INFECTION AND ImMUNITY, Feb. 1977, p. 417-422 Copyright e 1977 American Society for Microbiology
Vol. 15, No. 2
Printed in U.S.A.
Selective Response of Lymphocytes from Treponema pallidum-Infected Rabbits to Mitogens and Treponema reiteri CHARLES S. PAVIA, JOEL B. BASEMAN, AND JAMES D. FOLDS* Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 17 August 1976
The in vitro response of peripheral blood lymphocytes from rabbits infected with Treponema pallidum was examined using various mitogens and avirulent Treponema reiteri. For the first 4 weeks after treponemal infection, the response of lymphocytes from syphilitic rabbits to phytohemagglutinin and pokeweed mitogen was markedly reduced in comparison to uninfected controls. Lymphocytes from both groups of rabbits responded normally to class-specific immunoglobulin anti-sera (anti-immunoglobulin M and anti-immunoglobulin G) and T. reiteri. Although a variety of antibodies are produced during the course of Treponema pallidum infection, they seem to offer little, if any, protection since individuals who contract syphilis can progress to primary, secondary, and tertiary stages of disease (30). In addition, passive transfer of serum from infected rabbits to noninfected recipients before challenge with T. pallidum has been met with limited success (24, 28). Likewise, the role of cell-mediated immunity in syphilis has not been clearly elucidated. Evidence accumulated in several studies suggests that an impairment of thymus-dependent immunity occurs in humans during the early stages of treponemal infection (9, 13, 31). A reduced number of lymphocytes in the paracortical areas of lymph nodes has also been detected in primary and secondary human syphilitics (27). Similar abnormalities have been reported to occur in the T-cell-dependent areas of the spleens of infants dying of congenital syphilis (10). In addition, reduced levels of blastogenesis to phytohemagglutinin (PHA), treponemal, and nontreponemal antigens have been described in cases of human syphilis using the in vitro lymphocyte transformation test (9, 14). Although it has been suggested that T. pallidum infection in rabbits is an acute and selflimiting disease (15), the infectious spirochete localizes in the lymph nodes and remains there indefinitely as a form of latency (3). Schell and Musher (22) detected evidence of nonspecific cell-mediated immunity as measured by partial protection to Listeria monocytogenes in rabbits 3 to 6 weeks after T. pallidum infection. Fur-
thermore, a significant level of resistance to Listeria infection was observed after the transfer to normal rabbits of a mixture of viable T. pallidum and spleen cells from T. palliduminfected rabbits (23). It is unclear, however, whether this apparent development of cell-mediated immunity results from a specific response to T. pallidum antigens or from nonspecific stress conditions of infection which accompany the diseased state. We have recently shown that lymphocytes taken from rabbits during initial T. pallidum infection have a depressed in vitro response to the mitogen concanavalin A (Con A) (19). In this report we offer further evidence that T cell-dependent immunity in rabbits is selectively depressed during the early stages of T. pallidum infection by demonstrating reduced lymphocyte reactivity to PHA and pokeweed mitogen (PWM), whereas a normal response was obtained in the presence of anti-immunoglobulins and antigen(s) from Treponema reiteri. MATERIALS AND METHODS Animals. Outbred New Zealand white male rabbits (Pel-Freez) with a negative fluorescent treponemal antibody-absorption reaction were used. Animals weighing 7 to 8 pounds (ca. 3.18 to 3.63 kg) were kept at 16 to 18°C in individual cages before and during treponemal infection. Bacteria and infections. The virulent Nichols strain of T. pallidum was obtained through the courtesy of the Center for Disease Control, Atlanta, Ga. Rabbits were injected in each testis with 5 x 107 virulent T. pallidum. Rabbits injected with an equal amount of heat-killed T. pallidum (Nichols, 56°C for 1 h) were used as noninfected controls. T. reiteri was 417
418
PAVIA, BASEMAN, AND FOLDS
grown in spirolate broth at 350C for 3 days in an atmosphere of 10% H2, 35% N2, 5% C02, and 50% argon. After centrifugation of cultures at 20,000 x g for 15 min at 40C, the isolated organisms were washed three times with phosphate-buffered saline and resuspended in phosphate-buffered saline to yield 109 spirochetes/ml. Dilutions of organisms were performed and the protein concentration of each suspension was determined (11). Samples of each suspension were then tested for blastogenic activity and stored at -200C. Lymphocyte cultures. The technique for lymphocyte transformation was performed as previously described (19). Briefly, rabbit peripheral blood lymphocytes obtained at weekly intervals were purified using a modification of the technique of Boyum (2). Heparinized blood was layered onto a Methocel-Hypaque gradient and centrifuged at 650 x g for 20 min. The cells at the gradient interface were collected and washed twice in minimal essential medium (GIBCO), supplemented with 2 mmol of glutamine/ml, 100 U of penicillin/ml, and 100 ,ug of streptomycin/ml, and were suspended to a concentration of 5 x 106 cells/ml. A Wright stain showed that 95% of the cells were lymphocytes; 92 to 98% of the cells were viable as seen by exclusion of trypan blue. Using a modified technique of Williams (29), lymphocytes were cultured in flat-bottomed microtissue culture plates (Linbro) at a cell concentration of 5 x 105 cells/0.1 ml of minimal essential medium fortified with 10% heat-inactivated normal rabbit serum. Triplicate wells received 0.02 ml of the following mitogen or antigen stocks: phytohemagglutinin-P (PHA-P, Difco), 10 jug/ml; PWM (GIBCO), 60 gg/ml; goat anti-rabbit immunoglobulin M (IgM) (Miles Laboratories), 70 ,ug/ml; goat anti-rabbit IgG (Cappel Laboratories), 70 ug/ml; Reiter treponemal antigen, 60 ug/ml. These concentrations were predetermined as optimal for blastogenic reactivity (Table 1). Cultures were incubated in a total volume of 0. 12 ml at 370C for 60 h in a humidified chamber of 95% air and 5% C02. Then, 10 ul of medium containing 0.5 ,uCi of [3H]thymidine (Schwarz/Mann, Orangeburg, N.Y.) was added to each well for an additional 18 h. Lymphocytes were harvested as described by Williams (29) except that Econofluor (New England Nuclear) was used as scintillation fluid. Samples were counted for radioactivity in a liquid scintillation spectrometer. Statistical analysis of results. The data in counts per minute (cpm) are presented as group means and standard deviation of the mean of triplicate samples. This value is obtained by subtracting the mean cpm of unstimulated cultures from the mean cpm of mitogen- or antigen-stimulated cultures. Data were analyzed using the Student's t test.
