week chronically infected mice. Exposure to 1000 pg or more of SEA appeared to lead to production of antibodies against a major low-molecular-weight antigenic fraction of approximately 36,000 Da (Fig. 8). The antibody levels against most other components of SEA were low following high-dose exposure. DISCUSSION Immunologic tolerance to antigens has been classically induced by manipulation of the dose of the antigen, form of the antigen, and its route of administration. Multiple
MEAN 5
BSA IO
SPECIFIC
DTH
I5
20
IN Kj3CMkSEM 25
30
35
ELISA O.D. 0.53
I
0.640 0.312 0. I98 0.154
FIG. 4. Antibody levels by ELISA and ear thicknesspostchallengewith BSA in animals by shamthymectomy or with intrathymic saline or BSA.
417
TOLERANCE IN SCHISTOSOMIASIS MEAN 5 I
SEA SPECIFIC IO I
I5 ,
DTH 20 !
IN
10-3CM 5 SEM 25
30
3! 1
A
ELISA 0.D. 0.07 I 0. loo 0.208 0.223
0.275
FIG. 5. Antibody levels by ELBA and ear thickness postchallenge with SEA in animals treated by sham thymectomy or with intrathymic saline or SEA concentrations.
parenteral exposures to antigen at both low and high doseshave led to immunologic tolerance upon subsequent challenge ( 15, 16). Low dosesof antigen appear to induce tolerance by different mechanisms from that induced by high dosesof the sameantigen (17). Similarly, the use of monomeric forms of antigen as prepared by ultracentrifugation has been shown to facilitate the induction of tolerance ( 18, 19). Deliberate exposure to antigen via the gastrointestinal route is likewise an established means of producing systemic hyporesponsiveness to the same antigen when later administered in an immunogenic form (20, 21). Direct intrathymic injection of antigen has successfully induced tolerance in both rats (22-24) and mice (25).
MEAN 0 I
SEA 5cQqx
LUNG IO I
GRANULOMA 20 30 I I
VOLUME 40 I
IN MM3 x Id3 _+ SEM 50 60 70 I 1 I
00 I
I
FIG. 6. High dose tolerance. Mean lung granuloma volumes 8 days after tail vein egg injection (Day 0). SEA 500 pg X 1 is a positive sensitization control with SEA given at Day -7. Animals given 2 injections of SEA received 100,500, or 1000 pg SEA on Day -28 and Day - 14. *P < 0.05for starred groups compared to the SEA (500 pg X 1) control.
418
MC CURLEY ET AL.
0
I
ANTI 0.1
I
SEA ANTIBODY
0.2 -I
SEA
0.3 I
BY ELISA
0.4 I
@ticolDmsity)?
0.5 I
SEM
0.6 I
0.7 I
0.8 I
*
loopq12
FIG. 7. High dose tolerance. Anti-SEA antibody in the serum (1:500 dilution) detected by ELISA in the same groups as Fig. 6. *P i .05 for starred groups compared to SEA (500 pg Xl) control.
Many of these tolerogenic patternsof antigen presentation to the host could occur in chronic schistosomiasis.In human schistosomiasis,patients may well have exposure to SEA prior to their initial infections. Circulating schistosomal antigens are known to be present in schistosomiasis. They are found in serum, urine, and milk, and cross biological membranes in the kidney and lacteals (4, 5, 26). Some uninfected children born to mothers with chronic schistosomiasis have heightened skin test reactivity and demonstrable macrophage migration inhibition with schistosomal antigens relative to children born of uninfected mothers (27, 28). The effect of early exposure to antigen upon later infection is unknown though Gelfand has noted that the Katayama syn-
ABCDEF FIG. 8. Western blot showing reactivity of group sera with electrophoretically separated SEA antigenic components. Sera used to develop the separated SEA were obtained from infected mice or those injected with the materials stated below. (A) 16-weekchronically infected mouse, (B) PBS-injected control, (C) 500 pg Xl, (D) 100 pg SEA X2, (E) 500 pg SEA X2, (F) 1000 pg SEA X2. The positions of molecular weight standards are shown on the right.
