Workshop O: Animal Models of Immune Diseases

WORKSHOP 0 Animal Models of Immune Diseases

IAllg. und Exp. Pathologie, Innsbruck, Austria; 2Universitatskinderklinik, Freiburg, Germany

0.1 Immune pathogenesis of progressive systemic sclerosis in the UCD 200 chicken animal model: gamma/delta T cell receptor (TCR) positive cells in the initial paravascular infiltration S. BENSELER 1,2, M. GRUSCHWITZ 1, R. SGONC I , H. DIETRICH I, K. HALA\ M. COOPER, E. GERSHWIN,]. FORSTER 2, and G. WICK 1 Progressive Systemic Sclerosis (PSS) is an autoimmune disease of the skin and several internal organs. It starts with inflammation and edema and finally leads to severe fibrosis and loss of function in the afflicted organs. UCD 200 chicken inbred line spontaneously develop a chronically running disease resembling human PSS. In our sequential study we focused our main interest on the initial onset of inflammation in the skin by means of immunhistochemistry. The approach included in addition to the local immune reaction the characterization of the systemic immune response in peripheral blood, spleen and thymus by F ACS analysis. We further investigated the functional capacity of the gamma/ delta T cells in FACS and stimulation assays. Our approach revealed the initial local immune reaction in the skin to be a T cell response particularly of gamma/delta TCR positive cell. These cells were found surrounding dermal blood vessels at the age of 20 days, two weeks before skin lesions were noticed macroscopically. At that time of age in the peripheral blood a diminished amount of gamma/delta TCR positive cells was seen. Meanwhile up to 40 % of the splenocytes showed gamma/delta TCR on the surface. Up to 30 % of these cells were also CDS positive, none of them showed the helper/inducer subtype. At least in the PSS animal model UCD 200 chicken the pathogenetic role of T cells expressing gamma/delta TCR is prominent. The mechanism of inducing inflammation is part of our research. Further studies in human patients with PSS looking for a correlation functional role of gamma/delta T cells have to be done.

280 . 25th Meeting of the Society of Immunology Friedrich Schiller University, Institute of Pathology, Jen a, Germany

0.2 T cell-targeted immunosuppression of antigen-induced arthritis R. BRAuER, S. HENZGEN, P. PETROW, A. SCHREITER, K. THOSS, U. WUTZLER, and D. KATENKAMP The antigen-induced arthritis model in mice was used for testing the efficacy of T celldirected immunosuppressive treatments on joint inflammation and articular tissue degradation as well as on humoral and cellular immune responses. The chronic joint inflammation and cartilage destruction were clearly inhibited by monoclonal antibodies depleting murine CD4+ T cells (GK1.5, YTS 191.1) and by the immunosuppressive agents cyclosporin A, FK506 and rapamycin, which exert their effects by blocking events in T cell activation. Moreover, these treatments reduced the humoral and cellular immune responses to methylated bovine serum albumin (mBSA), which is used as specific antigen for the arthritis induction, and impaired the development of autoimmunity against connective tissue constituents such as collagen type I and II and cartilage proeteoglycans. The treatment efficacy was further verified by flow cytometry and immunohistological analysis of spleen and lymph nodes. The results underline the essential role of T cells in the pathogenesis of chronic joint inflammation.

Dept. of Pathology/Tumorimmunology, University of Regensburg, Regensburg; IDept. of Expt. Hematology, GSF, Munich, Germany

0.3 Increased mortality of mast cell-deficient mice after septic peritonitis is counteracted by exogenous TNF B. ECHTENACHER, D. N. MANNEL, and L. HDLTNER TNF depletion of infected animals has been shown to lead to aggravation in many experimental infections. Accordingly, survival of mice after sublethal CLP (cecal ligation and puncture) leading to peritonitis and sepsis depends critically on TNF as demonstrated earlier. Neutralization of endogenous TNF with anti-TNF antibodies was lethal in this infection model. Mast cells seem to be the only resident cells able to store preformed TNF in their granules. Therefore, kinetics of TNF appearance and serum TNF concentrations after systemic stimulation of TNF production was expected to be different in mast cell deficient W IWV mice compared to normallittermates if mast cell TNF significantly contributes to the serum TNF level. W/WV mice indeed showed a 4-8 fold decreased serum TNF level 30 minutes after Lipid A injection. The mast celldeficient W/W" mice that underwent CLP were more sensitive to CLP than normal mice resulting in 92 % mortality in W IWV vs. 8 % mortality in normallittermates. 60 % W IWV mice could be protected from lethal CLP by i.p. injection with recombinant mouse TNF after CLP. Reconstitution with cultured wild-type mast cells 2 weeks prior to CLP resulted in protection of 30 % of the reconstituted W IWV mice. Whether only the deficiency in mast cells is responsible for the observed effects and which mast cell function in particular affects survival will be investigated.