RESULTS Blastogenic response of rabbit lymphocytes to mitogens. Figures 1 and 2 show the lymphoblastic response to PHA and PWM in relation to the duration of infection. Lymphocytes from T. pallidum-infected rabbits responded poorly to PHA and PWM for the first 3 to 4 weeks of
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RESPONSE OF LYMPHOCYTES FROM INFECTED RABBITS
VOL. 15, 1977
WEEKS
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from the infected animals reached normal levels and was not statistically different from the control group. The incorporation of [3H]thymidine by lymphocytes from the noninfected rabbits varied little from week to week. It should be noted that the mean cell counts as measured by thymidine uptake for unstimulated control cultures showed little variation at each weekly interval (Fig. 1, dashed lines). Blastogenic response of rabbit lymphocytes to anti-immunoglobulins. As can be seen in Fig. 3 and 4, lymphocytes obtained from syphilitic rabbits and exposed to anti-rabbit IgM and
12
7 INFECTION
FIG. 1. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or heat-killed (0) T. pallidum and cultured in the presence (-) or absence (--) ofPHA . The data, representative of three separate experiments each using duplicate rabbits, are reported as group mean cpm of PHA-stimulated cultures after subtraction ofunstimulated controls. The standard deviation is shown by the brackets. Note that the initial cpm of thymidine incorporation (zero weeks) represents preinfected levels of stimulation.
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INFECTION
FIG. 3. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or heat-killed (0) T. pallidum and cultured in the presence
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Experimental condi-
tions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend ofFig. 1.
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FIG. 2. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) and heat-killed (0) T. pallidum and cultured in the presence ofPWM. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend ofFig. 1.
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infection. The mean uptake of [3H]thymidine in these lymphocytes was significantly reduced (P < 0.01) when compared to the levels of blastogenesis obtained from lymphocytes of rabbits injected with heat-killed T. pallidum. Thereafter, uptake of [3H]thymidine by lymphocytes
2
3
AFTER
4
T. PALLIDUM
6
1 712
INFECTION
FIG. 4. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or heat-killed (0) T. pallidum and cultured in the presence ofgoat anti-rabbit IgG. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend of Fig. 1.
420
PAVIA, BASEMAN, AND FOLDS
anti-rabbit IgG incorporated nearly the same amount of [3Hlthymidine as did those from control rabbits injected with heat-killed T. pallidum. In neither group of animals did the degree of lymphoblastic transformation vary significantly from week to week. The uptake of [3H]thymidine was slightly higher for cells cultured with anti-IgG than with anti-IgM. Blastogenic response of lymphocytes to T. reiteri. After incubation of lymphocytes with the Reiter treponemal preparation, the level of uptake of [3H]thymidine by lymphocytes from infected and noninfected rabbits was nearly identical for at least 3 months after infection (Fig. 5). At each weekly interval there were no significant differences between these two
INFECT. IMMUN.
