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Interleukin 2 responsive T cell clones from rheumatoid and normal subjects: Proliferative responses to connective tissue elements
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
50,
264-271 ( 1989)
BRIEF COMMUNICATION lnterleukin 2 Responsive T Cell Clones from Rheumatoid and Normal Subjects: Proliferative Responses to Connective Tissue Elements WILLIAM
A. OFOSU-APPIAH,*~
RICHARD J. WARRINGTON,*+$ AND JOHN A. WILKINS*~$~§~'
*Rheumatic Diseases Research Laboratory, Departments of flmmunology, itMedicine, and §Medical Microbiology, RR014 800 Sherbrook Street. Winnipeg, Manitoba, Canada R3A lM4 In vivo-activated interleukin 2 responsive T cell clones were generated from peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients and from normal control PB. The specificity of these clones was assessed by measuring proliferation induced by the connective tissue elements (CTE) collagen types I and II, native and denatured, proteoglycans, and irrelevant control antigens. The cloned T cells from RA patients but not from normal subjects responded in vitro with proliferation to all CTE but not to control antigens purified protein derivative, ovalbumin, or lysozyme. Proliferation occurred in the presence and absence of accessory cells (AC), but the responses were consistently higher in the presence of AC. Antibodies to HLA-DR abrogated the proliferative response to CTE suggesting that DR expression was necessary for the induction of proliferation. These findings demonstrate the existence of clonable T cells responsive to CTE in PB and SF of RA patients. Expression of reactivity to CTE may contribute to the chronicity of the inflammation in RA. Q 1989 Academic Press. hc.
INTRODUCTION
Previous studies have shown that an increased proportion of T cells infiltrating the synovial tissue (ST) and fluid (SF) (1, 2) and present in peripheral blood (PB) of rheumatoid arthritis (RA) patients (3,4) are activated as defined by the expression of HLA-DR (Ia) and Tat antigens. The nature of the antigen(s) that stimulates the T cells is unknown. There is evidence that cell-mediated autoimmunity may play a fundamental role in the chronic inflammation seen in RA. These cellular immune responses may develop as a result of autoimmunity to connective tissue elements (CTE). The induction of an intlammatory polyarthritis in rats (5) and mice (6) by injection of homologous type II collagen from cartilage demonstrates the importance of an immune response to cartilage components in the induction of experimental arthritis. This raises the possibility that a similar autoimmune process could contribute to the joint inflammation in RA. Cell-mediated autoimmunity to the interstitial collagens (7, 8) and proteoglycans (9) has been detected in RA patients using assays of lymphocyte transformation and lymphokine production such as leukocyte migration inhibition factor ’ To whom correspondence should be addressed. 264 0090-1229/89 $1.50 Copyright 0 1989 by Academic Press. Inc. AI1 rights of reproduction in any form reserved
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or leukocyte-derived monocyte chemotactic factor. However, these studies utilized bulk or uncloned lymphocyte populations, making it difficult to determine whether a single T cell was capable of responding to CTE or whether T cells with different specificities respond to the various CTE. The present study was undertaken to analyze the specificity and the mechanisms of response to CTE using in vivo-activated interleukin 2 (IL-2) responsive T cells. MATERIALS
AND METHODS
study groups. Ten patients with definite or classical RA according to American Rheumatism Association criteria and 10 normal subjects selected from the hospital staff were studied. The patients were being treated with nonsteroidal antiinflammatory drugs (NSAID) at the time of the study. Preparation of connective tissue elements. The CTE comprising native bovine types I and II collagens and proteoglycans were generous gifts from Drs. K. Morgan, University of Manchester, England, and A. R. Poole, Shriner’s Hospital, McGill University, Montreal, Canada. For the in vitro studies, native bovine types I and II collagens and proteoglycans were dissolved in 0.5 M acetic acid at a concentration of 2 mg/ml, dialyzed against 0.1 M/O. 15 M sodium chloride (NaCl), pH 3.0, at 4°C for 24 hr, and filtered through Millipore (0.8 pm). The inclusion of 0.01 M acetic acid kept the helical collagens in solution for the filtration to be achieved. The antigens were added to give a final concentration of 50 l&ml in the test wells. Control cultures received buffer alone. The use of antigens in 0.01 M acetic acid/O. 15 M NaCl buffer did not alter the pH of the medium or affect cell growth and proliferation. Denatured collagen was prepared by heating the collagens at 56°C for 30 min to unwind the (Ychains. Preparation of mononuclear cells (MNC). MNC were separated from PB and SF by centrifugation on a Ficoll-diatrizoate density gradient (LSM, Litton Bionetics, Kensington, MD) (10). The MNC (i.e., cells at the interface) were washed in saline and Hank’s balanced