25th Meeting of the Society of Immunology . 281 CIBA-GEIGY, Pharmaceuticals Research Division, Dept. of Inflammation, Basel, Switzerland

0.4 Therapeutic intervention with mycobacterial 65 kDa heatshock protein peptide 180-188 in adjuvant arthritis in Lewis rats U. FEIGE and]. GASSER Adjuvant arthritis (AA) in Lewis rats is induced by an injection of mycobacteria in oil. AA is a T cell mediated disease. Mycobacterial 65 kDa heat shock protein (hsp65) has been shown to be an antigen involved in the disease process of AA. In fact, hsp65 or peptide 180-188 of hsp65 (p 180-188) can be used to protect rats against an arthritogenic challenge with mycobacteria. However, therapy rather than prevention of disease appears to be the appropriate goal for intervention with disease in man. Therefore, we investigated whether p180-188 also exhibits therapeutic effects in AA. Treatment of rats with 0.1 to 1.0 mg of p180-188 in incomplete Freund's adjuvant i.p. at days 9 and 10 after the arthritogenic challenge with mycobacteria in oil completely inhibited the onset of AA (no paw swelling, weight loss or radiographical changes were present). Even treatment at days 12 and 13, when clinical symptoms of disease (paw swelling and weight loss) were already apparent, was effective. The extent of bone destruction was reduced; individual radiographical scores at day 35 were 0, 0, 15,72,78 in p180-18S treated rats and 44, 47, 57,75,101,119 in AA control rats. The data indicate that intervention with p180-188 brings the disease process to a halt and suggest that even in established disease the immune response is the major driving force of the disease process.

CIBA-GEIGY, Pharmaceuticals Research Division, Dept. of Inflammation, Basel, Switzerland

0.5 Antigen-induced nitric oxide production and apoptosis in spleen cell cultures of rats with adjuvant arthritis U. FEIGE, C. Ku\s,]. GASSER, N. BALOMATIS, M. MEReEI', and T. HALl. Adjuvant arthritis (AA) in Lewis rats is induced by an injection of mycobacteria in oil. AA is a T cell mediated disease. Mycobacterial 65 kD heat shock protein (hsp65) has been shown to be an antigen involved in the disease process. In fact, immunization with hsp65 or peptide 180-188 of hsp 65 protects rats against the arthritogenic challenge with mycobacteria. During the course of AA the cellular immune response to mycobacterial antigens follows a distinct kinetics. In the first 12 days after immunization proliferative spleen cell responses are observed to mycobacterial antigens in vitro. At around day 15, in these cultures antigen-induced suppression of proliferation, massive production of nitric oxide (NO) and apoptosis was observed; we found levels of NO up to 80 [l.molar in cultures of 5 x 10 5 spleen cells after 72 hours. In these cultures up to 90 %, of all splenic cells undergo apoptosis. The antigen-induced NO production can be inhbitited by Nmono-methyl-arginine, which also inhibits the antigen-induced apoptosis. We conclude that in these cultures NO is inducing apoptosis.

282 . 25th Meeting of the Society of Immunology Institute for Hygiene and Microbiology, University of Wurzburg, Wurzburg, Germany

0.6 Yersinia-induced arthritis in the rat model: the role of the complement system K. GAEDE, E. BAUMEISTER, and]. HEESEMANN Yersinia enterocolitica is an enteropathogenic pathogen, causing intestinal infections as well as immunopathological manifestations such as reactive arthritis (ReA). The pathomechanism of this sequel is still unknown. Patients with Yersinia-associated arthritis show high prevalence of HLA-B27, persistence of Yersinia-specific IgG- and IgA-antibodies, circulating Yersinia-specific immune complexes and chronic infection of intestinal mucosa. Since Y. enterocolitica 08 is also arthritogenic for rats we analyzed the pathogenesis of Yersinia-induced arthritis (YIA) in the rat model. In previous studies we demonstrated that arthritis-susceptible Lewis rats developed significantly higher Yersinia-specific antibody titers in the early state of the infection than arthritis-resistant Fischer rats. In the present study we focussed our interest on the role of the complement system in YIA. Our investigations demonstrated that in vivo complement depletion in Yersinia-infected Lewis rats by intraperitoneal injection of cobra venom factor suppressed the development of YIA clinically and histologically. Decomplementation did not affect the elimination of Yersiniae. These data suggest a functional role of the complement system in YIA in rats.

Institut fur Immunologie, Mainz, Germany

0.7 Administration of IL12 to mice immunized with protein antigens results in enhanced antibody production and altered Ig isotype pattern T. GERMANN, M. BONGARTZ, H. HESS, E. SCHMITT, and E. RUDE The influence of the cytokine IL12 on humoral immunity was studied in vivo. Mice immunized with protein antigens (KLH, PLA 2) adsorbed to Alum developed a Th2 like immune response (IgG1, IgE antibodies). IL12 promotes the development and the activation of Th1 cells. Thus, we investigated whether treatment with IL12 would downregulate antibody production or induce IgG2a, IgG2b and IgG3 antibody subclasses (Thllike). We observed that: 1) Administration of IL12 to mice together with protein antigens adsorbed to Alum stimulated the humoral immune response by increasing the synthesis of antibodies of the IgG3, IgG2b and IgG2a subclasses 10- to 1000-fold while the synthesis of IgGl was only slightly (2 to 5-fold) enhanced. 2) Production of the IgE isotype was (slightly) suppressed in mice treated with IL12 in most experiments. This suppression was mediated by IFNy and not by IL12 (directly) because the synthesis of IgE was highest in animals treated with IL12 plus anti-IFNy mAb in vivo. 3) Furthermore, an anti-IFNy mAb prevented most of the enhanced IgG2a production stimulated by IL12. 4) Mice treated with IL12 showed a strong (20-fold) upregulation of the synthesis of IFNy but no inhibition of IL5 production by ex vivo activated spleen cells. Antigen-specific cytokine synthesis by spleen cells was highest in animals treated with