with active syphilis. This depression, as in our studies, was greatest in the early stages of disease, whereas normal lymphocyte reactivity occurred later on in the infectious process. Several groups (4, 5, 17, 25, 26) have recently reported that the rabbit immune system contains distinct and separable populations of cells with functional properties analogous to murine and human thymus-derived (T) and Bursaequivalent (B) lymphocytes. It has been shown that rabbit T and B cells can be selectively stimulated by different mitogens. It is increasingly apparent that Con A, PHA, and PWM predominantly stimulate T cells, whereas antiimmunoglobulins principally transform B cells or immunoglobulin-bearing lymphocytes (4, groups. 18). In light of these reports we believe that our data represent selective impairment of T-cell DISCUSSION function, whereas B-cell activity appears unThe results obtained in this investigation changed during the course of syphilitic infecdemonstrate that peripheral blood lymphocytes tion. Since T. reiteri can be readily cultivated in from rabbits infected with T. pallidum have a depressed in vitro response to PHA and PWM vitro and shares common group antigens with during the first 3 to 4 weeks of infection. There- virulent T. pallidum, although their relationafter the blastogenic response of lymphocyte ship to T. pallidum infectivity is unknown (14), cultures from T. pallidum-infected rabbits to it was used as a source oftreponemal antigen in PHA and PWM returned to control levels. In our lymphocyte cultures. Musher et al. (14, 15) contrast, the stimulatory effects of anti-immu- demonstrated depressed levels of blastogenesis noglobulins and T. reiteri antigen on the lym- to several treponemal organisms, including T. phocyte population remained near control val- pallidum, T. refringens, and T. reiteri, in primary and secondary human syphilitics which, ues throughout the infection. These findings in part, contradicts our results. These inconare consistent with our earlier observation that lymphocytes from rabbits in the early stages of sistencies may be due to different preparations T. pallidum infection demonstrate decreased or concentrations of antigen used. Another possible explanation is that lymphocytes from hublastogenesis to concanavalin (Con A) (19). Levene et al. (9) found reduced levels of lym- man patients, unlike lymphocytes from inphocyte transformation to PHA in patients fected rabbits, are sensitive to a particular treponemal antigen(s). Musher et al., however, did not determine which lymphocyte population was adversely affected. How can we explain this apparent inhibition of thymus-dependent immunity and its relationship to the pathogenesis of the disease? Certain studies (8, 9) have shown that plasma or serum from patients with early syphilis decreased the in vitro response of normal lymphocytes to PHA. In our earlier report (19) we found that lymphocyte reactivity was maximally depressed when lymphocytes were culs, tured in the presence of serum obtained from rabbits 1 to 3 weeks after infection with T. 7 2 3 12 1 4 5 6 pallidum. It has been suggested (31) that antiWEEKS AFTER T. PALLIDUM INFECTION gen in the form of antigen-antibody complexes be responsible for the poor lymphocyte may FIG. 5. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or behavior. In human syphilitic glomeruloneheat-killed (0) T. pallidum and cultured in the pres- phritis, deposits of treponemal antigen-antitreponemal-antibody complexes have been shown ence of T. reiteri. Experimental conditions and unstimulated lymphocyte control values for both groups to contribute to tissue injury (7). Ryan et al. of rabbits are as reported in the legend ofFig. 1. (21) have demonstrated that antigen-antibody 10-
2-
0
VOL. 15, 1977
RESPONSE OF LYMPHOCYTES FROM INFECTED RABBITS
complexes profoundly inhibit the in vitro response to mitogens. Also, the blastogenic response to T-cell mitogens appears to be hampered in cases of defective cell-mediated immunity (17). Niklasson and Williams (16) reported that a poor lymphocyte response to PHA in patients with viral infections has been associated with reduced numbers of thymus-derived lymphocytes circulating in the blood. Also, decreased numbers of lymphocytes were found in the spleens of neonatal rabbits infected with T. pallidum (6). A similar change in the ratio of T and B lymphocytes in the peripheral blood of the rabbit during treponemal infection might account for the results obtained in our experiments. However, at the time of harvest of peripheral blood from T. pallidum-infected rabbits, the number of lymphocytes observed per volume of blood obtained 1 to 6 weeks after infection is approximately 20 to 40% greater than in uninfected rabbits (unpublished data). This increase in the cell population might be a direct result of the initiation of antibody synthesis to treponemal antigens. If increased numbers of circulating B lymphocytes during T. pallidum infection occur, one might anticipate a greater level of blast transformation by anti-immunoglobulins, which are selective mitogens for B cells. However, similar levels of stimulation in the lymphocyte population are observed in both control and infected rabbits (Fig. 3 and 4), possibly indicating a constant population of B cells in both groups. Alternatively, a significant increase in the number of B cells might not be detected because of modified sensitivities to the concentrations of mitogen used or to the presence of suppressor cells which could reduce the stimulatory effects of antiimmunoglobulins on the B-cell population. The production of a suppressor by lymphocytes has recently been described in rabbit peripheral blood and spleen. Luzzati and Lafleur (12) found that rabbit peripheral blood lymphocytes suppressed the secondary in vitro response of autologous spleen cells to sheep erythrocytes. In a similar fashion Redelman et al. (20) demonstrated that Con A-stimulated peripheral blood and splenic lymphocytes exerted an inhibitory effect on the in vitro anti-sheep erythrocyte plaque-forming cell response by spleen cells. Although the mechanisms of the suppressive effect in these studies remain unclear, it is believed that modifications of both T and B lymphocytes may be responsible for these activities. A large proportion of normal rabbit lymphocytes can be regularly induced to enlarge and proliferate in cultures containing either Con A,
421
PHA, PWM (18), anti-immunoglobulins (4), or appropriate antigen (1). The poor lymphocyte response to T-cell mitogens observed in the early stages of T. pallidum infection in rabbits may indicate a general impairment of thymusdependent immunity. Subpopulations of T cells or modulation of T-cell function by serum factors of host or treponemal origin which are generated during treponemal infection could elicit an immunosuppressive effect on T-cell blastogenesis. It should be realized, however, that these data are limited to in vitro studies on peripheral blood lymphocytes, and their regulation by serum and may not reflect the physiological interaction of factors in blood with peripheral blood or other tissue lymphocytes. Nonetheless, these data may explain in part the increased proliferation and establishment ofthe spirochete in selected host sites as well as the uniqueness of the host response to the disease. ACKNOWLEDGMENTS This work was supported by Public Health Service grants AI-11283 and research career development award 1K04-AI-00178 from the National Institute of Allergy and Infectious Diseases to J.B.B.
