Adoptively Transferred EAE in Mice Bearing thelprMutation

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 85, No. 3, December, pp. 315–319, 1997 Article No. II974450

RAPID COMMUNICATION Adoptively Transferred EAE in Mice Bearing the lpr Mutation1 Robert B. Clark,* Margaret Grunnet,† and Elizabeth G. Lingenheld* *Division of Rheumatic Diseases and †Department of Pathology, University of Connecticut School of Medicine, Farmington, Connecticut 06032

We have recently developed approaches for the generation of encephalitogenic T cell clones from mouse strains considered resistant to experimental allergic encephalomyelitis (EAE). By allowing for the direct use of knockout and mutant strains of mice, such clones allow for the efficient characterization of the relevance of specific gene products in the effector phase of EAE. Recent studies have suggested that Fas/ FasL-mediated cell death may play a role in the pathogenesis of MS. To assess the role of Fas/FasL in EAE, we have tested the ability of wild-type C57BL/6-derived, encephalitogenic T cell clones to mediate adoptively transferred EAE in Fas-deficient C57BL/6-lpr mice. We now report that mice with the lpr mutation are fully susceptible to the adoptive transfer of EAE. Our results suggest that Fas/FasL-mediated cell death in the central nervous system does not play an integral role in the effector phase of acute EAE. q 1997 Academic Press INTRODUCTION

Experimental allergic encephalomyelitis (EAE)2 is a well-characterized animal model for the human disease multiple sclerosis (MS). Because murine EAE is inducible in only a small number of mouse strains, most knockout mice and mice with naturally occurring mutations are on genetic backgrounds that are associated with resistance to EAE. While EAE can be studied in such mice that have been crossed onto genetic backgrounds that are susceptible to EAE (1–4), such breeding takes a significant amount of time and effort. We have recently described two approaches which allow for the efficient generation of encephalitogenic T cell lines and clones from EAE-resistant murine strains such as C57BL/6 and C3H/HeJ.3 By facilitating the 1 These studies were supported by a University of Connecticut Medical School Faculty Research Grant. 2 Abbreviations used: EAE, experimental allergic encephalomyelitis; MS, multiple sclerosis; PMBP, porcine myelin basic protein; MMBP, murine myelin basic protein; CNS, central nervous system; MM, mouse media. 3 Clark, R. B., Grunnet, M., and Lingenheld, E., The generation of encephalitogenic T cell lines from EAE-resistant strains of mice, Int. Immunol., in press.

study of EAE in knockout and mutant mice, these T cell lines and clones now make possible the identification of the relevance of many gene products in the effector phase of EAE. While EAE and MS are considered to be autoimmune in nature, the mechanisms underlying tissue injury in these diseases are not well understood. T cell cytokine production has been thought to play a role in the tissue injury seen in MS and EAE (5). In addition, there are reports in both EAE and MS suggesting that T cell cytotoxicity may be required in the pathogenesis of these diseases, although the specific mechanisms of the T cell cytotoxicity have not been identified (6, 7). Recently, studies have suggested that the Fas/FasL system, which has been demonstrated to be an important pathway for the induction of cell death by apoptosis, may play a role in the pathogenesis of MS (8, 9). We have now studied the role played by Fas/FasL in the effector phase of acute murine EAE and report that lpr mice, despite the lack of expression of Fas, are susceptible to adoptively transferred EAE. MATERIALS AND METHODS

Mice. Wild-type C57BL/6 mice and C57BL/6-lpr mice were purchased from Jackson Laboratories. Antigens. Porcine myelin basic protein (PMBP) was obtained as a gift from Eli Lilly Co. (Indianapolis, IN). Murine myelin basic protein (MMBP) was prepared in our laboratory as previously described (10). Generation of MBP-specific T cell lines and clones. MBP-specific T cell lines and clones were generated using methods that we have previously described (see footnote 3). Briefly, wild-type C57BL/6 mice were immunized using 100 mg of PMBP emulsified in CFA. Eight to 10 days later, lymph node suspensions were cultured in ‘‘mouse media’’ (MM) (RPMI 1640 with 10% fetal calf serum and 5 1 1005 M 2-mercaptoethanol) without IL-2, along with 25 mg/ml of PMBP and in the presence of irradiated (2600 R) syngeneic splenocytes. Three days after initiation, these cells were restimulated in MM with IL-2 (recombinant human IL-2, 16–