salt solution (HBSS) (GIBCO, Grand Island, NY) and were cultured in RPM1 1640 (GIBCO) containing L-glutamine, 25 rniV Hepes (KC Biologicals, Lenexa, KS), 100 pg/rnl penicillin G, 100 I&ml streptomycin (GIBCO), 50 l&ml gentamycin (Schering Corp., Kenilworth, NJ), and 10% fetal calf serum (FCS; BDH, Rexdale, Ontario) at 37°C in a humidified 5% CO2 atmosphere. Cloning of IL-2 responsive T lymphocytes. The procedure for cloning such cells is published in detail (11). Briefly, the in vivo-activated T cells from PB and SF were cloned at 12-1000 cells/well in the presence of lo4 autologous-irradiated (50 Gy) PB MNC filler in RPM1 1640 + 10% FCS containing 30 units of IL-2 (obtained from PHA-pulsed human tonsil lymphocytes) medium. Twenty-four replicate microcultures were set up for each cell concentration and the control consisted of irradiated filler cells alone. The cultures were fed on Day 7 with 100 ~1 of IL-Zcontaining medium. The cultures were microscopically scored for growth at Day 14 and those at a limiting
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dilution (12) were subcultured and expanded in partially purified fresh IL 2-containing medium (Electronucleonics Inc., Silver Spring, MD). Prol$2rution assay. T cell clones were examined for CTE-induced proliferation in a 96-hr assay by [‘H]TdR incorporation. Prior to testing, the T cell clones were “rested” for 14 days (i.e., they had not received irradiated filler cells) to clear them of contaminating filler cells. The clones were washed twice in RPM1 1640 t 10% FCS to remove all IL-2. The cellular composition of the rested cultures was checked by staining cytocentrifuge smears of the cells for nonspecific esterase (13). Esterase-positive accessory cell contamination was not seen within the “rested” T cell clones indicating that residual macrophages were not present in the cultures. T cell clones (5 x 104/well) were stimulated with 50 p,g/ml of native and denatured CTE and irrelevant control antigens such as purified protein derivative, ovalbumin, and lysozyme or with 0.1% PHA. These concentrations were chosen because previous studies showed them to be optimum doses. Stimulation was carried out in the presence of irradiated (35 Gy) autologous PB MNC as accessory cells (AC) or in their absence, in 96-well round-bottomed microtiter plates in a total volume of 200 p,l of RPM1 1640 + 10% FCS in the absence of IL-2. A positive control of 3% IL-2 and a negative control of medium (RPM1 1640 + 10% FCS) alone were included in each assay. After a 96-hr incubation, the cultures were pulse-labeled with 0.2 uCi of t3H]TdR for 18 hr and were then harvested onto glass fiber discs with an automatic multiple harvester (PHD System, Cambridge, MA). The level of incorporated radioactivity was assessed by liquid scintillation spectroscopy. The results were expressed as mean counts per minute (cpm) ? standard deviation (SD) of triplicate cultures. In some experiments, the effects of mouse Ia alloantiserum (Cedarlane Labs Ltd., Hornby, Ontario, Canada) which recognizes HLA-DR on the proliferative response to CTE were determined. The anti-Ia antibody was used at I:50 final dilution and was added at the initiation of the culture and left through the culture period. The 1:50 dilution was chosen because it represents the optimum dilution that modulates off the HLA-DR molecules from the surface of the clones. Normal mouse serum (NMS) was used as control and was also diluted 1:50. RESULTS
Specificity ofT cell clones to CTE. The tine specificity of CTE reactivity of IL-2 dependent T cell clones was analyzed by assessment of proliferative responses. As shown in Fig. 1, all the RA T cell clones, irrespective of origin, but not the normal T cell clones, responded polyspecifically to the stimulating antigen, i.e., individual clones responded to all CTE. This polyspecificity may be due to the presence of common or cross-reacting epitopes on the complex multideterminant CTE which the clones recognize or to non-T cell receptor-mediated interactions which induce proliferation. T cell clones were responsive to CTE in the absence of autologous AC. The responses to denatured antigens were greater than negative antigens (Fig. 1). However, with both native and denatured antigens, the ability of T cell clones to stimulate proliferation was consistently lower than that of cultures containing
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FIG. 1. CTE-induced proliferation by IL-2 dependent T cell clones without (A) or with (B) autologous antigen-presenting cells. Triplicate cultures of T cell clones (5 x 104) were stimulated with soluble CTE in the presence of autologous irradiated cells (50 Gy, 104/well) as the source of APC or in the absence of APC in 200 ~1 total volume. Mouse Ia alloantiserum or normal mouse serum 150 final dilution was added to some cultures. After a %-hr incubation, the cells were pulsed-labeled with 0.2 uCi of [3H]TdR for 18 hr, harvested, and counted. The results are expressed as mean counts per minute (cpm). Controls comprised T cell clones cultured in medium alone or in 3% IL-2. SF clones (m), RAPB clones (A), and NPB clones (0).