LITERATURE CITED 1. Benezra, D., I. Gery, and A. M. Davies. 1969. The relationship between lymphocyte transformation and immune responses. 11. Correlations between transformation and humoral and cellular responses. Clin. Exp. Immunol. 5:155-161. 2. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21(Suppl. 97):1-91. 3. Brown, W. H., and L. Pearce. 1921. Latent infections with the demonstration of Spirochaeta pallida in lymphoid tissues of the rabbit. Am. J. Syph. 5:1-8. 4. Calkins, C. E., H. Ozer, and B. H. Waksman. 1975. Bcells in the appendix and other lymphoid organs of the rabbit: stimulation of DNA synthesis by antiimmunoglobulin. Cell. Immunol. 18:187-198. 5. Daguillard, F., and M. Richter. 1970. Cells involved in the immune response. XII. The differing responses of normal rabbit lymphoid cells to PHA, goat anti-rabbit immunoglobulin antiserum and allogeneic and xenogeneic lymphocytes. J. Exp. Med. 130:1187-1208. 6. Festenstein, H., C. Abrahams, and V. Bokkenheuser. 1967. Runting syndrome in neonatal rabbits infected with Treponema pallidum. Clin. Exp. Immunol. 7:311-320. 7. Gambel, C.N., and J. B. Reardon. 1975. Immunopathogenesis of syphilitic glomerular immune-complex disease. N. Engl. J. Med. 292:449-454. 8. Kantor, F. S. 1975. Infection, anergy and cell mediated
immunity. N. Engl. J. Med. 292:629-634. 9. Levene, G. M., J. L. Turk, D. J. M. Wright, and A. G. S. Grimble. 1969. Reduced lymphocyte transformation due to a plasma factor in patients with active syphilis. Lancet ii:246-247. 10. Levene, G. M., D. J. M. Wright, and J. L. Turk. 1971. Cell-mediated immunity and lymphocyte transformation in syphilis. Proc. R. Soc. Med. 64:14-16. 11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin
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PAVIA, BASEMAN, AND FOLDS
phenol reagent. J. Biol. Chem. 193:265-275. 12. Luzzati, A. L., and L. Lafleur. 1976. Suppressor cells in rabbit peripheral blood. Eur. J. Immunol. 6:125-129. 13. Marshak, L. C., and S. Rothman. 1951. Skin testing with a purified suspension of Treponema pallidum. Am. J. Syph. 35:35-41. 14. Musher, D. M., R. F. Schell, and J. M. Knox. 1974. In vitro lymphocyte response to Treponema refringens in human syphilis. Infect. Immun. 9:654-657. 15. Musher, D. M., R. F. Schell, R. H. Jones, and A. M. Jones. 1975. Lymphocyte transformation in syphilis: an in vivo correlate of immune suppression in vovo. Infect. Immun. 11:1261-1264. 16. Niklasson, P. M., and R. C. Williams, Jr. 1974. Studies of peripheral blood T- and B-lymphocytes in acute infections. Infect. Immun. 9:1-7. 17. Oppenheim, J. J. 1968. Relationship of in vitro lymphocyte transformation to delayed hypersensitivity in guinea pigs and man. Fed. Proc. 27:21-27. 18. Ozer, H., and B. H. Waksman. 1974. The response of rabbit lymphocytes to mitogens and alloantigens: evidence of T-cell heterogeneity. J. Immunol. 113:17801792. 19. Pavia, C. S., J. D. Folds, and J. B. Baseman. 1976. Depression of lymphocyte response to concanavalin A in rabbits infected with Treponema pallidum. Infect. Immun. 14:320-322. 20. Redelman, D., C. B. Scott, H. W. Sheppard, and S. Sell. 1976. In vitro studies of the rabbit immune system. V. Suppressor T cells activated by concanavalin A block the proliferation, not the induction of antierythrocyte plaque-forming cells. J. Exp. Med. 143:919-936. 21. Ryan, J. L., R. D. Arbeit, H. B. Dickler and P. A. Henkart. 1975. Inhibition of lymphocyte mitogenesis by immobilized antigen-antibody complexes. J. Exp. Med. 142:814-826.