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20 units/ml, Genzyme, Inc.), using irradiated syngeneic splenocytes (5 1 106/well) and PMBP (25 mg/ml). The T cell lines were then propagated from these cultures using a 2-week growth cycle involving antigenic stimulation (APC and PMBP) followed by a week in IL-2, followed by a week of culture in the absence of IL-2. Adoptive transfer of EAE. The T cell line C-A-46 and the T cell clone A-C-2-4 were used to adoptively transfer EAE as we have previously described (10, 11). Unirradiated, naive, wild-type C57BL/6 or C57BL/6lpr mice were the recipients. T cells were injected via intracardiac puncture. The number of T cells injected was 12–13 1 106 for A-C-2-4 and 16 1 106 cells for CA-46. Mice received one injection of T cells and then received no other treatments or interventions. All recipient C57BL/6 wild-type mice were females and were 4–8 weeks old when studied. All recipient C57BL/6lpr mice were males and were 6–12 weeks old when studied. All mice were followed for signs of EAE for a minimum of 3 months after the single injection of T cells. EAE was graded as previously described (10): Grade 1, flaccid tail; grade 2, leg weakness and gait abnormality; grade 3, hind leg dragging; grade 4, hind leg dragging with front leg weakness; grade 5, death. Evaluation of CNS histopathology. The pathological specimens were obtained and the CNS histopathology evaluated blindly as we have previously described (11). Antigen-specific proliferative assays. The T cell line and clone were assayed for antigen-specific proliferation in 3-day [3H]thymidine assays as previously described (10, 11). Assay for cytokine secretion. T cells (0.5 1 106/ml) were plated in MM in the absence of IL-2 in 24well plates that had been coated with 10 mg/ml antimurine CD3 monoclonal antibody (Pharmingen, Inc.). After 3 days, supernatants from these cultures were harvested, filtered, and frozen until assayed. Assays for murine interferon- g and murine TNF-a were performed using cytokine-specific ELISAs (Genzyme, Inc.). RESULTS

Characterization of Encephalitogenic T Cell Lines and Clones We have recently generated myelin basic proteinreactive, encephalitogenic T cell lines from the EAEresistant, C57BL/6 mice (see footnote 3). In the present studies we have utilized either one T cell subline (CA-46) or one T cell clone (A-C-2-4) to mediate the transfer of EAE. Both C-A-46 and A-C-2-4 were derived from encephalitogenic T cell lines generated from a wildtype C57BL/6 mouse immunized with porcine MBP