25th Meeting of the Society of Immunology . 283 IL12 plus anti-IFNy mAb in vivo. According to these results the predominant Th2 like humoral immune response is converted into a mixed (ThO like) response - suggesting that IL12 can be a potent adjuvant for inducing humoral immunity to protein antigens adsorbed to Alum.

Institut fur Immunologie, Mainz, Germany

0.8 IL12 in combination with type" collagen induces arthritis in DBAl1 mice T. GERMANN,]. SZELIGA, H. HESS, E. SCHMITT, and E. RODE Immunization of DBAl1 mice with type II collagen emulsified with Mycobacterium tuberculosis in oil results in severe arthritis associated with a strong anti-collagen immune response of the Th1 type. IL12 is a cytokine implicated in the development of Th1 cells. It's production by macrophages is triggered by several microorganisms including mycobacteria. Our assumption was, that IL12 induced by bacteria might be involved in the development of the anti-collagen response. Therefore, we tested whether IL12 could replace mycobacteria and cause arthritis of DBAl1 mice immunized with type II collagen in oil. Immunization of DBAl1 mice with type II collagen in oil alone resulted in a weak immune response and only a few animals developed arthritis (3/20). Administration of IL12 simultaneously to immunization strongly enhanced the anticollagen immune response as shown by an about 10-fold enhancement of collagenspecific IFNy synthesis by ex vivo activated spleen cells and a 10- to 100-fold upregulation of collagen-specific IgG2a and IgG2b antibody production. The incidence of arthritic mice was very high (18/19) in the group immunized with collagen + IL12. A neutralizing anti-IFNy mAb blocked most of the enhanced IgG2a production and prevented the development of arthritis (0/20) when given in combination with IL12 and collagen. Thus, endogenous IFNy is involved in the enhancement of anti-collagen immunity induced by IL12. In conclusion, our data show that in vivo administered IL12 can profoundly uprcgulate an immune response directed to a potential autoantigen resulting in arthritis of the animals.

National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Immunopathology and Laboratory of Parasitic Diseases, Bethesda, MD, USA

0.9 In vivo treatment with IL-12 protects mice from immune abnormalities observed during murine AIDS (MAIDS) N. A. GIESE, R. T. GAZZINELLI, and H. C. MORSE III. Lymphoproliferation, chronic B cell activation, hypergammaglobulinemia, and profound immunodeficiency of cell mediated functions are prominent features of a retrovirusinduced syndrome designated mouse. AIDS (MAIDS). In vivo treatment of infected

284 . 25th Meeting of the Society of Immunology mice with recombinant IL-12 beginning at the time of infection or up to 9 wk after virus inoculation markedly inhibited the development of lymphoproliferative disease, chronic activation of B-lymphocytes and elevations of serum IgG and IgM levels. Treatment with IL-12 also had major effects in preventing immunodeficiency, restoring IFN-y and IL-2 production as well as proliferative responses to various stimuli. The therapeutic effects of IL-12 on the immune system of mice with MAIDS were associated with reduced expression of the retrovirus that causes this disease and independent of ecotropic MuL V. The involvement of IFN-y in the control of BM5 def replication and related immunopathology is also suggested since IL-12 treatment was not effective in IFN-y knockout mice or infected mice treated simultaneously with IL-12 and anti IFN -yo These results demonstrate that the pathogenesis involved in the progression of MAIDS is antagonized by IL-12 and IFN -y and may serve as an experimental basis for developing treatments for retrovirus induced immune disorders with similar immunopathogenic mechanisms.

lDept. of Immunology and 2Clinic and Policlinic for General Surgery, Georg August University, Giittingen, Germany

0.10 Multiple rat recipient pretreatment with cis-urocanic acid (cis-UCA) further improves the outcome after allogeneic small-bowel transplantation (S8T) R. K. H. GIESELER\ R. SCHLEMMINGER2, T. STOjANOVIC 2 , F. KLEMP2, and]. H. PETERS 1 Deamination of L-histidine and UV -B irradiation of the trans-isomeric product yields cis-UCA. This substance was shown to mount specific immunosuppression towards a wide range of tested antigens (1). We recently showed for the first time that a single pretreatment of organ recipients with cis-UCA 7 days before experimental allografting [Brown Norway (BN; RT1n) ~ Lewis (LEW; RT11)] prolongs survival after heterotopic SBT: While untreated controls survived for only 8.5 days, i.v. injection of cis-UCA resulted in 16 days (2) and i.p. treatment in even 27 days of survival (3). Using the same strain combination, these results could further be improved when the recipient was repeatedly injected with cis-UCA. However, the outcome largely depended on the pretreatment regimen. The most promising results were gained when LEW recipients were administered three consecutive i.p. injections of 2 mg/ml cis-UCA at 1 ml each, given 21,14, and 7 days before SBT (n=3). The animals survived for 48 ± 8 days, the grafts showed almost no ectatic alterations and cellular infiltrates, and a graft-enclosing fibrous capsule was never observed. Other multiple injection protocols with higher doses, a dosage enhancement, or daily follow-up injections yielded inferior or inconsistent results only. We propose a pretreatment with cis-UCA for combinatorial regimens with other immunosuppressants. 1. NORVAL, M. et al. 1989. Photochem. Photobiol. 50: 267. 2. GIESELER, R. K. H. et al. 1994. Transplant. Proc., in press. 3. SCHLEMMINGER, R. et al. 1994. Transplant. Proc., in press. Supported by the Deutsche Forschungsgemeinschaft (SFB 330, Grant D/9).