INFECT. IMMUN. 22. Schell, R. F., and D. M. Musher. 1974. Detection of nonspecific resistance to Listeria monocytogens in rabbits infected with Treponema pallidum. Infect. Immun. 9:658-662. 23. Schell, R. F., D. M. Musher, K. Jacobson, and P. Schwethelm. 1975. Induction of acquired cellular resistance following transfer of thymus-derived lymphocytes from syphilitic rabbits. J. Immunol. 114:550-553. 24. Sepetjian, M., D. Salvssola, and J. Thivolet. 1973. Attempt to protect rabbits against experimental syphilis by passive immunization. Br. J. Vener. Dis. 49:335-337. 25. Sheppard, H., D. Redelman, and S. Sell. 1976. In vitro studies of the rabbit immune system: differential mitogen responses of isolated T and B cells. Cell. Immunol. 24:34-44. 26. Theodorescu, M., E. P. Mayer, H. Reiter, and S. Dray. 1976. Rabbit lymphocyte subpopulations. I. Separation of Ig- and Ig+ cells and their interaction in cultures stimulated by mitogens. Cell. Immunol. 22:66-75. 27. Turner, D. R., and D. J. M. Wright. 1973. Lymphadenopathy in early syphilis. J. Pathol. 110:305-308. 28. Turner, T. B., P. H. Hardy, B. Newman, and E. E. Nell. 1973. Effects of passive immunization on experimental syphilis in the rabbit. Johns Hopkins Med. J. 133:241-251. 29. Williams, R. M. 1973. DNA synthesis by cultured lymphocytes: a modified method for measuring 3H-thymidine incorporation. Cell. Immunol. 9:435-444. 30. World Health Organization. 1970. Treponematoses research. Tech. Rep. Ser No. 455. World Health Organization, Geneva. 31. Wright, D. J. M., and A. G. S. Grimble. 1974. Why is the infectious stage of syphilis prolonged? Br. J. Vener. Dis. 50:45-49.
Vol. 15, No. 2
Printed in U.S.A.
Selective Response of Lymphocytes from Treponema pallidum-Infected Rabbits to Mitogens and Treponema reiteri CHARLES S. PAVIA, JOEL B. BASEMAN, AND JAMES D. FOLDS* Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 17 August 1976
The in vitro response of peripheral blood lymphocytes from rabbits infected with Treponema pallidum was examined using various mitogens and avirulent Treponema reiteri. For the first 4 weeks after treponemal infection, the response of lymphocytes from syphilitic rabbits to phytohemagglutinin and pokeweed mitogen was markedly reduced in comparison to uninfected controls. Lymphocytes from both groups of rabbits responded normally to class-specific immunoglobulin anti-sera (anti-immunoglobulin M and anti-immunoglobulin G) and T. reiteri. Although a variety of antibodies are produced during the course of Treponema pallidum infection, they seem to offer little, if any, protection since individuals who contract syphilis can progress to primary, secondary, and tertiary stages of disease (30). In addition, passive transfer of serum from infected rabbits to noninfected recipients before challenge with T. pallidum has been met with limited success (24, 28). Likewise, the role of cell-mediated immunity in syphilis has not been clearly elucidated. Evidence accumulated in several studies suggests that an impairment of thymus-dependent immunity occurs in humans during the early stages of treponemal infection (9, 13, 31). A reduced number of lymphocytes in the paracortical areas of lymph nodes has also been detected in primary and secondary human syphilitics (27). Similar abnormalities have been reported to occur in the T-cell-dependent areas of the spleens of infants dying of congenital syphilis (10). In addition, reduced levels of blastogenesis to phytohemagglutinin (PHA), treponemal, and nontreponemal antigens have been described in cases of human syphilis using the in vitro lymphocyte transformation test (9, 14). Although it has been suggested that T. pallidum infection in rabbits is an acute and selflimiting disease (15), the infectious spirochete localizes in the lymph nodes and remains there indefinitely as a form of latency (3). Schell and Musher (22) detected evidence of nonspecific cell-mediated immunity as measured by partial protection to Listeria monocytogenes in rabbits 3 to 6 weeks after T. pallidum infection. Fur-
thermore, a significant level of resistance to Listeria infection was observed after the transfer to normal rabbits of a mixture of viable T. pallidum and spleen cells from T. palliduminfected rabbits (23). It is unclear, however, whether this apparent development of cell-mediated immunity results from a specific response to T. pallidum antigens or from nonspecific stress conditions of infection which accompany the diseased state. We have recently shown that lymphocytes taken from rabbits during initial T. pallidum infection have a depressed in vitro response to the mitogen concanavalin A (Con A) (19). In this report we offer further evidence that T cell-dependent immunity in rabbits is selectively depressed during the early stages of T. pallidum infection by demonstrating reduced lymphocyte reactivity to PHA and pokeweed mitogen (PWM), whereas a normal response was obtained in the presence of anti-immunoglobulins and antigen(s) from Treponema reiteri. MATERIALS AND METHODS Animals. Outbred New Zealand white male rabbits (Pel-Freez) with a negative fluorescent treponemal antibody-absorption reaction were used. Animals weighing 7 to 8 pounds (ca. 3.18 to 3.63 kg) were kept at 16 to 18°C in individual cages before and during treponemal infection. Bacteria and infections. The virulent Nichols strain of T. pallidum was obtained through the courtesy of the Center for Disease Control, Atlanta, Ga. Rabbits were injected in each testis with 5 x 107 virulent T. pallidum. Rabbits injected with an equal amount of heat-killed T. pallidum (Nichols, 56°C for 1 h) were used as noninfected controls. T. reiteri was 417