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(PMBP). The T cell line C-A-46 was derived by limiting dilution cultures plated at 5 cells per well. The T cell clone A-C-2-4 was derived by limiting dilution cultures plated at 1 cell per well. Both C-A-46 and A-C-2-4 are CD4-positive T cells (data not shown). C-A-46 and AC-2-4 proliferate in vitro when stimulated by PMBP in the context of C57BL/6 APC. In addition, both C-A-46 and A-C-2-4 also proliferate in vitro to the autoantigen, murine MBP (MMBP) (Table 1). Both C-A-46 and AC-2-4 secrete tumor necrosis factor and interferon-g, making them Type 1 helper T cells (Table 1). Adoptively Transferred EAE We have utilized C-A-46 and A-C-2-4 to compare the mediation of adoptively transferred EAE in wild-type C57BL/6 versus C57BL/6-lpr mice. Using an anti-murine Fas antibody, we first confirmed that cells derived from our C57BL/6-lpr mice lacked Fas expression (data not shown). We compared the mediation of EAE in wild-type C57BL/6 mice versus C57BL/6-lpr mice after a single intracardiac injection of either C-A-46 or A-C2-4. As can be seen in Fig. 1, the intracardiac injection of either C-A-46 or A-C-2-4 led to consistent and significant clinical signs of EAE in the wild-type C57BL/ 6 mice. These clinical signs ranged from significant leg weakness with an abnormal gait (grade 2.5 EAE) to death. The average maximal EAE clinical grade of the wild-type mice in these studies was 3.78. Significantly, the T cell line and clone also mediated severe EAE in the C57BL/6-lpr mice. The clinical signs in the C57BL/ 6-lpr mice ranged from tail weakness (grade 1 EAE) to death. The adoptive transfer of the encephalitogenic T cells into the C57BL/6-lpr recipients resulted in death (grade 5 EAE) in almost one-half of the C57BL/6-lpr mice studied (Fig. 1). The average maximal EAE clinical grade of EAE in C57BL/6-lpr mice was 3.12. The small difference in the average maximal clinical grade between the wild-type C57BL/6 mice and C57BL/6-lpr mice was possibly related to the fact that the lpr mice were on average about 4 weeks older and larger than the wild-type mice. Both C-A-46 and A-C-2-4 mediate EAE in wild-type and C57BL/6-lpr mice after a single intracardiac injection of 12–16 1 106 cells, and do not require irradiation or any treatment of the naive, recipient mouse. The EAE in the C57BL/6-lpr mice manifested with clinical signs that were identical to those seen in the wild-type mice in terms of day of onset and clinical progression. In both wild-type and C57BL/6-lpr mice the day of onset of clinical signs was between days 5 and 7 after injection of the encephalitogenic T cells. In both wild-type and C57BL/6-lpr mice the clinical signs began with tail weakness and progressed in order (depending on the individual mouse) through hind leg weakness, hind leg paralysis, front leg weakness, and death. Clinical signs reached their maximum level be-

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TABLE 1 C57BL/6-Derived Encephalitogenic T Cells 3

H cpm (a)

T cell

Line/clone

APC (b)

APC/PMBP (c)

APC/MMBP (c)

TNF (d)

INF-g (d)

C-A-46 A-C-2-4

Line Clone

401 743

104,441 36,449

48,246 13,713

59 61

852 628

Note. Antigen-specific proliferation assay: (a) 3H cpm, mean counts per minute of 3H of triplicate wells of 5 1 104 T cells stimulated by (b) 5 1 105 irradiated C57BL/6 splenocytes (APC) alone and (c) APC together with 25 mg/ml of PMBP or MMBP. Standard error of the means were all less than 15%. (d) Concentration (ng/ml) of tumor necrosis factor-a (TNF) or interferon-g (INF-g) secreted after stimulation of 0.5 1 106/ml T cells with immobilized anti-CD3 antibody.

tween days 8 and 12 after injection of the T cells in both groups of mice. In both wild-type and C57BL/6lpr mice this maximum level of clinical disease was either maintained for months or the signs of EAE slowly lessened after approximately day 14. Tail weakness, however, usually persisted indefinitely. Mice were observed for up to 4 months after injection and a second exacerbation of EAE was not seen in any of our wild-type or C57BL/6-lpr mice. Histopathology The central nervous system (CNS) histopathology of typical wild-type and C57BL/6-lpr mice with EAE was examined by a blinded observer. No qualitative or quantitative differences were found in the EAE CNS histopathology between C57BL/6-lpr mice and wildtype mice (Fig. 2). Both wild-type C57BL/6 and C57BL/ 6-lpr mice with EAE were found to have classic CNS

FIG. 1. Clinical EAE after adoptive transfer into wild-type and C57BL/6-lpr mice. Wild-type C57BL/6 mice or C57BL/6-lpr mice were injected, via an intracardiac route, with 16 1 106 cells (C-A46) or 12 1 106 cells (A-C-2-4). Mice were observed for 4 weeks and scored for clinical EAE as under Materials and Methods. Points represent maximal clinical scores achieved for individual mice studied. ‘‘c’’ indicates that the mouse was injected with the T cell line C-A46. All other mice were injected with the T cell clone, A-C-2-4.