TOLERANCE IN SCHISTOSOMIASIS
419
drome, a hypersensitivity reaction, occurs in schistosomeinfected Europeans in Africa but not in the indigenous African population (29). In mice, at least one study suggests that offspring of infected mothers have reduced delayed type hypersensitivity reactions to injected eggs compared to offspring of uninfected controls (30). Similarly, once infected, the host may be exposed to circulating schistosomal egg antigen which is biofiltered by liver (15). Furthermore, it is feasible that chronic intravascular antigen exposure could lead to a breach of the thymic/blood barrier. Whether these exposure patterns contribute to the establishment of the immunoregulation of cell-mediated responses (DTH and granuloma formation) to SEA seen in mice with 5’. mansoni (l-3) is unknown. Several studies have shown that oral feeding of antigen may lead to diminished humoral and cell-mediated responses to that antigen on subsequent challenge (20, 21). Many of these studies have been performed with relatively simple antigens such as TNCB or ovalbumin. Perrotto showed that in neonatal CFl mice, oral feeding of schistosome eggslead to augmented delayed type hypersensitivity responses(granuloma formation) on subsequent challenge with SEA (3 1). With adult mice, Perrotto found augmented DTH (granuloma formation on Day -8) responsesin animals given schistosome eggsin NaHC03, but not in animals given SEA or eggsin PBS. Recently, Weinstock has shown that direct injection of 25,000 eggsinto the cecal pouch of mice infected for 4 weeks leads to modulation of hepatic granuloma formation which may be adoptively transferred to infected animals (32). With intragastrically administered SEA in either PBS and NaHC03 vehicles or eggsgiven orally in PBS, we were unable to seeeither increased or diminished responses(DTH and humoral) to later challenge with parenterally administered eggs.No differences were detected at either Day -8 or Day - 16 following challenge. In the 1960sDresser demonstrated that intraperitoneal injection of small quantities of deaggregatedbovine -y-globulin produced specific inhibition of antibody production in adult CBA mice on subsequent challenge ( 18). This unresponsiveness was specific and was observed only with the ultracentrifuged fraction of the antigen. We wondered if ultracentrifuged preparations of SEA would produce tolerance to subsequent challenge with immunogenic forms of SEA. We studied the effect of multiple exposures to ultracentrifuged SEA at a variety of doses.Subsequent challenge with intravenously injected eggsdemonstrated similar humoral and DTH responsesin all groups of animals. Preexposure to deaggregatedpreparations of SEA did not influence later responsiveness. Direct intrathymic injection of antigen is a relatively unique method to induce specific tolerance (22-25). However, it was considered that long-term, intravascular exposure to eggsand SEA might lead to crossing of the thymic/blood barrier during chronic intravascular infection. Our studies with BSA provide a positive control for this intrathymic form of induction of humoral and cell-mediated tolerance. However, parallel regimens using SEA failed to develop tolerance to SEA. It is possible that the antigenic heterogeneity of crude SEA is such that critical concentrations of any given component are not achieved in the amounts available. Hang et al., demonstrated hyporesponsiveness to granuloma formation in both adult and neonatal mice treated with large doses of SEA (6). Similar treatment with lower doses resulted in sensitization to granuloma formation in both groups. In our studies high dosesof SEA (2 1000 pg) also resulted in a significant reduction in DTH as measured by lung granuloma diameters. Lower dosesproduced consistent sensiti-
420
MC CURLEY ET AL.
