pallidum-infected Rabbits to Mitogens and

Transcription

1 INFECTION AND ImMUNITY, Feb. 1977, p 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 ofbacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 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) Ȧlthough 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. Furthermore, 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 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

2 418 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% eq c argon. After centrifugation of cultures at 20,000 x g = LO If for 15 min at 40C, the isolated organisms were washed three times with phosphate-buffered saline Z o and resuspended in phosphate-buffered saline to e LO 0 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 3 c activity and stored at -200C. 2 ZOX Lymphocyte cultures. The technique for lympho- 2 :_ cyte transformation was performed as previously o0.. t.,-+ described (19). Briefly, rabbit peripheral blood lym- h Mcu ctphocytes obtained at weekly intervals were purified *QX using a modification of the technique of Boyum (2). % g Heparinized blood was layered onto a Methocel-Hy- X paque gradient and centrifuged at 650 x g for 20 min. The cells at the gradient interface were col- _ ' - lected and washed twice in minimal essential me- M +.u 1t+dium (GIBCO), supplemented with 2 mmol of - e+i +I +I +I +I glutamine/ml, 100 U of penicillin/ml, and 100,ug of X I" 0 I"tu E streptomycin/ml, and were suspended to a concen-.# ce M 3 co tration of 5 x 106 cells/ml. A Wright stain showed cq that 95% of the cells were lymphocytes; 92 to 98% of i t the cells were viable as seen by exclusion of trypan blue. Using a modified technique of Williams (29), o 0 c' lymphocytes were cultured in flat-bottomed micro- X E ru tissue culture plates (Linbro) at a cell concentration +Io+ +I+I+ of 5 x 105 cells/0.1 ml of minimal essential medium 5 t- I" + +4eq fortified with 10% heat-inactivated normal rabbit. I" co M' serum. Triplicate wells received 0.02 ml of the fol- X lowing mitogen or antigen stocks: phytohemaggluti- 0 nin-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 3 8u (Cappel Laboratories), 70 ug/ml; Reiter treponemal antigen, 60 ug/ml. These concentrations were pre Z Z +1 determined as optimal for blastogenic reactivity X, c c (Table 1). Cultures were incubated in a total volume > e nce of ml at 370C for 60 h in a humidified chamber of cq 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 addi-,0 tional 18 h. Lymphocytes were harvested as de- 929l + 9n scribed by Williams (29) except that Econofluor i. zzzz (New England Nuclear) was used as scintillation X. 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 Sc standard deviation of the mean of triplicate sam- 7 5q ples. This value is obtained by subtracting the mean E +1 cpm of unstimulated cultures from the mean cpm of LO+ 11~~~ 0 mitogen- or antigen-stimulated cultures. Data were analyzed using the Student's t test. RESULTS Blastogenic response of rabbit lymphocytes 0 to mitogens. Figures 1 and 2 show the lympho- 76 blastic response to PHA and PWM in relation be to 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 INFECT. IMMUN.

3 VOL. 15, 1977 RESPONSE OF LYMPHOCYTES FROM INFECTED RABBITS 419 4t-4' >-.e* 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 FIG. 1. Deoxyribonucleic acid synthesis in lymphocytes (-) 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 of unstimulated controls. The standard deviation is shown by the brackets. Note that the initial cpm of thymidine incorporation (zero weeks) represents preinfected levels of stimulation ' M 6-0 x 4 " 2' * FIG. 3. Deoxyribonucleic acid synthesis in lymphocytes of goat anti-rabbit IgM. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend of Fig I-1-0-if FIG. 2. Deoxyribonucleic acid synthesis in lymphocytes from rabbits injected with viable (0) and ofpwm. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend offig. 1. 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 0 x LI o FIG. 4. Deoxyribonucleic acid synthesis in lymphocytes 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.

4 420 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 groups. DISCUSSION The results obtained in this investigation demonstrate that peripheral blood lymphocytes from rabbits infected with T. pallidum have a depressed in vitro response to PHA and PWM during the first 3 to 4 weeks of infection. Thereafter the blastogenic response of lymphocyte cultures from T. pallidum-infected rabbits to PHA and PWM returned to control levels. In contrast, the stimulatory effects of anti-immunoglobulins and T. reiteri antigen on the lymphocyte population remained near control values throughout the infection. These findings are consistent with our earlier observation that lymphocytes from rabbits in the early stages of T. pallidum infection demonstrate decreased blastogenesis to concanavalin (Con A) (19). Levene et al. (9) found reduced levels of lymphocyte transformation to PHA in patients s, FIG. 5. Deoxyribonucleic acid synthesis in lymphocytes of T. reiteri. Experimental conditions and unstimulated lymphocyte control values for both groups of rabbits are as reported in the legend of Fig. 1. 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, 18). In light of these reports we believe that our data represent selective impairment of T-cell function, whereas B-cell activity appears unchanged during the course of syphilitic infection. Since T. reiteri can be readily cultivated in vitro and shares common group antigens with virulent T. pallidum, although their relationship to T. pallidum infectivity is unknown (14), it was used as a source of treponemal antigen in our lymphocyte cultures. Musher et al. (14, 15) demonstrated depressed levels of blastogenesis to several treponemal organisms, including T. pallidum, T. refringens, and T. reiteri, in primary and secondary human syphilitics which, in part, contradicts our results. These inconsistencies may be due to different preparations or concentrations of antigen used. Another possible explanation is that lymphocytes from human patients, unlike lymphocytes from infected 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 ofthe 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 cultured in the presence of serum obtained from rabbits 1 to 3 weeks after infection with T. pallidum. It has been suggested (31) that antigen in the form of antigen-antibody complexes may be responsible for the poor lymphocyte behavior. In human syphilitic glomerulonephritis, deposits of treponemal antigen-antitreponemal-antibody complexes have been shown to contribute to tissue injury (7). Ryan et al. (21) have demonstrated that antigen-antibody

5 VOL. 15, 1977 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 ofsuppressor 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, RESPONSE OF LYMPHOCYTES FROM INFECTED RABBITS 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 and research career development award 1- K04-AI from the National Institute of Allergy and Infectious Diseases to J.B.B. LITERATURE CITED 1. Benezra, D., I. Gery, and A. M. Davies The relationship between lymphocyte transformation and immune responses. 11. Correlations between transformation and humoral and cellular responses. Clin. Exp. Immunol. 5: Boyum, A Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21(Suppl. 97): Brown, W. H., and L. Pearce Latent infections with the demonstration of Spirochaeta pallida in lymphoid tissues of the rabbit. Am. J. Syph. 5: Calkins, C. E., H. Ozer, and B. H. Waksman B- cells in the appendix and other lymphoid organs of the rabbit: stimulation of DNA synthesis by antiimmunoglobulin. Cell. Immunol. 18: Daguillard, F., and M. Richter Cells involved in the immune response. XII. 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