418
PAVIA, BASEMAN, AND FOLDS
grown in spirolate broth at 350C for 3 days in an atmosphere of 10% H2, 35% N2, 5% C02, and 50% argon. After centrifugation of cultures at 20,000 x g for 15 min at 40C, the isolated organisms were washed three times with phosphate-buffered saline and resuspended in phosphate-buffered saline to yield 109 spirochetes/ml. Dilutions of organisms were performed and the protein concentration of each suspension was determined (11). Samples of each suspension were then tested for blastogenic activity and stored at -200C. Lymphocyte cultures. The technique for lymphocyte transformation was performed as previously described (19). Briefly, rabbit peripheral blood lymphocytes obtained at weekly intervals were purified using a modification of the technique of Boyum (2). Heparinized blood was layered onto a Methocel-Hypaque gradient and centrifuged at 650 x g for 20 min. The cells at the gradient interface were collected and washed twice in minimal essential medium (GIBCO), supplemented with 2 mmol of glutamine/ml, 100 U of penicillin/ml, and 100 ,ug of streptomycin/ml, and were suspended to a concentration of 5 x 106 cells/ml. A Wright stain showed that 95% of the cells were lymphocytes; 92 to 98% of the cells were viable as seen by exclusion of trypan blue. Using a modified technique of Williams (29), lymphocytes were cultured in flat-bottomed microtissue culture plates (Linbro) at a cell concentration of 5 x 105 cells/0.1 ml of minimal essential medium fortified with 10% heat-inactivated normal rabbit serum. Triplicate wells received 0.02 ml of the following mitogen or antigen stocks: phytohemagglutinin-P (PHA-P, Difco), 10 jug/ml; PWM (GIBCO), 60 gg/ml; goat anti-rabbit immunoglobulin M (IgM) (Miles Laboratories), 70 ,ug/ml; goat anti-rabbit IgG (Cappel Laboratories), 70 ug/ml; Reiter treponemal antigen, 60 ug/ml. These concentrations were predetermined as optimal for blastogenic reactivity (Table 1). Cultures were incubated in a total volume of 0. 12 ml at 370C for 60 h in a humidified chamber of 95% air and 5% C02. Then, 10 ul of medium containing 0.5 ,uCi of [3H]thymidine (Schwarz/Mann, Orangeburg, N.Y.) was added to each well for an additional 18 h. Lymphocytes were harvested as described by Williams (29) except that Econofluor (New England Nuclear) was used as scintillation fluid. Samples were counted for radioactivity in a liquid scintillation spectrometer. Statistical analysis of results. The data in counts per minute (cpm) are presented as group means and standard deviation of the mean of triplicate samples. This value is obtained by subtracting the mean cpm of unstimulated cultures from the mean cpm of mitogen- or antigen-stimulated cultures. Data were analyzed using the Student's t test.
RESULTS Blastogenic response of rabbit lymphocytes to mitogens. Figures 1 and 2 show the lymphoblastic response to PHA and PWM in relation to the duration of infection. Lymphocytes from T. pallidum-infected rabbits responded poorly to PHA and PWM for the first 3 to 4 weeks of
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RESPONSE OF LYMPHOCYTES FROM INFECTED RABBITS
VOL. 15, 1977
WEEKS
4
AFTER
5
T. PALLIDUM
6
from the infected animals reached normal levels and was not statistically different from the control group. The incorporation of [3H]thymidine by lymphocytes from the noninfected rabbits varied little from week to week. It should be noted that the mean cell counts as measured by thymidine uptake for unstimulated control cultures showed little variation at each weekly interval (Fig. 1, dashed lines). Blastogenic response of rabbit lymphocytes to anti-immunoglobulins. As can be seen in Fig. 3 and 4, lymphocytes obtained from syphilitic rabbits and exposed to anti-rabbit IgM and
12
7 INFECTION
FIG. 1. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or heat-killed (0) T. pallidum and cultured in the presence (-) or absence (--) ofPHA . The data, representative of three separate experiments each using duplicate rabbits, are reported as group mean cpm of PHA-stimulated cultures after subtraction ofunstimulated controls. The standard deviation is shown by the brackets. Note that the initial cpm of thymidine incorporation (zero weeks) represents preinfected levels of stimulation.
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T. PALLIDUM
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INFECTION
FIG. 3. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or heat-killed (0) T. pallidum and cultured in the presence
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Experimental condi-
tions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend ofFig. 1.
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T. PALLIDUM
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FIG. 2. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) and heat-killed (0) T. pallidum and cultured in the presence ofPWM. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend ofFig. 1.