25th Meeting of the Society of Immunology . 285 'Dept. of Immunology and 2Clinic and Policlinic for General Surgery, Georg August University, Gottingen, Germany

0.11 Combinatorial regimen of dose-reduced cyclosporin with cis-urocanic acid (ucis-UCA) allows indefinite survival of small-bowel (58) allograft recipients

Survival after fully allogeneic SB transplantation could be prolonged to 48 days when the recipients were pretreated i.p. with cis-UCA on days 21, 14, and 7 before heterotopic organ grafting (1). In the same animal model [Brown Norway (BN; RTln) ---> Lewis (LEW; RTll )], indefinite survival was established with daily S.c. injections of cyclosporin (CsA) at 15 mg/kg BW on days 0 to 14 and 10 mg/kg BW on days 15 to 28 after transplantation (2). Here, we present a new combinatorial therapy of graft rejection: LEW recipients were firstly subjected to the aforementioned cis-UCA protocol. Following the heterotopic transplantation of an entire BN SB, six groups (n = 3) of allograft recipients were then administered different dosages of CsA S.c. (d 0-14/d 15-28), i.e. 15/ 10; 15/5; 10/10; 10/5; 7.5/5; and 5/5 mg/kg BW. Cis-UCA pretreatment was omitted in the control groups (n=3). Transfer of the heterotopic SB into the orthotopic functional position on day 28 and indefinite survival (> 150 days) was now already achievable at reduced CsA concentrations of 10 (d 0-14) and 5 (d 15-28) mg/kg BW S.c. The control group without cis-UCA pretreatment survived for 33 days only. Since this combinatorial regimen allows for a dose reduction of total CsA by 40 %, such a treatment might significantly reduce the toxic side effects of CsA (3). 1. GIESELER, R. K. H. et al. 1994. Immunobiol. (this volume). 2. SCHLEMMINGER, R. et al. 1993. Res. Exp. Med. 193: 379. 3. SIGAL, N. H. and F. J. DUMONT. 1992. Annu. Rev. Immunol. 10: 519.

Supported by the Deutsche Forschungsgemeinschaft (SFB 330, Grant D/9).

Max-Planck-Institut fiir Immunbiologie, Freiburg, Germany

0.12 Functional analysis of bovine peripheral blood mononuclear cells after transfer into SCID mice G. HASCH, N.JOSWIG, U. STAUFFER, and H. MOSSMANN The transfer of human lymphoid cells into SCID mice was studied extensively with respect to the development of the immune system, to the pathogenesis of diseases and drug testing. There are only few reports describing immunological chimeras with other species than humans. We have transferred different numbers of bovine peripheral blood mononuclear cells (PBMC) into SCID mice by intraperitoneal, intraveneous and subcutaneous injection. While the subcutaneous route failed to reconstitute SCID mice, bovine IgG was present after i.p. and i.v. administration of PBMC. After i.p. transfer of 2 x 10 7 bovine PBMC increasing bovine IgG and IgM levels were found in bovine PBMC/SCID-mice sera, reaching a maximum around week 5 (2-6 mg/ml IgG) post

286 . 25th Meeting of the Society of Immunology reconstitution. Even after more than 20 weeks low bovine IgG serum levels « 50 flg/ml) were detectable in half of the tested animals. Bovine CD4+ and CDS+ cells could be detected by flowcytometry in the peritoneal cavity for more than twelve weeks. Five weeks after cell transfer CD4 and CDS positive cells could be detected in spleen, liver, lung, abdominal lymph nodes, thymus and bone marrow. Immunohistology showed CD4+ and CDS+ cells to be present in recipient spleen sections after 16 weeks post transfer. After immunization of calves against KLH and DNP, respectively, 2 x 10 7 presensitized PBMC were injected i.p. into SCID mice. Secondary bovine IgG responses to both antigens were observed in sera of recipient mice after challenge. It is shown that SCID mice can be reconstituted with bovine PBMC, that the transferred cells are able to seed lymphoid and non lymphoid mouse organs and that presensitized PBMC can be restimulated specifically in the SCID mouse environment. These data suggest that the bovine PBMC/SCID-mouse could serve as a small animal model for bovine lymphopoiesis and infectious diseases of cattle. Acknowledgment:

J.

NAESSENS, ILRAD, Kenya for supporting mAB.

