CELLULARIMMUNOLOGY17, 306--309 (1975)
Selective Immunodepression in Mice by Trichinella spiralis Extracts and Infections O ~ A R O . BARRIGA
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174
Received December 12, 1974 C57BL/6J mice pretreated with saline, Trichinella spiraIis extract, or bovine serum albumin, or infected with T. spiralis were immunized with either polyvinylpyrrolidone (PVP) or sheep red blood cells (SRBC). Hemagglutinating anti-PVP titers were similar in all groups but anti-SRBC titers were markedly lower in infected mice, and were undeteetable in extraet-pretreated mice. These results suggest that only the activity of T Iymphocytes was affected by the infection and the inoculation of extract. Circumstantial evidence suggests that the immunodepression was independent of phagocytic activity and different from that achieved by pretreatment with anti-lymphocyte serum. INTRODUCTION
Trichinella spiralis infections in mice interfere with both cell-mediated and humoral immune responses to unrelated antigens ( 1 - 3 ) , but whether this immunodepression is due to antigenic competition, a physiological deterioration of diseased animals, or a specific effect on the immune system remains unknown. The experiment reported here shows that an extract of T. spiralis possesses immunodepressive properties and suggests that the immunodepression is restricted to the T-cell activity.
MATERIALS AND METHODS Eighty C57BL/6J female mice, 6-wk old, were divided into 4 groups of 20 and were treated as follows: group N, serving as normal control, received 7 daily intraperitoneal injections of 0.15 M NaC1; group Ts received 40 viable T. splralis larvae per mouse orally ; group Ag and groups B S A received 7 daily intraperitoneal inoculations of T. spiralis larval extract containing 0.2 mg of protei n or 0.2 mg of five times crystallized bovine serum albumin (BSA, Pentex, Kankakee, Illinois), respectively. The larval extract was prepared by high speed homogenization of clean larvae followed by centrifugation at 12,000g for 30 min at 2°C. Protein estimations were done by the Biuret technique. Each group was then divided into two subgroups of ten mice; one subgroup was immunized intraperitoneally with 10 r sheep red blood cells ( S R B C ) and the other subgroup was injected intraperitoneally with 0.5 >g of polyvinylpyrrolidone K-90 ( P V P K-90; av M W 360,000 daltons, Matheson Coleman and Bell, Nor306 Copyright ~) 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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256" 128" 64' 32" 16"
!i~il 8" 4' 7"
Ts
k[
BSA
MICE
FIG. l. Anti-sheep red blood cell (SRBC) titers of mice infected with Trichlnella spiralis (Ts) or inoculated with saline (N), T. spiralls extract (Ag), or bovine serum albumin (BSA) and immunized subsequently with SRBC. wood, Ohio). Immunizations were performed on the seventh day of infection and on day 1 after the injections of saline solution, T. spira.lis extract and BSA. All animals were killed and bled on the eighth day after immunization with SRBC and P V P . The sera from each subgroup were pooled and examined repeatedly by direct hemagglutination ( H A ) with SRBC and by indirect hemagglutination ( I H A ) with erythrocytes coated with P V P K-15 (av M W 10,000 daltons) by treatment with tannic acid (4). All sera were inactivated at 56°C for 30 rain and those utilized for I H A were also absorbed with SRBC. RESULTS The serum titers of SRBC-immunized mice, when reacted with SRBC, are presented in Fig. 1. IJnabsorbed pooled sera of the PVP-immunized mice were used as negative controls; only one group showed weak hemagglutination at 1:2 dilution. The serum titers of PVP-immunized mice when reacted with the homologous antigen are presented in Fig. 2. The specificity of the reaction was determined by I H A inhibition with soluble antigen. Pooled sera of the SRBC-immunized mice, used as negative controls, only showed positive I H A to 1:4 dilution. Both 2561286432..........
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FIG. 2. An ti-polyvinylpyrrolidone (PVP) titers of mice infected with Trichinella spiralis (Ts) or inoculated with saline (N), T. spiralis extract (Ag), or bovine serum albumin (BSA) and immunized subsequently with PVP.