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perivascular cuffing and tissue inflammation of the spinal cord (Figs. 2A and 2B). DISCUSSION

While EAE and MS are considered to be autoimmune in nature, the mechanisms underlying tissue injury in the CNS are not well understood. Recently, the possible role of Fas/FasL-mediated cell death in the pathogenesis of MS has begun to be investigated. To assess the role of Fas-mediated cell death in MS, Dowling et al. searched for Fas and FasL expression in acute and chronic MS plaques obtained from autopsy material. These investigators demonstrated that FasL is widely present on glial cells and macrophages in many chronic MS plaques (8). In addition, Fas expression was also found to be upregulated in MS tissue. These investigators demonstrated that apoptotic cells in MS lesions colocalized with the expression of FasL. They concluded that the expression of both Fas and FasL in MS lesions and the colocalization with apoptotic cells suggest that the Fas death system may contribute to the pathogenesis of MS. In a similar study, D’Souza et al. found that CNS tissue from MS patients demonstrated Fas expression on oligodendrocytes and FasL expression on microglia and infiltrating lymphocytes (9). D’Souza et al. found that, in vitro, Fas ligation of oligodendrocytes using anti-Fas-antibody led to cell death, although, surprisingly, this oligodendrocyte death was not through apoptosis (9). These authors also suggested that FasFasL interaction in the CNS may be a potential mechanism for oligodendrocyte injury in MS. In the present studies, we asked whether Fas/FasLmediated cell death played a role in the pathogenesis of adoptively transferred, acute EAE. We have recently described two approaches for the efficient generation of encephalitogenic, MBP-specific T cell lines and clones from strains of mice normally considered to be resistant to the induction of all forms of EAE (see footnote 3). This has now allowed us to readily examine the role played by Fas/FasL in the effector mechanisms involved in adoptively transferred EAE by studying

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FIG. 2. Histopathology of EAE in wild-type and C57BL/6-lpr mice. Nine to 12 days after adoptive transfer of encephalitogenic T cells, mice were euthanized and spinal cords were removed for pathological examination. (A) Spinal cord of a wild-type C57BL/6 mouse adoptively transferred with A-C-2-4, demonstrating perivascular cuffing and tissue inflammation. (B) Spinal cord of a C57BL/6-lpr mouse adoptively transferred with C-A-46, demonstrating perivascular cuffing and tissue inflammation.

C57BL/6-lpr mice. In mice bearing the lpr mutation, an early transposable element is inserted into intron 2 of the Fas gene. This causes aberrant splicing and

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premature termination of the Fas transcript, resulting in essentially no surface expression of Fas in such mice (12). On the MRL background, the lpr mutation results

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in the accumulation of an abnormal population of nonmalignant CD4-negative, CD8-negative T cells in the spleen and lymph nodes. Such mice also produce many autoantibodies and suffer from a clinical autoimmune disease resembling systemic lupus erythematosus. On the C57BL/6 background, the lpr mutation results in no spontaneous clinical autoimmune disease. However, in such mice, there is the development of autoantibodies and the accumulation of an abnormal T cell population at a late age (13). We are aware of only one previous report of EAE in mice bearing the lpr mutation. In this report, a novel EAE therapeutic regimen was tested (4). To study the mechanisms of action of this therapy, Elliot et al. bred the lpr gene onto the SJL/J background by crossing SJL/J mice with MRL lpr/lpr mice, back-crossing to SJL/J for two generations, and then intercrossing (4). They found that primary EAE could be induced in SJL/ J mice bearing the lpr mutation through immunization with a proteolipid protein peptide. Our present results, demonstrating that C57BL/6-lpr mice are susceptible to adoptively transferred EAE, now confirm these findings in an MBP-driven system. Furthermore, we have also further dissected the question of the role of Fas/ FasL by studying the effector phase of EAE. Our findings, together with those of Elliot et al., strongly suggest that Fas/FasL-mediated apoptosis does not play a significant role in the pathogenesis of acute murine EAE. However, our results do not allow us to draw conclusions concerning the relevance of Fas/ FasL-mediated cell death in the pathogenesis of chronic, relapsing–remitting EAE or the chronic CNS pathology seen in MS. Nevertheless, our model of adoptive transfer of EAE involves the penetration of T cells across the endothelium representing the blood–brain barrier, the entry of T cells into the parenchyma of the CNS, and the mediation of tissue injury leading to significant clinical signs and death. As such, our finding that Fas/FasL does not appear to play a role in these disease-initiating and early-phase effector events of acute EAE suggests that Fas/FasL may also not be involved in the early, initiating pathogenesis of MS. ACKNOWLEDGMENTS