autologous AC under similar conditions. The control antigens did not cause proliferation, indicating a CTE-restricted response. The kinetics of the CTE-induced proliferation by the T cell clones peaked at 96 hr (data not shown), thus all subsequent proliferation studies were harvested at 96 hr in culture. Proliferative response to PHA and IL-2. The proliferative responses to PHA and IL-2 are also shown in Fig. 1. It can be seen that there was no detectable response to PHA by 96 hr in culture. This lack of PHA response was not due to the inability of the T cell clones to respond to the mitogen, but rather due to the kinetics of the response, for the optimum proliferation to PHA occurred between 24 and 48 hr in culture (Fig. 2). There was no significant difference between RA PB and SF clones in their ability to respond to PHA except that the RA PB clone responses to PHA peaked at 24 hr while SF clone responses peaked at 48 hr. Normal PB T cell clone responses to PHA also peaked at 48 hr. The responses to exogenous IL-2 were similar between normal PB and RA T cell clones, but there was a tendency for SF T cell clones to have higher proliferative responses. EfSect of anti-la on CTE-induced proliferation. As an approach to determining the mechanism of proliferation, anti-Ia antibody (150 dilution) was added to the
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IN CULTURE
FIG. 2. Kinetics of PHA response of T cell clones. T cell clones (5 x 104/200 p.1) were cultured in the presence of 0.1% PHA-P in round-bottomed microtiter plates. On the indicated hours of culture, the cultures were pulsed-labeled with 0.2 pCi of [‘H]TdR for 6 hr, harvested, and counted. Data are expressed as mean counts per minute (cpm) -t standard deviation (SD) of triplicate cultures. Control consisted of T cells clones cultured in medium alone with cpm values of 700-1000 SF clones (W), RAPB clones (A), and NP clones (0). N = 3 in each group.
denatured type II collagen (DII)-stimulated T cells in the presence or absence of AC. The DII collagen was chosen because it represents the antigen that induces the strongest response. The addition of anti-Ia antibody to the culture system abrogated the proliferative response to DII collagen (Fig. I), implying that HLADR molecules are critically involved in the CTE-induced proliferative response. No inhibition was seen with a control normal mouse serum or with the Ia alloantiserum in the mitogenic response induced by PHA. DISCUSSION
Considerable evidence has indicated the existence of autoreactive T cells for CTE in RA patients (7-9) and normal subjects (14). However, these experiments used uncloned lymphocytes and demonstrated polyspecific responses to CTE, but it was not clear whether a single cell type was recognizing and responding to CTE or if cells with different specificities were responding to the CTE. The present studies were undertaken to determine the fine specificity of in Go-activated, IL-2 responsive T cells to CTE. The rationale for using spontaneously activated, IL-2 responsive T cells was that as these cells have been activated in viva, definition of
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their specificities might provide evidence as to the inducing stimuli. Our results demonstrate that RA T cell clones proliferated polyspecifically to CTE either in the presence or absence of AC, with denatured antigens giving stronger stimuli than native antigens. We did not detect any proliferative responses to CTE in the normal T cell clones. Previous studies, however, showed that CTE can stimulate both RA and normal T cell clones to secrete interferon-y with RA clones showing the greatest reactivity (15). The lack of proliferation of normal T cell clones to CTE suggests that antigen-induced proliferation may either be a less sensitive measurement of CTE reactivity than lymphokine production or that the two processes do not necessarily reflect the same activation pathway. Indeed, other investigators have reported the determination of lymphocyte transformation as a measure of CTE reactivity is, on the whole, less sensitive than lymphokine production (7,9). Alternatively, it is also possible that functional activation of T cells, such as the release of lymphokines and proliferation, may relate to the number of signals required. The present results also further suggest that on the basis of precursor frequency data, the pool of CTE-reactive T cells in the PB and SF of RA patients is larger than those in normal donor PB. The fact that all RA T cell clones, irrespective of origin, proliferated to CTE suggests the existence of common or cross-reacting epitopes on the CTE or that a non-T cell receptor-mediated process is required for stimulation. If the CTE were mitogenic for the T cell clones, it would appear that only a subpopulation of T cells are responsive because normal T cell clones did not proliferate following exposure to CTE. It is noteworthy that recent studies by Sanders et al. (16) have indicated differences in responsiveness of memory and primary human T cells to antigenic or mitogenic stimulus. It is of interest that the proliferation of the RA clones was apparently Ia dependent. Various Ia+ T cell types are capable of presenting antigen (17) and recent reports indicate that Ia + T cells can present a variety of antigens. Ben-Nun et al. (18) showed that irradiated Ia+ T cell clones functioned effectively to stimulate beef insulin-specific T cell clones. Similarly, Triebel et al. (19) also showed that diphtheria toxoid (DT)-specific T cell clones expressing