Institute of Immunology, University of Heidelberg, Heidelberg, Germany

0.13 Protective role of C059 in a pig-to-human for hyperacute rejection

in vitro model

B. HECKL-OSTREICHER, R. BINDER, and M. KIRSCHfiNK Hyperacute rejection which is mediated by natural antibodies and complement develops within minutes and is a major obstacle to xenotransplantation of vascularized organs. One strategy to effectively inhibit host complement might be to introduce complement inhibitory proteins of the recipient species into the endothelial cell membranes of the donor organ. This hypothesis was tested by several investigators in an in vitro model of hyperacute rejection consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and complement. Recently, a protective role has been shown for the human complement regulatory membrane proteins decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) that inhibit an early step in the complement reaction. So far, no protection was seen with purified human CD 59, an inhibitor of the late complement components that was incorporated into pig endothelial cells. In our study we introduced the CD59 molecule by transfection into porcine aortic endothelial cells. Expression of CD 59 was detected by indirect immunofluorescence, and positive transfectants were tested for complement resistancy in a lactate dehydrogenase (LDH)-release assay. Incubation of the cells with 10 % and 20 % human serum resulted in an inhibition of lysis of 90 and 70 %, respectively. The inhibitory effect seen could be abolished by preincubation of the cells with monoclonal antibodies to CD59. An application of this approach might be to produce transgenic pigs expressing human membrane associated inhibitors of complement, such as DAF, MCP and CD59.

25th Meeting of the Society of Immunology . 287 IMax-Planck-Institut fur Immunobiologie, Preiburg, Germany; 2Dept. Molecular Microbiology, Washington University, St. Louis, MO, USA; 3Abteilung fur angewandte Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany

0.14 Biochemical and functional analysis of the Borrelia burgclorferi B cell mitogens N. HONARVAR I, U. E. SCHAIBLF2 , R. WALLlCH 3, and M. M. SIMONI Infection of humans with the spirochete Borrelia burgdorferi (B. burgdorferi) leads to the induction of specific immune responses. However, in many cases, patients develop various clinical symptoms. Studies in the mouse model of B. burgdorferi infection suggest that Band T cells playa role both in protection and pathogenesis of arthritis and carditis. Recently, we and others have demonstrated that spirochetes induce similar levels of proliferation and antibody production in unselected spleen cells from naive and immune mice, suggesting the presence of a B cell mitogen(s). Quantitative analyses of the B. burgdorferi-mediated poly clonal activation showed that B cells are stimulated to produce IgM and IgG with frequencies similar to those found for LPS. Characterization of the responsible structures revealed that the mitogenic activity can be attributed to the outer surface proteins A and B (OspA; OspB) as well as a phenol-chloroform-petroleum ether (PCP) extract of B. burgdorferi which lacks OspA and OspB. SDS-PAGE analysis showed that the PCP extract consists of two major components with molecular masses of 14 kDa and ~ 3 kDa respectively, which could be enriched by gel electrophoresis. Biochemical studies with radiolabelled palmitate demonstrated that both structures are lipidated. In addition, the 14 kDa but not the ~ 3 kDa structure was shown to be i) protease sensitive, ii) stained by coomassie and iii) to incorporate tritiated amino acids. Functional analysis of the two isolated preparations showed that both structures are mitogenic for B cells and that the activities were not affected by their prior treatment with proteinase K. Application of sonicated B. burgdorferi, OspA, PCP extract or the isolated 14 kDa- and ~3 kDa-structures in vivo resulted in the induction of high numbers of immunoglobulin producing cells, synthesizing mainly IgM. Injection of sonicated preparations of B. burgdorferi lead in addition to an increase in the number of IgG2a, IgG2b and IgG3 producing B-cells. The data show that B. burgdorferi contain at least 4 distinct structures with mitogenic activity for B cells in vitro and in vivo. The biological significance of these mitogens in protection against the infection and in the development of arthritis and carditis is presently under investigation.

IInstitute for Biomedical Aging Research, Austrian Academy of Sciences, 2Institute for General and Experimental Pathology, and 3Institute for Physiology, University of Innsbruck, Medical School, Innsbruck, Austria

0.15 Heat-shock protein 65 induced arteriosclerotic lesions regress in normocholesterolemic rabbits

Previous studies have shown that arteriosclerotic lesions can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein (hsp) 65. To investigate changes of lesions in time, 63 New Zealand White rabbits were treated either

288 . 25th Meeting of the Society of Immunology by immunization with Freund's complete adjuvant containing Mycobacterium tuber-

culosis (= hsp65-rich material) by administration of a 0.2 % cholesterol-diet only, or by a combination of both immunization and cholesterol-rich diet. Sixteen weeks after the first immunization, half of the animals were killed and severe atherosclerotic lesions in the intima of the aortic arch were found in 8 out of 10 immunized animals. The other half of the animals were killed 32 weeks after the beginning of the experiment, keeping them on a normal, non cholesterol enriched diet. Only 3 out of 10 rabbits immunized still showed moderate lesions in their aortae 32 weeks after the first immunization. Microscopically, most of the intima appeared normal in serial cross-sections of the aortic arch. Titers of serum antibodies to hsp65 cross reacting with mammalian hsp60, as well as proliferative responses of lymphocytes derived from peripheral blood of immunized animals to hsp65 decreased significantly between 16 and 32 weeks. In contrast, atherosclerotic lesions induced by cholesterol-rich diet, or by immunization plus cholesterol-rich diet did not show regression between 16 and 32 weeks. Nevertheless, proliferative responses of peripheral blood lymphocytes and serum antibody titers to hsp65 revealed a significant decrease in double treated animals. In conclusion, the early stages of arteriosclerotic lesions induced by immunization are able to regress in the absence of additional risk factors for atherosclerosis. Supported by the Austrian Research Council (project no. 8925).