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infection. The mean uptake of [3H]thymidine in these lymphocytes was significantly reduced (P < 0.01) when compared to the levels of blastogenesis obtained from lymphocytes of rabbits injected with heat-killed T. pallidum. Thereafter, uptake of [3H]thymidine by lymphocytes
2
3
AFTER
4
T. PALLIDUM
6
1 712
INFECTION
FIG. 4. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or heat-killed (0) T. pallidum and cultured in the presence ofgoat anti-rabbit IgG. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend of Fig. 1.
420
PAVIA, BASEMAN, AND FOLDS
anti-rabbit IgG incorporated nearly the same amount of [3Hlthymidine as did those from control rabbits injected with heat-killed T. pallidum. In neither group of animals did the degree of lymphoblastic transformation vary significantly from week to week. The uptake of [3H]thymidine was slightly higher for cells cultured with anti-IgG than with anti-IgM. Blastogenic response of lymphocytes to T. reiteri. After incubation of lymphocytes with the Reiter treponemal preparation, the level of uptake of [3H]thymidine by lymphocytes from infected and noninfected rabbits was nearly identical for at least 3 months after infection (Fig. 5). At each weekly interval there were no significant differences between these two
INFECT. IMMUN.
with active syphilis. This depression, as in our studies, was greatest in the early stages of disease, whereas normal lymphocyte reactivity occurred later on in the infectious process. Several groups (4, 5, 17, 25, 26) have recently reported that the rabbit immune system contains distinct and separable populations of cells with functional properties analogous to murine and human thymus-derived (T) and Bursaequivalent (B) lymphocytes. It has been shown that rabbit T and B cells can be selectively stimulated by different mitogens. It is increasingly apparent that Con A, PHA, and PWM predominantly stimulate T cells, whereas antiimmunoglobulins principally transform B cells or immunoglobulin-bearing lymphocytes (4, groups. 18). In light of these reports we believe that our data represent selective impairment of T-cell DISCUSSION function, whereas B-cell activity appears unThe results obtained in this investigation changed during the course of syphilitic infecdemonstrate that peripheral blood lymphocytes tion. Since T. reiteri can be readily cultivated in from rabbits infected with T. pallidum have a depressed in vitro response to PHA and PWM vitro and shares common group antigens with during the first 3 to 4 weeks of infection. There- virulent T. pallidum, although their relationafter the blastogenic response of lymphocyte ship to T. pallidum infectivity is unknown (14), cultures from T. pallidum-infected rabbits to it was used as a source oftreponemal antigen in PHA and PWM returned to control levels. In our lymphocyte cultures. Musher et al. (14, 15) contrast, the stimulatory effects of anti-immu- demonstrated depressed levels of blastogenesis noglobulins and T. reiteri antigen on the lym- to several treponemal organisms, including T. phocyte population remained near control val- pallidum, T. refringens, and T. reiteri, in primary and secondary human syphilitics which, ues throughout the infection. These findings in part, contradicts our results. These inconare consistent with our earlier observation that lymphocytes from rabbits in the early stages of sistencies may be due to different preparations T. pallidum infection demonstrate decreased or concentrations of antigen used. Another possible explanation is that lymphocytes from hublastogenesis to concanavalin (Con A) (19). Levene et al. (9) found reduced levels of lym- man patients, unlike lymphocytes from inphocyte transformation to PHA in patients fected rabbits, are sensitive to a particular treponemal antigen(s). Musher et al., however, did not determine which lymphocyte population was adversely affected. How can we explain this apparent inhibition of thymus-dependent immunity and its relationship to the pathogenesis of the disease? Certain studies (8, 9) have shown that plasma or serum from patients with early syphilis decreased the in vitro response of normal lymphocytes to PHA. In our earlier report (19) we found that lymphocyte reactivity was maximally depressed when lymphocytes were culs, tured in the presence of serum obtained from rabbits 1 to 3 weeks after infection with T. 7 2 3 12 1 4 5 6 pallidum. It has been suggested (31) that antiWEEKS AFTER T. PALLIDUM INFECTION gen in the form of antigen-antibody complexes be responsible for the poor lymphocyte may FIG. 5. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) or behavior. In human syphilitic glomeruloneheat-killed (0) T. pallidum and cultured in the pres- phritis, deposits of treponemal antigen-antitreponemal-antibody complexes have been shown ence of T. reiteri. Experimental conditions and unstimulated lymphocyte control values for both groups to contribute to tissue injury (7). Ryan et al. of rabbits are as reported in the legend ofFig. 1. (21) have demonstrated that antigen-antibody 10-
2-
0
VOL. 15, 1977
RESPONSE OF LYMPHOCYTES FROM INFECTED RABBITS