zation similar to that of positive controls. Paradoxically, these reduced cell-mediated reactions following high doses of SEA were seen in animals with significantly higher levels of antibody relative to the positive control. Western Blot analysis of this altered antibody response showed that antibody was directed predominantly against lowermolecular-weight components of SEA with a pattern similar to, but more intense than that of positive controls. We attempted to elicit tolerance to schistosomal antigens in mice by patterns of SEA administration paralleling potential natural exposures. We were unsuccessful in inducing tolerance with oral or intrathymic introduction of SEA, or with parenteral deaggregatedforms of the antigen. However, tolerance in terms of delayed-type hypersensitivity was successfully induced with high dosesof SEA. Interestingly, antibody levels were high in these animals suggesting a divergence of anti-SEA humoral and DTH responses. This observation generally reflects the situation observed during chronic murine infection (3). Our studies suggestthat with complex, poorly defined antigens such as SEA, many of the classic methods of eliciting immunologic tolerance fail. This inability may not accurately reflect the situations that occur during chronic infections in endemic populations. Certainly our ability to reproduce the conditions, duration, amount and presentation of SEA exposure may not parallel those of chronically infected mice or patient populations. These studies indicate that certain approaches to the study of SEA-induced tolerance will be experimentally difficult. This may well rest on the current complexity of the highly heterogeneousmaterials (8) termed SEA. Later studies using purified antigens or antigens produced by recombinant DNA technology may alleviate these problems. As previously reported (6), upon use of extraordinary amounts of SEA, immunomanipulation can yield exogenously induced granuloma modulation or tolerance/immunoregulation. The biologic significance of the form of tolerance inducible by high doses of SEA remains to be defined. REFERENCES 1. Andrade, Z. A., and Warren, K. S., Trans. Roy. Sot. Trop. Med. Hyg. S&53, 1964. 2. Domingo, E. O., and Warren, K. S., Amer. J. Pathol. 52, 369, 1968. 3. Colley, D. G., J. Zmmunol. 115, 150, 1975. 4. Deedler, A. M., KIappc, H. T. M., and Van Den Aardweg, G. J. M. J., Exp. Parasitol. 40,435, 1977. 5. Santoro, F., Carher, Y., Borojevic, R., Bout, D., Tachon, P., and Chapron, A., Ann. Trop. Med. Parasitol. 71, 121, 1977. 6. Hang, L. M., Boros, D. L., and Warren, K. S., J. Infect. Dis. 130, 515, 1974. 7. Bores, D. L., and Warren, K. S., J. Exp. Med. 132,488, 1970. 8. Carter, C. E., and Colley, D. G., J. Parasitol. 64,385, 1978. 9. Lowry, 0. H., Rosebrough, N. J., Farr, L., and Randall, R. J., J. Biol. Chem. 193,265, 1951. 10. Little, J., Carter, C. E., and Colley, D. G., J. Parasitol. 68, 5 19, 1982. 11. Colley, D. G., J. Exp. Med. 43, 696, 1976. 12. Tsang, V. C. W., Peralta, J. M., and Simons, A. R., In “Methods in Enzymology” (J. J. Langone and H. Van Vunakis, Eds.), Vol. 92, p. 377. Academic Press,Orlando, FIa., 1983. 13. Titus, R. G., and Chiller, J. M., J. Zmmunol. Methods 45, 65, 1981. 14. Colley, D. G., Proc. Sac. Exp. Biol. Med. 140,772, 1972. 15. Battisto, J. R., and Miller, J., Proc. Sot. Exp. Biol. Med. 111, 111, 1962. 16. Dixon, F. J., and Maurer, P. H., .Z.Exp. Med. 101,245, 1955. 17. Green, D. R., Hood, P. M., and Gershon, R. K., In “Annual Review of Immunology” (Paul, W. E., Fatbman, C. G., and Metzger, H. Eds.), pp. 439-464, Annual Reviews, Inc., Palo Alto, 1983. 18. Dresser, D. W., Zmmunology5,378, 1962. 19. Gamble, C. N., Znt. Arch. Allergy 30, 446, 1966.
TOLERANCE IN SCHISTOSOMIASIS 20. 21. 22. 23. 24. 25. 26.
421
Tomasi, T. B., Transplantation 29, 353, 1980. Stokes, C. R., and Swarbick, E. T., Biochem. Sot. Trans. 5, 1573, 1977. Horiuchi, A., and Waksman, B. H., J. Immunol. 101, 1322, 1968. Horiuchi, A., and Waksman, B. H., J. Immunol. 100,914, 1968. Waksman, B. H., Clin. Exp. Immunol. 28, 363, 1977. Shim& M., Kimura, T., Kakinuma, M., and G’Hara, T., J. Immunol. Methods 31,41, 1979. Carlier, Y., Bout, D., Bina, J. C., Camus, D., Figueiredo, J. F. M., and Capron, A., Amer. J. Trop. Med. Hyg. 24, 949, 1975. 27. Camus, D., Carlier, Y., Bina, J. C., Borojevic, R., Prata, A., and Capron, A., J. Infect. Dis. 134, 405, 1976. 28. Tachon, P., and Borojevic, R., Trans. R. Sot. Trop. Med. Hyg. 72,605, 1978. 29. Gelfand, M., In “Bilharziasis” (F. K. Mostofi, Ed.), p. 104-I 15, Springer Verlag, New York, 1967. 30. Lewert, R. M., and Mandlowitz, S., Nature (London) 224, 1030, 1969. 31. Perrotto, J. L., Hang, L. M., Iselbacher, K. J., and Warren, K. S., J. Exp. Med. 140, 296, 1974. 32. Weinstock, J. V., Blum, A. M., and Kassab, J. T., J. Immunol. 135, 560, 1985.