HLA-DR, DQ, DP, and Tat antigens can stimulate in mixed lymphocyte reactions, autologous mixed lymphocyte reactions, primed lymphocyte testing, and can present antigen in DT-specific proliferative responses. These proliferative responses were inhibited by preincubation of the T cells with anti-HLA-DR antibody, suggesting that the stimulatory molecules were predominantly DR antigens or that these antigens were essential to the response. The ability of the Ia+ , IL-2 responsive T cell clones to proliferate in response to denatured antigen more effectively than native antigen suggests that the T cells may not be able to process soluble antigens as efficiently as classical AC. Thus, although the T cell clones could respond to both native and denatured collagen in the absence of AC, the responses were consistently less than those seen with autologous AC under identical conditions. Our data are in accordance with the findings of Gerrard et al. (20) who showed that activated T cells presented denatured key hole limpet hemocyanin and tetanus toxoid more effectively than native antigens and that the responses were always less than those induced by conventional AC; as well as those of Stuart et al. (8) that immune reactivity in collagen
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in RA patients was usually greater to denatured than native collagen, suggesting that the responses to collagen may develop secondarily to tissue degradation. There have been some suggestions that denatured antigen may mimic antigen processing in that certain sites on the antigen molecule, which were previously masked, will be unmasked. Indeed, work by Allen and Unanue (21) indicated that denatured lysozyme bypasses the need for antigen processing in presentation to lysozyme-specific T cell hybridomas. Thus, changes in protein configuration, such as the unfolding of native antigens as a result of denaturation, rather than further reduction in size of the native molecule by proteolytic cleavage is critical in determining the need for antigen processing (22). It is conceivable that the la + , IL-2 responsive T cell clones responded to native soluble antigens with the release of proteolytic enzymes or that interaction of the native antigen with HLA-DR (Ia) molecules partially denatures the antigen. It is also possible that the T cells were responding to low levels of denatured antigens that were present in the native antigen during the antigenic preparation. The present results do not allow for the differentiation between an antigenspecific phenomenon mediated by the T cell receptor complex or for a process in which a nonclonally restricted receptor(s) for extracellular matrix is expressed. The plausibility of the latter suggestion would seem to be enhanced by the recent description of a series of structurally related cell surface antigens of the VLA family (23). These antigens are related to receptors for extracellular matrix components and are known to be expressed in a nonclonally restricted fashion on leukocytes. However, evidence of a functional consequence of the interaction of these receptors with ligand has not been reported. Regardless of the nature of the extracellular matrix recognition by lymphocytes, it is clear that the interaction results in the generation of a functional signal. The ability of Ia+ T cells to effectively respond to denatured collagen, which is abundant in the inflamed joint, may have important implications in the perpetuation of the chronic inflammation in RA. Studies have shown that the majority of nonlymphoid synovial cells, despite the presence of large amounts of surface HLA-DR (Ia) antigens, are not potent inducers of T cell proliferation but rather strong suppressors of polyclonal T cell activation (24). Thus, Ia+ T cells may complement synovial cells in the T cell proliferation seen in synovial tissues, thereby functioning to amplify the immune response to CTE in the joint. In conclusion, we have shown that Ia-t-, IL-2 responsive T cell clones derived from PB and the joint in RA respond to CTE polyspecifically by proliferating in the presence or absence of AC, although the ability of the T cell clones to enhance CTE-induced proliferation was always less than with classical AC. This ability of Ia+ T cells to respond to antigens may represent an important amplification mechanism in the inflammatory process in the joint. The importance of this cellular pathway in the pathogenesis of RA remains to be more clearly defined. ACKNOWLEDGMENTS We thank Drs. F. Baragar, 1. Chalmers, and T. Hunter of the Rheumatic Diseases Unit, University of Manitoba, for providing the synovial fluid samples. We also thank Mrs. Theresa Coulson for typing the manuscript. This work was supported by a grant from the Canadian Arthritis Society.
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REFERENCES 1. Burmester, G. R., Yu, D. T., Irani. A. M., Kunkel, H. G.. and Winchester, R. J., Arthritis Rheum. 24, 1370, 1981. 2. Forre, O., Doubloug, J. H., and Natvig, J. B., Scnnd. J. Zmmunol. 15, 227, 1982. 3. Kluin-Nelemans, H. C., Vander-Linder, J. A., Gmelig-Meylig, F. H. J., and Schuman, H. J., J. Rheum. 11, 272, 1984. 4. Burmester, G. R., John, B., Gramatzki, M., Zaker. J., and Kalden, J. R., J. Zmmunol. 133, 1984. 5. Trentham, D. W., Toews, A. S., and Kang. A. H., J. Exp. Med. 146, 857, 1977. 6. Courtenay, J. S., Dallman, M. J., Dayan, A. D.. Martin, A.. and Mosedale, B., Nature (London) 283, 666, 1980. 7. Trentham, D. E., Dynesius, R. A., Rocklin, R. E.. and David, J. R., N. Engl. J. Med. 229, 327, 1978.
8. 9. 10. 11. 12. 13.