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HA and IHA tests were repeated several times and the results were remarkably consistent. There was no difference between the anti-SRBC titers (Fig. 1) of the normal and the BSA-inoculated mice. This suggests that overload by an unrelated protein is not the cause of immunodepression. Infected animals showed much lower titers than the controls, and the extract-treated mice revealed no titers. These results demonstrate that the living parasite is unnecessary to induce immunodepression. The difference in the degree of depression induced by the moderate infection and by the inoculation of a large amount of extract suggests a dose-dependent effect. The anti-PVP titers (Fig. 2) revealed no large difference among normal, antigen-treated, and BSA-treated mice. The small difference between the control and the Trichinella-infected mice is negligible and may be attributed either to variation inherent to the assay or to deterioration of the normal physiology caused by the infection. DISCUSSION Although the design of the experiment did not allow estimation of the dispersion of the titers within groups, it is unlikely that individual idiosyncracies could account for the differences registered between groups of ten highly inbred mice subjected to identical conditions of management. This is further supported for the PVP system by the remarkably consistent humoral response obtained with similar doses in the same (4) or a different (5) strain of mice. The humoral response to SRBC in mice depends on T and B cell cooperation, while the response to PVP is dependent on B cells solely (6, 7). Therefore, the results suggest that the immunodepressive ability of Trichinella affects mainly the T-cell activity. It is not clear, however, whether this effect is mediated by a direct action on the T lymphocytes or indirectly by deterioration of the macrophage activity. Fauber and Tanner (8) found that the bone marrow cells from Trichinella infected mice were inefficient in reconstituting anti-SRBC responsiveness in thymectomized, irradiated mice; since irradiation does not affect the uptake of antigen by macrophages (9) the phagocytic activity of the recipient mice must have been preserved, therefore the immunodepression displayed must have been independent of the phagocytic activity. It is also known that PVP is normally stored in the phagocytic cells for long periods (10, 11) ; decreased uptake by these cells should result in persistence of the antigen in the circulation with a corresponding diminution of the antibody titers due to immune-elimination. No difference was found, however, between the anti-PVP titers in antigen-treated mice and in BSA-inoculated mice. These two facts point to a preservation of the macrophagie function in the Trichinella-immunodepressed mice. It has also been found that depletion of thymus-derived lymphocytes by administration of anti-lymphocyte serum enhances the humoral response to PVP (12). The fact that such an enhancement was not detected in the animals receiving Trichinella antigen suggests that the immunodepression caused by the parasite is more restricted than the simple elimination of T cells. The demonstration of selective immunodepression by T. spiralis may provide an important clue for further studies on the phenomenon of modification of the immune response by parasitic infections and may lead to new models to investigate
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the composition, functions, and interrelations of the different cellular compartments in the imnmne response. ACKNOWLEDGMENT This work was supported by NSF Institutional Grant GU 3702.
REFERENCES 1. Svet-Moldavsky, G. J. et al., Transplantation 9, 69, 1970. 2. Fauber, G., and Tanner, C. E., Exp. Parasit. 30, 120, 1971. 3. Cypess, R. H., Lubiniecki, A. S., and I-Iammon, W. M., Proc. Soc. Exp. Biol. 143, 469, 1973.
4. 5. 6. 7. 8. 9. 10. 11. 12.
Andersson, B., J. Immunol. 102, 1309, 1969. Kerbel, R. S., and Eidinger, D., Eur. J. Immunol. 2, 114, 1972. Andersson, B., and Blomgren, H., Cell. Immunol. 2, 411, 1971. Kruger, J., and Gershon, R. K., :. Immunol. 108, 581, 1972. Faubert, G. M., and Tanner, C. E., Immunology 27, 501, 1974. Unanue, E. R., Advan. Immunol. 15, 95, 1972. Ravin, I-I. A., Seligman, A. M., and Fine, f., N. Engl. Y. Med. 247, 921, 1952. Steele, R., Van Slyke, D. D., and Plazin, J., Ann. N.Y. Acad. Sci. 55, 479, 1952. Kebel, R. S., and Eidinger, D., J. Immunol. 106, 917, 1971.