Institut fur Virologie und Immunbiologie der Universitat Wurzburg, Wurzburg, Germany

0.16 Superantigen induced responses of C04+ and C08+ T cells in IL-2 -/- mice B. KNEITZ, T. HERRMANN, and A. SCHIMPL Injection of Superantigen (SAg) into normal mice leads to rapid induction of IL-2 production and a V~ specific increase in both CD4+ and CD8+ T cells which reaches its maximum around day 2 post injection. At later times, the numbers of SAg reactive cells decline again to levels below those observed before SAg application, the residual cells are no longer stimulated by the homologous SAg. To investigate whether expansion is not only accompanied by but also dependent on IL-2 production, IL-2 -/- mice were injected with SEB or SEA and the numbers of V~8+ or VI:lll + cells in the lymph nodes determined by FACS analysis on different days. On day 2, numbers of SAg- reactive CD4+ and CD8+ cells were increased to levels very similar to those seen in IL-2 producing littermates. Deletion of SAg reactive-CD4+ cells, however, at later timepoints was less pronounced in IL2-/- mice than in controls. Also, lymph node cells from the IL-2 deficient mice taken at late times after SAg injection responded to the homologous SAg as measured by thymidine incorporation while cells from IL-2 producing controls had become anergic. The data show that in vivo expansion of SAg reactive cells can proceed in the absence of IL-2, while deletion and anergy induction, directly or indirectly, depend on IL-2. A failure to properly terminate immune responses could either be causative for the lymphadenopathy observed in IL-2 deficient mice or be the consequence of an accumulation of T cells with a memory phenotype detectable in these mutant mice.

25th Meeting of the Society of Immunology . 289 Dept. of Immunology, University Hospital, Hamburg; 1Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany

0.17 Elucidation of the genomic structure of the rat RT6 gene G. KUHLENBAUlI1ER, F. HAAC, F. NOLTE, E. WINGENDER 1, and H.-G. THIELE The RT6 antigens are phosphatidylinositol (gPI) anchored membrane proteins that recently were shown to be members of the family of ADP-Ribosyltransferases. RT6 expression is restricted to mature T lymphocytes. The two main allotypes RT6a and RT6b are different alleles of a single gene. Expression of RT6 appears to be under tight transcriptional control. In Diabetes Prone Bio-Breeding (DP-BB) rats diabetes and lymphopenia are associated with a RT6 expression defect. Molecular analysis and crossbreeding studies have shown that the coding region of the DP-BB rat RT6 gene is structurally intact and that the gene is expressible. For these reasons the underlying defect seems to be a malfunction in the transcriptional control system of the DP-BB rat. To enable us to study the transcriptional regulation of the RT6 gene we decided to elucidate the genomic structure with the aim of identifying potential cis-acting regulatory elements. 5'Rapid amplification of cDNA ends Polymerase chain reaction (5'Race PCR) analysis showed that 5' of the first exon previously identified in cDNA clones one further exon exists. Sequence analysis of the 5'Race PCR products revealed alternative splicing in the 5' region. Repeated screening of a genomic lambda library yielded four overlapping clones covering the whole RT6b gene. Restriction mapping showed that the transcribed region of the RT6 gene is approximately 18 kb, the largest intron 7.5 kb, long. Sequence analysis of the potential promotor region and of some of the intron regions allowed us to identify a wealth of potential transcription factor binding sites including TATAA and CCAAT boxes. Our next aim is to prove the functional significance of the putative promotor and enhancer elements through reporter gene assays.

INSERM U.283, H6pital Cochin, Paris, France

0.18 Macrophage-inactivating interleukin-13 suppresses experimental autoimmune encephalomyelitis (EAE) in rats O. ROTT and E. CASH EAE is initiated by myelin basic protein (MBP) specific Thl cells which subsequently trigger the invasion of monocytes/macrophages (M functions in vitro, including an inhibition of the production of the proinflammatory cytokines IL-l and TNF, a simultaneous enhancement of MHC class II and CD4 receptor expression and a diminution of nitric oxide (NO) production by microglial cells. In addition, hrIL-13 could slightly but in a highly reproducible manner inhibit the proliferative response of MBP-specific Thl cells in the presence of thymic APCs. Upon in vivo application of hrIL-13-secreting vector cells into MBP-immunized animals, the cytokine could markedly suppress the induction of clinical EAE. This suppression coincided with no

290 . 25th Meeting of the Society of Immunology significant alteration in either autoreactive T cell or B cell responses. We infer from these results that a strictly Th 1-initiated disease can be efficiently attenuated by a cytokine which primarily targets cell of the monocyte/M lineage and seems to exert no undesirable general suppression on either T cell or B cell reactivity in vivo.