14. IS. 16. 17. 18. 19. 20.
Stuart, J. M., Postlethwaite, A. E., Townes, A. S., and Kang, A. H., Amer. J. Med. 69, 13, 1980. Glant, T., Csongor, J., and Szucs, T., Stand. J. Zmmunol. 11, 247, 1980. Boyum, A., Stand. J. Clin. Lab. Invest. 21, Suppl. 97, 77, 1968. Ofosu-Appiah, W. A., McKenna, R. M., Warrington. R. J., and Wilkins, J. A., Clin. Exp. Zmmunol. 64, 555, 1986. Lefkovits, I., and Waldmann, R., Cambridge Univ. Press, Cambridge, 1979. Yam, L. T., Li, C. Y., and Crosby, W. H., Amer. J. Clin. Pathol. 55, 283, 1971. Solinger, A. M., Bhatnagar, R., and Stobo, J. D., Proc. Natl. Acad. Sci. USA 78, 3877, 1981. Ofosu-Appiah, W. A., Warrington, R. J., Morgan, K., and Wilkins, J. A., J. Zmmunol., submitted for publication, 1988. Sanders, M. E., and Makgoba, M. W.. Sharrow, S. 0.. Stephany, D., Springer, T. A., Young, H. A.. and Shaw, S., J. Zmmunol. 140, 1401, 1988. Unaue, E., Beller, D. I., Lu, C. Y., and Allen, P. M., J. Zmmunol. 132, 1, 1948. Ben-Nun, A., Strauss, W., Leeman, S. A., Cohn, L. E., Murre, C., Duby. A., Seidman, J. G., and Glimcher, L. H., Immunogenetics 22, 123, 1985. Triebel, F., DeRoguefeuil, S., Blanc, C., Charron, D. J., and Debre, P., Hum. Zmmunol. 15, 202, 1986. Gerrard, T. L., Volkman, D. J., Jurgensen, C. H., and Fauci, A. S.. Hum. Zmmunol. 17, 416, 1986.
21. Allen, P. M., and Unanue, E. R., J. Zmmunol. 132, 1077, 1984. 22. Streicher, H. Z., Berkower, I. J., Busch, M., Gurd, F. R. N., and Berzofsky, J. A.. Proc. Narl. Acad. Sci. USA 81, 6831, 1984. 23. Hemler. M. E., Zmmunol. Today 9, 109, 1988. 24. Burmester, G. R.. Schneeberger, J., Gramatzki, J. B., Zacher, J., and Kalden, J. R., Rheumatol. Znt.4,
31, 1984.
Received July 5, 1988; accepted with revision October 28. 1988
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
50,
264-271 ( 1989)
BRIEF COMMUNICATION lnterleukin 2 Responsive T Cell Clones from Rheumatoid and Normal Subjects: Proliferative Responses to Connective Tissue Elements WILLIAM
A. OFOSU-APPIAH,*~
RICHARD J. WARRINGTON,*+$ AND JOHN A. WILKINS*~$~§~'
*Rheumatic Diseases Research Laboratory, Departments of flmmunology, itMedicine, and §Medical Microbiology, RR014 800 Sherbrook Street. Winnipeg, Manitoba, Canada R3A lM4 In vivo-activated interleukin 2 responsive T cell clones were generated from peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients and from normal control PB. The specificity of these clones was assessed by measuring proliferation induced by the connective tissue elements (CTE) collagen types I and II, native and denatured, proteoglycans, and irrelevant control antigens. The cloned T cells from RA patients but not from normal subjects responded in vitro with proliferation to all CTE but not to control antigens purified protein derivative, ovalbumin, or lysozyme. Proliferation occurred in the presence and absence of accessory cells (AC), but the responses were consistently higher in the presence of AC. Antibodies to HLA-DR abrogated the proliferative response to CTE suggesting that DR expression was necessary for the induction of proliferation. These findings demonstrate the existence of clonable T cells responsive to CTE in PB and SF of RA patients. Expression of reactivity to CTE may contribute to the chronicity of the inflammation in RA. Q 1989 Academic Press. hc.
INTRODUCTION
Previous studies have shown that an increased proportion of T cells infiltrating the synovial tissue (ST) and fluid (SF) (1, 2) and present in peripheral blood (PB) of rheumatoid arthritis (RA) patients (3,4) are activated as defined by the expression of HLA-DR (Ia) and Tat antigens. The nature of the antigen(s) that stimulates the T cells is unknown. There is evidence that cell-mediated autoimmunity may play a fundamental role in the chronic inflammation seen in RA. These cellular immune responses may develop as a result of autoimmunity to connective tissue elements (CTE). The induction of an intlammatory polyarthritis in rats (5) and mice (6) by injection of homologous type II collagen from cartilage demonstrates the importance of an immune response to cartilage components in the induction of experimental arthritis. This raises the possibility that a similar autoimmune process could contribute to the joint inflammation in RA. Cell-mediated autoimmunity to the interstitial collagens (7, 8) and proteoglycans (9) has been detected in RA patients using assays of lymphocyte transformation and lymphokine production such as leukocyte migration inhibition factor ’ To whom correspondence should be addressed. 264 0090-1229/89 $1.50 Copyright 0 1989 by Academic Press. Inc. AI1 rights of reproduction in any form reserved
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or leukocyte-derived monocyte chemotactic factor. However, these studies utilized bulk or uncloned lymphocyte populations, making it difficult to determine whether a single T cell was capable of responding to CTE or whether T cells with different specificities respond to the various CTE. The present study was undertaken to analyze the specificity and the mechanisms of response to CTE using in vivo-activated interleukin 2 (IL-2) responsive T cells. MATERIALS
AND METHODS