Selective Immunodepression in Mice by Trichinella spiralis Extracts and Infections O ~ A R O . BARRIGA
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174
Received December 12, 1974 C57BL/6J mice pretreated with saline, Trichinella spiraIis extract, or bovine serum albumin, or infected with T. spiralis were immunized with either polyvinylpyrrolidone (PVP) or sheep red blood cells (SRBC). Hemagglutinating anti-PVP titers were similar in all groups but anti-SRBC titers were markedly lower in infected mice, and were undeteetable in extraet-pretreated mice. These results suggest that only the activity of T Iymphocytes was affected by the infection and the inoculation of extract. Circumstantial evidence suggests that the immunodepression was independent of phagocytic activity and different from that achieved by pretreatment with anti-lymphocyte serum. INTRODUCTION
Trichinella spiralis infections in mice interfere with both cell-mediated and humoral immune responses to unrelated antigens ( 1 - 3 ) , but whether this immunodepression is due to antigenic competition, a physiological deterioration of diseased animals, or a specific effect on the immune system remains unknown. The experiment reported here shows that an extract of T. spiralis possesses immunodepressive properties and suggests that the immunodepression is restricted to the T-cell activity.
MATERIALS AND METHODS Eighty C57BL/6J female mice, 6-wk old, were divided into 4 groups of 20 and were treated as follows: group N, serving as normal control, received 7 daily intraperitoneal injections of 0.15 M NaC1; group Ts received 40 viable T. splralis larvae per mouse orally ; group Ag and groups B S A received 7 daily intraperitoneal inoculations of T. spiralis larval extract containing 0.2 mg of protei n or 0.2 mg of five times crystallized bovine serum albumin (BSA, Pentex, Kankakee, Illinois), respectively. The larval extract was prepared by high speed homogenization of clean larvae followed by centrifugation at 12,000g for 30 min at 2°C. Protein estimations were done by the Biuret technique. Each group was then divided into two subgroups of ten mice; one subgroup was immunized intraperitoneally with 10 r sheep red blood cells ( S R B C ) and the other subgroup was injected intraperitoneally with 0.5 >g of polyvinylpyrrolidone K-90 ( P V P K-90; av M W 360,000 daltons, Matheson Coleman and Bell, Nor306 Copyright ~) 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
307
COMMUNICATIONS
256" 128" 64' 32" 16"
!i~il 8" 4' 7"
Ts
k[
BSA
MICE
FIG. l. Anti-sheep red blood cell (SRBC) titers of mice infected with Trichlnella spiralis (Ts) or inoculated with saline (N), T. spiralls extract (Ag), or bovine serum albumin (BSA) and immunized subsequently with SRBC. wood, Ohio). Immunizations were performed on the seventh day of infection and on day 1 after the injections of saline solution, T. spira.lis extract and BSA. All animals were killed and bled on the eighth day after immunization with SRBC and P V P . The sera from each subgroup were pooled and examined repeatedly by direct hemagglutination ( H A ) with SRBC and by indirect hemagglutination ( I H A ) with erythrocytes coated with P V P K-15 (av M W 10,000 daltons) by treatment with tannic acid (4). All sera were inactivated at 56°C for 30 rain and those utilized for I H A were also absorbed with SRBC. RESULTS The serum titers of SRBC-immunized mice, when reacted with SRBC, are presented in Fig. 1. IJnabsorbed pooled sera of the PVP-immunized mice were used as negative controls; only one group showed weak hemagglutination at 1:2 dilution. The serum titers of PVP-immunized mice when reacted with the homologous antigen are presented in Fig. 2. The specificity of the reaction was determined by I H A inhibition with soluble antigen. Pooled sera of the SRBC-immunized mice, used as negative controls, only showed positive I H A to 1:4 dilution. Both 2561286432..........
16-
::;:i::
l::~*2l
l
TS
::e~:=
A|
JSA
NrCE
FIG. 2. An ti-polyvinylpyrrolidone (PVP) titers of mice infected with Trichinella spiralis (Ts) or inoculated with saline (N), T. spiralis extract (Ag), or bovine serum albumin (BSA) and immunized subsequently with PVP.