Institut fiir Klinische Immunologie und Transfusionsmedizin, and! Klinik fiir Nuklearmedizin, Universitatsklinikum, Leipzig, Germany

0.19 The delayed phase: chronic process in hu/mu SCID arthritis U. SACK, s. HEILMANN,J. LEHMANN, 1. KAMPFER!, M. GENEST, and F. EMMRICH In hu/mu scm arthritis, particles of human rheumatoid (RA) synovial membrane (SM) are transferred into the knee joint of mice with severe combined immunodeficiency (SCID) in direct contact with murine cartilage. An unspecific postoperative inflammation develops during the first 48 hours immediatedly after surgery. Subsequently, a mixed human/murine pannus tissue is formed presenting typical features of an arthritis such as hyperplasia of lining cell layer, chondroid metaplasia, presence of multinuclear giant cells, and interstitial fibroblast-like cells detected by CD68, while tissues such as healthy SM, normal lymph node tissue, or granulomatous tissue did not show these characteristics. The model provides an interesting tool to investigate effects of immunological mediators and new therapeutic agents on human tissue in vivo. Caused by the highly heterogeneous cellular composition of RA-SM, reliable parameters are required which are representative for destruction and for inflammation. Therefore, we examined human and murine interleukin-6 and human IgM as well as IgG in murine serum. Radioisotope scanning was performed using technetium 99m diphosphonate which indicates reactive bone formation in parallel to immunohistological examination. The acute postoperative arthritic process decreases during the first four weeks and comes to a non-active state. Unexpectedly, after a three months delay there were detactable foci of arthritic processes in the primary affected knee joint as well as in the hips and contralateral knee. This indicated an active process of chronification following the acute phase of hu/mu SCID arthritis (1).

1.]. Rheumatol. 21 (1994): 10-16.

Max-Planck-Society, Clinical Research Unit for Rheumatology/Immunology at the University of Erlangen-Niirnberg, Erlangen, Germany

0.20 Cytokine-detection by semiquantitative PCR in synovial membranes of rats with antigen-induced arthritis (AlA) A. SIEGLING, R. W. KINNE, E. BUCHNER, and F. EMMRICH Antigen-induced arthritis (AlA) is an animal model for rheumatoid arthritis induced by injection of the antigen (mBSA) into knee joints of immunized rats. T helper cells may play an important role in AlA, as evidenced by the possibility to induce AlA using CD4-

25th Meeting of the Society of Immunology . 291 positive T cell clones together with their respective antigen or else by the success of cyclosporin A- and anti-a[:I-T cell receptor treatment. Macrophages in AlA may directly participate in joint destruction and contribute to the induction phase of arthritis by acting as antigen-presenting cells; on the other hand, they may also provide the second signal required for full activation of T cells. Total RNA was extracted from inflamed synovial tissues of AlA rats (0 h, 6 h, d 1, d 3, d 6), reverse transcribed into cDNA, and adjusted with regard to equal beta-actin content. The amount of mRNA for IL-1~, IL-6, IL-2 and IL-5 was determined by semiquantitative competitive PCR using a control fragment. The mRNA levels of the monokines IL-l~ and IL-6 (lOO-lOOO-fold) are massively increased during the first 6 h after induction of arthritis and are decreasing until day 6 (10-100 fold). The gene expression of the Thl-like cytokine IL-2 sharply rose until6h (10.000 fold), decreased rapidly until day 1 (10-fold) and plateaued thereafter. The mRNA levels of the Th2-like cytokine IL-5 increased only marginally (maximally 10-fold). Massively increased mRNA levels of the monokines IL-6 and IL-1~ and the Thl-like cytokine IL-2 indicate that macrophages and Th I-cells may actively contribute to the inflammatory process in AlA.

Dept. of Neurology, University ofWurzburg, Wurzburg, Germany

0.21 Anti-ICAM treatment partially inhibits adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE): a clinical and immunohistochemical study C. SIMONIS, U. BERNTHALER, U. ZETTL, S. MORRISSEY, J. ARCHELOS, S. lUNG, K. V. TOYKA, and H.-P. HARTUNG

The present study investigates the influence of anti-I CAM treatment on the course of AT-EAE and its influence on cell infiltration and blood-brain-barrier. Our group recently reported successful suppression of active EAE in rats treated with anti-ICAM-I (lA29); however, there are conflicting data about its efficacy in AT-EAE. AT-EAE was induced by injecting 4 x 106 MBP-specific CD4+ T cells into female Lewis rats. Starting on day 0, a group of animals received daily 5 mg of purified monoclonal antibody against rCAM-l. Animals were perfused on day 7 and the brains processed for immunohistochemistry using monoclonal antibodies against T cells, macrophages (EDl) and albumin. Clinically, anti-ICAM treated animals were significantly less affected than the control EAE-group. Immunohistochemistry revealed only 57 % of T cells and 44 'Yo of macrophages in the treated group compared to control EAE animals. Also the albumin staining tended to be less pronounced in treated animals. These data suggest a therapeutic role of anti-rCAM treatment on the course of AT-EAE by partially inhibiting the disease as evidenced by clinical and immunohistochemical findings.