study groups. Ten patients with definite or classical RA according to American Rheumatism Association criteria and 10 normal subjects selected from the hospital staff were studied. The patients were being treated with nonsteroidal antiinflammatory drugs (NSAID) at the time of the study. Preparation of connective tissue elements. The CTE comprising native bovine types I and II collagens and proteoglycans were generous gifts from Drs. K. Morgan, University of Manchester, England, and A. R. Poole, Shriner’s Hospital, McGill University, Montreal, Canada. For the in vitro studies, native bovine types I and II collagens and proteoglycans were dissolved in 0.5 M acetic acid at a concentration of 2 mg/ml, dialyzed against 0.1 M/O. 15 M sodium chloride (NaCl), pH 3.0, at 4°C for 24 hr, and filtered through Millipore (0.8 pm). The inclusion of 0.01 M acetic acid kept the helical collagens in solution for the filtration to be achieved. The antigens were added to give a final concentration of 50 l&ml in the test wells. Control cultures received buffer alone. The use of antigens in 0.01 M acetic acid/O. 15 M NaCl buffer did not alter the pH of the medium or affect cell growth and proliferation. Denatured collagen was prepared by heating the collagens at 56°C for 30 min to unwind the (Ychains. Preparation of mononuclear cells (MNC). MNC were separated from PB and SF by centrifugation on a Ficoll-diatrizoate density gradient (LSM, Litton Bionetics, Kensington, MD) (10). The MNC (i.e., cells at the interface) were washed in saline and Hank’s balanced salt solution (HBSS) (GIBCO, Grand Island, NY) and were cultured in RPM1 1640 (GIBCO) containing L-glutamine, 25 rniV Hepes (KC Biologicals, Lenexa, KS), 100 pg/rnl penicillin G, 100 I&ml streptomycin (GIBCO), 50 l&ml gentamycin (Schering Corp., Kenilworth, NJ), and 10% fetal calf serum (FCS; BDH, Rexdale, Ontario) at 37°C in a humidified 5% CO2 atmosphere. Cloning of IL-2 responsive T lymphocytes. The procedure for cloning such cells is published in detail (11). Briefly, the in vivo-activated T cells from PB and SF were cloned at 12-1000 cells/well in the presence of lo4 autologous-irradiated (50 Gy) PB MNC filler in RPM1 1640 + 10% FCS containing 30 units of IL-2 (obtained from PHA-pulsed human tonsil lymphocytes) medium. Twenty-four replicate microcultures were set up for each cell concentration and the control consisted of irradiated filler cells alone. The cultures were fed on Day 7 with 100 ~1 of IL-Zcontaining medium. The cultures were microscopically scored for growth at Day 14 and those at a limiting
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dilution (12) were subcultured and expanded in partially purified fresh IL 2-containing medium (Electronucleonics Inc., Silver Spring, MD). Prol$2rution assay. T cell clones were examined for CTE-induced proliferation in a 96-hr assay by [‘H]TdR incorporation. Prior to testing, the T cell clones were “rested” for 14 days (i.e., they had not received irradiated filler cells) to clear them of contaminating filler cells. The clones were washed twice in RPM1 1640 t 10% FCS to remove all IL-2. The cellular composition of the rested cultures was checked by staining cytocentrifuge smears of the cells for nonspecific esterase (13). Esterase-positive accessory cell contamination was not seen within the “rested” T cell clones indicating that residual macrophages were not present in the cultures. T cell clones (5 x 104/well) were stimulated with 50 p,g/ml of native and denatured CTE and irrelevant control antigens such as purified protein derivative, ovalbumin, and lysozyme or with 0.1% PHA. These concentrations were chosen because previous studies showed them to be optimum doses. Stimulation was carried out in the presence of irradiated (35 Gy) autologous PB MNC as accessory cells (AC) or in their absence, in 96-well round-bottomed microtiter plates in a total volume of 200 p,l of RPM1 1640 + 10% FCS in the absence of IL-2. A positive control of 3% IL-2 and a negative control of medium (RPM1 1640 + 10% FCS) alone were included in each assay. After a 96-hr incubation, the cultures were pulse-labeled with 0.2 uCi of t3H]TdR for 18 hr and were then harvested onto glass fiber discs with an automatic multiple harvester (PHD System, Cambridge, MA). The level of incorporated radioactivity was assessed by liquid scintillation spectroscopy. The results were expressed as mean counts per minute (cpm) ? standard deviation (SD) of triplicate cultures. In some experiments, the effects of mouse Ia alloantiserum (Cedarlane Labs Ltd., Hornby, Ontario, Canada) which recognizes HLA-DR on the proliferative response to CTE were determined. The anti-Ia antibody was used at I:50 final dilution and was added at the initiation of the culture and left through the culture period. The 1:50 dilution was chosen because it represents the optimum dilution that modulates off the HLA-DR molecules from the surface of the clones. Normal mouse serum (NMS) was used as control and was also diluted 1:50. RESULTS
Specificity ofT cell clones to CTE. The tine specificity of CTE reactivity of IL-2 dependent T cell clones was analyzed by assessment of proliferative responses. As shown in Fig. 1, all the RA T cell clones, irrespective of origin, but not the normal T cell clones, responded polyspecifically to the stimulating antigen, i.e., individual clones responded to all CTE. This polyspecificity may be due to the presence of common or cross-reacting epitopes on the complex multideterminant CTE which the clones recognize or to non-T cell receptor-mediated interactions which induce proliferation. T cell clones were responsive to CTE in the absence of autologous AC. The responses to denatured antigens were greater than negative antigens (Fig. 1). However, with both native and denatured antigens, the ability of T cell clones to stimulate proliferation was consistently lower than that of cultures containing
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FIG. 1. CTE-induced proliferation by IL-2 dependent T cell clones without (A) or with (B) autologous antigen-presenting cells. Triplicate cultures of T cell clones (5 x 104) were stimulated with soluble CTE in the presence of autologous irradiated cells (50 Gy, 104/well) as the source of APC or in the absence of APC in 200 ~1 total volume. Mouse Ia alloantiserum or normal mouse serum 150 final dilution was added to some cultures. After a %-hr incubation, the cells were pulsed-labeled with 0.2 uCi of [3H]TdR for 18 hr, harvested, and counted. The results are expressed as mean counts per minute (cpm). Controls comprised T cell clones cultured in medium alone or in 3% IL-2. SF clones (m), RAPB clones (A), and NPB clones (0).