We thank Dr. Steven Padula, and Dr. Leo Lefranc¸ois for their reading of the manuscript. We also thank Ms. Felicia Ledger for her technical assistance and Ms. Sue Roman and Mr. John Kane for their assistance in preparing the manuscript.

REFERENCES 1. Ferber, I. A., Brocke, S., Taylor-Edwards, C., Ridgway, W., Dinisco, C., Steinman, L., Dalton, D., and Fathman, C. G., Mice with a disrupted IFN-g gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156, 5–7, 1996. 2. Koh, D., Ho, A., Rahemtulla, A., Penninger, J., and Mak, T., Experimental allergic encephalomyelitis in mice lacking CD4/ T cells. Eur. J. Immunol. 24, 2250, 1994. 3. Grewal, I. S., Foellmer, H. G., Grewal, K. D., Xu, J., Hardardottir, F., Baron, J. L., Janeway, C. A., and Flavell, R. A., Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273, 1864, 1996. 4. Elliot, E. A., McFarland, H. I., Nye, S. H., Cofiell, R., Wilson, T. M., Wilkins, J. A., Squinto, S. P., Matis, L. A., and Mueller, J. P., Treatment of experimental allergic encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein. J. Clin. Invest. 98(7), 1602–1612, 1996. 5. Merrill, J., Kono, D., Clayton, J., Ando, D., Hinton, D., and Hofman, F., Inflammatory leukocytes and cytokines in the peptideinduced disease of experimental allergic encephalomyelitis in SJL and B10.PL mice. Proc. Natl. Acad. Sci. USA 89, 574–579, 1992. 6. Kuchroo, U. K., Martin, C. A., Greer, J. M., Ju, S. T., Sobel, R. A., and Dorf, M. E., Cytokines and adhesion molecules contribute to the ability of myelin proteolipid protein-specific T cell clones to mediate experimental allergic encephalomyelitis. J. Immunol. 151, 4371–4382, 1993. 7. Beraud, E., Balzano, C., Zamora, A. J., Bernard, D., and BenNun, A., Pathogenic and non-pathogenic T lymphocytes specific for the encephalitogenic epitope of myelin basic protein: Functional characteristics and vaccination properties. J. Neuroimmunol. 47, 41–53, 1993. 8. Dowling, P., Shang, G., Raval, S., Menonna, J., Cook, S., and Husar, W., Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain. J. Exp. Med. 184, 1513–1518, 1996. 9. D’Souza, S. D., Bonetti, B., Balasingam, V., Cashman, N. R., Barker, P. A., Troutt, A. B., Raine, C. S., and Antel, J. P., Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J. Exp. Med. 184, 2361–2370, 1996. 10. Padula, S. J., Lingenheld, E. G., Stabach, P. R., Chou, C-H. J., Kono, D. H., and Clark, R. B., Identification of encephalitogenic V-4-bearing T cells in SJL mice. Further evidence for the Vregion disease hypothesis? J. Immunol. 146, 879–883, 1991. 11. Ruddle, N. H., Bergman, C. M., McGrath, K. M., Lingenheld, E. G., Grunnet, M. L., Padula, S. J., and Clark, R. B., An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J. Exp. Med. 172, 1193–1200, 1990. 12. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Nagata, S., Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314–317, 1992. 13. Lynch, D. H., Ramsdell, F., and Alderson, M. R., Fas and FasL in the homeostatic regulation of immune responses. Immunol. Today 16, 569–574, 1995.

Received July 31, 1997; accepted August 12, 1997

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