complexes profoundly inhibit the in vitro response to mitogens. Also, the blastogenic response to T-cell mitogens appears to be hampered in cases of defective cell-mediated immunity (17). Niklasson and Williams (16) reported that a poor lymphocyte response to PHA in patients with viral infections has been associated with reduced numbers of thymus-derived lymphocytes circulating in the blood. Also, decreased numbers of lymphocytes were found in the spleens of neonatal rabbits infected with T. pallidum (6). A similar change in the ratio of T and B lymphocytes in the peripheral blood of the rabbit during treponemal infection might account for the results obtained in our experiments. However, at the time of harvest of peripheral blood from T. pallidum-infected rabbits, the number of lymphocytes observed per volume of blood obtained 1 to 6 weeks after infection is approximately 20 to 40% greater than in uninfected rabbits (unpublished data). This increase in the cell population might be a direct result of the initiation of antibody synthesis to treponemal antigens. If increased numbers of circulating B lymphocytes during T. pallidum infection occur, one might anticipate a greater level of blast transformation by anti-immunoglobulins, which are selective mitogens for B cells. However, similar levels of stimulation in the lymphocyte population are observed in both control and infected rabbits (Fig. 3 and 4), possibly indicating a constant population of B cells in both groups. Alternatively, a significant increase in the number of B cells might not be detected because of modified sensitivities to the concentrations of mitogen used or to the presence of suppressor cells which could reduce the stimulatory effects of antiimmunoglobulins on the B-cell population. The production of a suppressor by lymphocytes has recently been described in rabbit peripheral blood and spleen. Luzzati and Lafleur (12) found that rabbit peripheral blood lymphocytes suppressed the secondary in vitro response of autologous spleen cells to sheep erythrocytes. In a similar fashion Redelman et al. (20) demonstrated that Con A-stimulated peripheral blood and splenic lymphocytes exerted an inhibitory effect on the in vitro anti-sheep erythrocyte plaque-forming cell response by spleen cells. Although the mechanisms of the suppressive effect in these studies remain unclear, it is believed that modifications of both T and B lymphocytes may be responsible for these activities. A large proportion of normal rabbit lymphocytes can be regularly induced to enlarge and proliferate in cultures containing either Con A,
421
PHA, PWM (18), anti-immunoglobulins (4), or appropriate antigen (1). The poor lymphocyte response to T-cell mitogens observed in the early stages of T. pallidum infection in rabbits may indicate a general impairment of thymusdependent immunity. Subpopulations of T cells or modulation of T-cell function by serum factors of host or treponemal origin which are generated during treponemal infection could elicit an immunosuppressive effect on T-cell blastogenesis. It should be realized, however, that these data are limited to in vitro studies on peripheral blood lymphocytes, and their regulation by serum and may not reflect the physiological interaction of factors in blood with peripheral blood or other tissue lymphocytes. Nonetheless, these data may explain in part the increased proliferation and establishment ofthe spirochete in selected host sites as well as the uniqueness of the host response to the disease. ACKNOWLEDGMENTS This work was supported by Public Health Service grants AI-11283 and research career development award 1K04-AI-00178 from the National Institute of Allergy and Infectious Diseases to J.B.B.
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immunity. N. Engl. J. Med. 292:629-634. 9. Levene, G. M., J. L. Turk, D. J. M. Wright, and A. G. S. Grimble. 1969. Reduced lymphocyte transformation due to a plasma factor in patients with active syphilis. Lancet ii:246-247. 10. Levene, G. M., D. J. M. Wright, and J. L. Turk. 1971. Cell-mediated immunity and lymphocyte transformation in syphilis. Proc. R. Soc. Med. 64:14-16. 11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin
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PAVIA, BASEMAN, AND FOLDS
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INFECT. IMMUN. 22. Schell, R. F., and D. M. Musher. 1974. Detection of nonspecific resistance to Listeria monocytogens in rabbits infected with Treponema pallidum. Infect. Immun. 9:658-662. 23. Schell, R. F., D. M. Musher, K. Jacobson, and P. Schwethelm. 1975. Induction of acquired cellular resistance following transfer of thymus-derived lymphocytes from syphilitic rabbits. J. Immunol. 114:550-553. 24. Sepetjian, M., D. Salvssola, and J. Thivolet. 1973. Attempt to protect rabbits against experimental syphilis by passive immunization. Br. J. Vener. Dis. 49:335-337. 25. Sheppard, H., D. Redelman, and S. Sell. 1976. In vitro studies of the rabbit immune system: differential mitogen responses of isolated T and B cells. Cell. Immunol. 24:34-44. 26. Theodorescu, M., E. P. Mayer, H. Reiter, and S. Dray. 1976. Rabbit lymphocyte subpopulations. I. Separation of Ig- and Ig+ cells and their interaction in cultures stimulated by mitogens. Cell. Immunol. 22:66-75. 27. Turner, D. R., and D. J. M. Wright. 1973. Lymphadenopathy in early syphilis. J. Pathol. 110:305-308. 28. Turner, T. B., P. H. Hardy, B. Newman, and E. E. Nell. 1973. Effects of passive immunization on experimental syphilis in the rabbit. Johns Hopkins Med. J. 133:241-251. 29. Williams, R. M. 1973. DNA synthesis by cultured lymphocytes: a modified method for measuring 3H-thymidine incorporation. Cell. Immunol. 9:435-444. 30. World Health Organization. 1970. Treponematoses research. Tech. Rep. Ser No. 455. World Health Organization, Geneva. 31. Wright, D. J. M., and A. G. S. Grimble. 1974. Why is the infectious stage of syphilis prolonged? Br. J. Vener. Dis. 50:45-49.