autologous AC under similar conditions. The control antigens did not cause proliferation, indicating a CTE-restricted response. The kinetics of the CTE-induced proliferation by the T cell clones peaked at 96 hr (data not shown), thus all subsequent proliferation studies were harvested at 96 hr in culture. Proliferative response to PHA and IL-2. The proliferative responses to PHA and IL-2 are also shown in Fig. 1. It can be seen that there was no detectable response to PHA by 96 hr in culture. This lack of PHA response was not due to the inability of the T cell clones to respond to the mitogen, but rather due to the kinetics of the response, for the optimum proliferation to PHA occurred between 24 and 48 hr in culture (Fig. 2). There was no significant difference between RA PB and SF clones in their ability to respond to PHA except that the RA PB clone responses to PHA peaked at 24 hr while SF clone responses peaked at 48 hr. Normal PB T cell clone responses to PHA also peaked at 48 hr. The responses to exogenous IL-2 were similar between normal PB and RA T cell clones, but there was a tendency for SF T cell clones to have higher proliferative responses. EfSect of anti-la on CTE-induced proliferation. As an approach to determining the mechanism of proliferation, anti-Ia antibody (150 dilution) was added to the
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FIG. 2. Kinetics of PHA response of T cell clones. T cell clones (5 x 104/200 p.1) were cultured in the presence of 0.1% PHA-P in round-bottomed microtiter plates. On the indicated hours of culture, the cultures were pulsed-labeled with 0.2 pCi of [‘H]TdR for 6 hr, harvested, and counted. Data are expressed as mean counts per minute (cpm) -t standard deviation (SD) of triplicate cultures. Control consisted of T cells clones cultured in medium alone with cpm values of 700-1000 SF clones (W), RAPB clones (A), and NP clones (0). N = 3 in each group.
denatured type II collagen (DII)-stimulated T cells in the presence or absence of AC. The DII collagen was chosen because it represents the antigen that induces the strongest response. The addition of anti-Ia antibody to the culture system abrogated the proliferative response to DII collagen (Fig. I), implying that HLADR molecules are critically involved in the CTE-induced proliferative response. No inhibition was seen with a control normal mouse serum or with the Ia alloantiserum in the mitogenic response induced by PHA. DISCUSSION
Considerable evidence has indicated the existence of autoreactive T cells for CTE in RA patients (7-9) and normal subjects (14). However, these experiments used uncloned lymphocytes and demonstrated polyspecific responses to CTE, but it was not clear whether a single cell type was recognizing and responding to CTE or if cells with different specificities were responding to the CTE. The present studies were undertaken to determine the fine specificity of in Go-activated, IL-2 responsive T cells to CTE. The rationale for using spontaneously activated, IL-2 responsive T cells was that as these cells have been activated in viva, definition of
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their specificities might provide evidence as to the inducing stimuli. Our results demonstrate that RA T cell clones proliferated polyspecifically to CTE either in the presence or absence of AC, with denatured antigens giving stronger stimuli than native antigens. We did not detect any proliferative responses to CTE in the normal T cell clones. Previous studies, however, showed that CTE can stimulate both RA and normal T cell clones to secrete interferon-y with RA clones showing the greatest reactivity (15). The lack of proliferation of normal T cell clones to CTE suggests that antigen-induced proliferation may either be a less sensitive measurement of CTE reactivity than lymphokine production or that the two processes do not necessarily reflect the same activation pathway. Indeed, other investigators have reported the determination of lymphocyte transformation as a measure of CTE reactivity is, on the whole, less sensitive than lymphokine production (7,9). Alternatively, it is also possible that functional activation of T cells, such as the release of lymphokines and proliferation, may relate to the number of signals required. The present results also further suggest that on the basis of precursor frequency data, the pool of CTE-reactive T cells in the PB and SF of RA patients is larger than those in normal donor PB. The fact that all RA T cell clones, irrespective of origin, proliferated to CTE suggests the existence of common or cross-reacting epitopes on the CTE or that a non-T cell receptor-mediated process is required for stimulation. If the CTE were mitogenic for the T cell clones, it would appear that only a subpopulation of T cells are responsive because normal T cell clones did not proliferate following exposure to CTE. It is noteworthy that recent studies by Sanders et al. (16) have indicated differences in responsiveness of memory and primary human T cells to antigenic or mitogenic stimulus. It is of interest that the proliferation of the RA clones was apparently Ia dependent. Various Ia+ T cell types are capable of presenting antigen (17) and recent reports indicate that Ia + T cells can present a variety of antigens. Ben-Nun et al. (18) showed that irradiated Ia+ T cell clones functioned effectively to stimulate beef insulin-specific T cell clones. Similarly, Triebel et al. (19) also showed that diphtheria toxoid (DT)-specific T cell clones expressing HLA-DR, DQ, DP, and Tat antigens can stimulate in mixed lymphocyte reactions, autologous mixed lymphocyte reactions, primed lymphocyte testing, and can present antigen in DT-specific proliferative responses. These proliferative responses were inhibited by preincubation of the T cells with anti-HLA-DR antibody, suggesting that the stimulatory molecules were predominantly DR antigens or that these antigens were essential to the response. The ability of the Ia+ , IL-2 responsive T cell clones to proliferate in response to denatured antigen more effectively than native antigen suggests that the T cells may not be able to process soluble antigens as efficiently as classical AC. Thus, although the T cell clones could respond to both native and denatured collagen in the absence of AC, the responses were consistently less than those seen with autologous AC under identical conditions. Our data are in accordance with the findings of Gerrard et al. (20) who showed that activated T cells presented denatured key hole limpet hemocyanin and tetanus toxoid more effectively than native antigens and that the responses were always less than those induced by conventional AC; as well as those of Stuart et al. (8) that immune reactivity in collagen
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in RA patients was usually greater to denatured than native collagen, suggesting that the responses to collagen may develop secondarily to tissue degradation. There have been some suggestions that denatured antigen may mimic antigen processing in that certain sites on the antigen molecule, which were previously masked, will be unmasked. Indeed, work by Allen and Unanue (21) indicated that denatured lysozyme bypasses the need for antigen processing in presentation to lysozyme-specific T cell hybridomas. Thus, changes in protein configuration, such as the unfolding of native antigens as a result of denaturation, rather than further reduction in size of the native molecule by proteolytic cleavage is critical in determining the need for antigen processing (22). It is conceivable that the la + , IL-2 responsive T cell clones responded to native soluble antigens with the release of proteolytic enzymes or that interaction of the native antigen with HLA-DR (Ia) molecules partially denatures the antigen. It is also possible that the T cells were responding to low levels of denatured antigens that were present in the native antigen during the antigenic preparation. The present results do not allow for the differentiation between an antigenspecific phenomenon mediated by the T cell receptor complex or for a process in which a nonclonally restricted receptor(s) for extracellular matrix is expressed. The plausibility of the latter suggestion would seem to be enhanced by the recent description of a series of structurally related cell surface antigens of the VLA family (23). These antigens are related to receptors for extracellular matrix components and are known to be expressed in a nonclonally restricted fashion on leukocytes. However, evidence of a functional consequence of the interaction of these receptors with ligand has not been reported. Regardless of the nature of the extracellular matrix recognition by lymphocytes, it is clear that the interaction results in the generation of a functional signal. The ability of Ia+ T cells to effectively respond to denatured collagen, which is abundant in the inflamed joint, may have important implications in the perpetuation of the chronic inflammation in RA. Studies have shown that the majority of nonlymphoid synovial cells, despite the presence of large amounts of surface HLA-DR (Ia) antigens, are not potent inducers of T cell proliferation but rather strong suppressors of polyclonal T cell activation (24). Thus, Ia+ T cells may complement synovial cells in the T cell proliferation seen in synovial tissues, thereby functioning to amplify the immune response to CTE in the joint. In conclusion, we have shown that Ia-t-, IL-2 responsive T cell clones derived from PB and the joint in RA respond to CTE polyspecifically by proliferating in the presence or absence of AC, although the ability of the T cell clones to enhance CTE-induced proliferation was always less than with classical AC. This ability of Ia+ T cells to respond to antigens may represent an important amplification mechanism in the inflammatory process in the joint. The importance of this cellular pathway in the pathogenesis of RA remains to be more clearly defined. ACKNOWLEDGMENTS We thank Drs. F. Baragar, 1. Chalmers, and T. Hunter of the Rheumatic Diseases Unit, University of Manitoba, for providing the synovial fluid samples. We also thank Mrs. Theresa Coulson for typing the manuscript. This work was supported by a grant from the Canadian Arthritis Society.
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Received July 5, 1988; accepted with revision October 28. 1988