292 . 25th Meeting of the Society of Immunology Basel Institute for Immunology, Basel, Switzerland

0.22 A contribution of genes from NZW mice is not necessary for the SLE like disease in crosses with NZB mice T. H. WINKLER, F. MELCHERS, and A. ROLINK (NZBxNZW)F1 mice serve as a model for SLE. Whereas in these mice high levels of IgG anti-dsDNA antibodies and severe glomerulonephritis are found, the parental NZB mice show only IgM autoantibodies and no signs of nephritis. We were interested in the genetic contribution of the NZB mice for the disease and therefore analyzed a series of recombinant inbred strains developed from NZB and SM/J mice. One of the lines, the line NXSM-X, showed all the traits from the NZB mouse namely hyper-IgM in the serum. IgM anti-dsDNA antibodies as well as anti-erythrocyte autoantibodies. Surprisingly high titres of IgG anti-dsDNA antibodies early in life and to some extent nephritis at one year old female mice were observed in the NXSM-X mice. In a cross of the NXSM-X line with NZB the FI mice developed IgG autoantibodies as well as severe proteinuria in kinetics and severity indistinguishable from (NZBxNZW)F1 mice that were analyzed in parallel. We conclude from these data that in the recombinant inbred line NXSM-X the essential genes for the autoimmune disease from NZB are inherited. In addition dominant gene(s) of the SM/J mouse lead to isotype switch of the autoantibodies and to a SLE like disease comparable to (NZBxxNZW)F1 mice. One possible interpretation is that in the NZB mouse a recessive gene suppressing the switch of the autoantibodies is expressed. Currently backcross (NZB NNXSM-X) NNZB mice are analyzed for disease and genetically typed using simple sequence length polymorphisms. Supported by the Deutsche Forschungsgemeinschaft (Wi 1183/1-1).

Basel Institute for Immunology, Basel, Switzerland

0.23 Transfer of pre-B cells from NZBIW mice into SCID mice gives rise to selective clonal expansion and activation of autoreactive B cells T. H. WINKLER, F. MELCHERS, and A. ROLINK The B cell compartment of SCID or RAG-2 T mice were repopulated with pre-B cells from long-term cultures from (NZBxNZW)F1 mice (NZB/W), which develop an autoimmune disease resembling systemic lupus erythematosus. The repopulated mice showed several manifestations of the endogenous NZB/W disease: B cell hyperactivity with IgM and IgG hypergamma-globuminemia and anti-nuclear autoantibodies. We were interested to see whether a clonal selection and expansion of anti-DNA reactive B cell can occur in this model and whether the NZB/W -derived B cells, that spontaneously switch in vivo in the absence of T cells can also hypermutate. The VH genes from 23 IgM and IgG anti-DNA hybridomas from repopulated mice three months after transfer of pre-B cells were analyzed. 21 hybridomas used VH genes that have been described in IgM and IgG anti-DNA hybridomas from diseased NZB/W mice. Within our collection of

25th Meeting of the Society of Immunology . 293 the hybridomas a remarkable restriction in the VH gene usage was observed and two cases of identical VWD-JH rearrangements in independent anti-DNA hybridomas from individual mice were obtained. This strongly suggests clonal origin. There was no somatic mutation in any of the 21 sequences where germline genes could be assigned. We conclude that in the absence of T cells anti-DNA reactive B lymphocytes from NZB/W mice can get activated as well as expanded in a clonally selective way and undergo isotype switch but cannot hypermutate and therefore cannot undergo affinity maturation. Supported by the Deutsche Forschungsgemeinschaft (Wi 1183/1-1).

Dept. of Neurology, J ulius-Maximilians- U niversitat, Wurzburg, Germany

0.24 Cytokines activate nitric oxide synthase in rat and mouse striated muscle cells

J. ZIELASEK, H. REICHMANN, K. V. TOYKA, and H. P. HARTUNG Nitric oxide (NO) is a toxic molecule which is produced by a wide variety of cells and plays a role in the pathogenesis of autoimmune arthritis and host defense against parasites. Inflammatory myopathies are characterized by infiltrates of immune cells, which produce cytokines locally. We studied the induction of nitric oxide synthase (NOS) by treating cultured adult rat and mouse skeletal myoblasts with cytokines. Cultures were more than 99 'Yo pure myoblasts when assayed by immunocytochemistry with antibodies against striated muscle antigens. NOS activation was assessed by measuring nitrite, a stable end-product of NO oxidation in aqueous solutions, with the Griess reaction. We found that both rat and mouse myoblasts secreted nitrite after cytokine-treatment in a dose- and time-dependent manner. This could be blocked by Nmonomethyl-arginine, an inhibitor of both the cytokine-inducible and the constitutive isoforms of NOS, but not with N-nitro arginine, which selectively inhibits the constitutive isoforms of NOS. Since NO inhibits respiratory chain enzymes in vitro, locally produced NO may impair muscle function in inflammatory myopathies.