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HA and IHA tests were repeated several times and the results were remarkably consistent. There was no difference between the anti-SRBC titers (Fig. 1) of the normal and the BSA-inoculated mice. This suggests that overload by an unrelated protein is not the cause of immunodepression. Infected animals showed much lower titers than the controls, and the extract-treated mice revealed no titers. These results demonstrate that the living parasite is unnecessary to induce immunodepression. The difference in the degree of depression induced by the moderate infection and by the inoculation of a large amount of extract suggests a dose-dependent effect. The anti-PVP titers (Fig. 2) revealed no large difference among normal, antigen-treated, and BSA-treated mice. The small difference between the control and the Trichinella-infected mice is negligible and may be attributed either to variation inherent to the assay or to deterioration of the normal physiology caused by the infection. DISCUSSION Although the design of the experiment did not allow estimation of the dispersion of the titers within groups, it is unlikely that individual idiosyncracies could account for the differences registered between groups of ten highly inbred mice subjected to identical conditions of management. This is further supported for the PVP system by the remarkably consistent humoral response obtained with similar doses in the same (4) or a different (5) strain of mice. The humoral response to SRBC in mice depends on T and B cell cooperation, while the response to PVP is dependent on B cells solely (6, 7). Therefore, the results suggest that the immunodepressive ability of Trichinella affects mainly the T-cell activity. It is not clear, however, whether this effect is mediated by a direct action on the T lymphocytes or indirectly by deterioration of the macrophage activity. Fauber and Tanner (8) found that the bone marrow cells from Trichinella infected mice were inefficient in reconstituting anti-SRBC responsiveness in thymectomized, irradiated mice; since irradiation does not affect the uptake of antigen by macrophages (9) the phagocytic activity of the recipient mice must have been preserved, therefore the immunodepression displayed must have been independent of the phagocytic activity. It is also known that PVP is normally stored in the phagocytic cells for long periods (10, 11) ; decreased uptake by these cells should result in persistence of the antigen in the circulation with a corresponding diminution of the antibody titers due to immune-elimination. No difference was found, however, between the anti-PVP titers in antigen-treated mice and in BSA-inoculated mice. These two facts point to a preservation of the macrophagie function in the Trichinella-immunodepressed mice. It has also been found that depletion of thymus-derived lymphocytes by administration of anti-lymphocyte serum enhances the humoral response to PVP (12). The fact that such an enhancement was not detected in the animals receiving Trichinella antigen suggests that the immunodepression caused by the parasite is more restricted than the simple elimination of T cells. The demonstration of selective immunodepression by T. spiralis may provide an important clue for further studies on the phenomenon of modification of the immune response by parasitic infections and may lead to new models to investigate
SHORT COMMUNICATIONS
309
the composition, functions, and interrelations of the different cellular compartments in the imnmne response. ACKNOWLEDGMENT This work was supported by NSF Institutional Grant GU 3702.
REFERENCES 1. Svet-Moldavsky, G. J. et al., Transplantation 9, 69, 1970. 2. Fauber, G., and Tanner, C. E., Exp. Parasit. 30, 120, 1971. 3. Cypess, R. H., Lubiniecki, A. S., and I-Iammon, W. M., Proc. Soc. Exp. Biol. 143, 469, 1973.
4. 5. 6. 7. 8. 9. 10. 11. 12.
Andersson, B., J. Immunol. 102, 1309, 1969. Kerbel, R. S., and Eidinger, D., Eur. J. Immunol. 2, 114, 1972. Andersson, B., and Blomgren, H., Cell. Immunol. 2, 411, 1971. Kruger, J., and Gershon, R. K., :. Immunol. 108, 581, 1972. Faubert, G. M., and Tanner, C. E., Immunology 27, 501, 1974. Unanue, E. R., Advan. Immunol. 15, 95, 1972. Ravin, I-I. A., Seligman, A. M., and Fine, f., N. Engl. Y. Med. 247, 921, 1952. Steele, R., Van Slyke, D. D., and Plazin, J., Ann. N.Y. Acad. Sci. 55, 479, 1952. Kebel, R. S., and Eidinger, D., J. Immunol. 106, 917, 1971.
