- Email: [email protected]
Workshop G Autoimmunity
Workshop G Autoimmunity
III. Medizinische u. Poliklinik, Universitat, 6300 Giessen, Germany
G.t Organ specific autoimmune markers in type I diabetes mellitus F. BECKER, K. KRAUTER, S. SCHERER, H. SAUER, and K. FEDERLIN
The use of islet-cell antibodies (ICA) and insulin antibodies (IAA) has made the recognition of the long pre-diabetic period in Type I diabetes mellitus possible. Testing for these antibody specificities in susceptible individuals appears to make prediction of future diabetes possible. In the present study, more than 750 healthy first degree relatives of Type I diabetic patients were followed prospectively for almost 6 years and more than 8000 serum samples screened for the presence of ICA and IAA. The overall prevalence for parents and siblings are 5.1 and 2.8 % for ICA and 7.8 resp. 4.9 % for IAA in this group. While fluctuation of ICA was only observed in two cases with very low titre « 5 JDF-units) ICA, this could be seen in 7 out of 15 IAA-positive patients tested over the whole observation period. The titres of these fluctuating IAA tended to be lower than average. Only one seroconversion was observed with respect to both ICA and IAA each. There was a strong correlation between high titre ICA and the presence of IAA. So far, 4 individuals from the study population have turned diabetic with both tests identifying 3 of these 4 persons. Metabolic testing revealed a faster decline of insulin secretion in individuals with high titre ICA and those with both antibody specificities. Though there is a strong link between the two tests, testing for both ICA and IAA enlarges the probability to identify subjects at risk to develop IDDM.
Institute of Diabetes, 2201 Karlsburg, Institute of Med. Immunology, Humboldt-Universitat, 1040 Berlin, and Institute of Immunology, Free University, 1000 Berlin, Germany
G.2 Tolerance induction after anti-CD25 therapy of diabetic BB/OK rats H.
J. HAHN, B.
KUTILER, A. DUNGER, H. D. YOLK, and T. DIAMANTSTEIN
The autoaggressive disease «insulin-dependent diabetes» of BB/OK rats can be cured by a short-term (10 d) anti-CD25/CyclosporinA (CS-A) therapy, since the majority (> 70 %) of treated rats maintained normoglycaemia for life. In the experiments to be presented we investigated the induction of a possible tolerance 30, 120 or 240 days after treatment. Diabetic BB/OK rats were syngeneically grafted with BB/OK rat islets and treated for 10 d with 1.0 mg ART-18/kg b.w. and 1.5 mg CS-A/kg b.w. 30,120 and 240d later the first graft was removed
XXIInd Meeting of the Society of Immunology . 203 and after relapse into hyperglycaemia a second syngeneic islet graft was given without any immunotreatment into the BB/O K rats. In a second series of experiments the spleen cells of grafted normoglycaemic BB/OK rats were prepared 30,120 or 240d after treatment and given to a diabetic BB/OK rat with a syngeneic islet graft. Neither the recipients (age of diabetes diagnosis, hyperglycaemia, pancreatic insulin content) nor the grafts (islet number and insulin content) differed significantly. Whereas 30 d after the primary islet transplantation only 9 of 14 recipients (64 %) accepted a secondary graft, the secondary graft acceptance increased to 100 % in the other animals investigated. The lymphocytes transfused (no differences in the phenotypically characterized subsets) prevented the graft destruction in 64 %, 66 % or 50 % of the animals. The results let us assume that a lifelong tolerance induction can be achieved within 30d.
Institute of Clinical Immunology and Rheumatology, Dept. of Medicine III, University Erlangen-Niirnberg, 8520 Erlangen, Germany
G.3 Analysis of retroviral genes and transcripts in patients with autoimmune diseases M. HERRMANN, F. E. KRAPF, and J. R. KALDEN
Although the etiology of SLE is unknown, the detection of lentivirus interferon and of antibodies directed against retroviral gene products suggest an involvement of retroviruses in the etiopathogenesis. Recently, GARRY et al. described the detection of a human intracisternal A-type retroviral particle which is antigenic ally related to HIV I in patients with Sjogren's syndrome [1]. We investigated ubiquitous retroviral genes and their transcripts in patients with autoimmune diseases. PCR primers (DONEHOWER et al. [2]) suitable to amplify pol-genes of most known infectious and of many endogenous retroviruses were used to generate pol fragment libraries from lymphocytes from patients with SLE and normal healthy controls. Sequence analysis of a limited number of clones revealed homology to retroviral pol genes in most cases. Moreover, open reading frames were present in 50 % of the clones. To select clones for Northern and Southern Blot analyses the retroviral pol fragment library was screened by reverse Dot Blot hybridization. PCR amplified (universal pol primers) extrachromosomal DNA was used as a probe to detect potential retroviral replication intermediates. Retroviral transcripts were detected by PCR amplified cDNA. To exclude false positive reactions by contaminating genomic DNA in the cDNA preparations, a modified PCR protocol was used for the generation of probes. A pol gene specific upstream primer in addition to a cDNA specific downstream primer was employed. Northern and Southern Blot analysis of selected clones will be presented. 1. GARRY, R. F. et al.: Science 250, 1127-1129 (1990). 2. DONE HOWER, L. A. et al.: J. Virol. Meth. 28, 33-46 (1990). Supported by the Deutsche Forschungsgemeinschaft SFB 263.
204 . XXIInd Meeting of the Society of Immunology Institut fur Med. Immunologie, Charite, Humboldt-Universitat, 1040 Berlin, Germany
G.4 Natural autoantibodies and autoimmune disease S. ]AHN, B. NIEMANN, G. KOTINER, S. T. KIESSIG, and R. VON BAEHR
The healthy organism may possess autoantibody producing B cells. Natural (physiological) autoantibodies encoded by germline genes were often found to be polyspecific, i.e. react with different non-self and autoantigens. From the immortalization of splenic B cells from a patient with idiopathic thrombocytopenia hybridomas were obtained producing polyspecific antibodies recognizing autoantigens (ssDNA, thrombocytes) and non-self material (Lipid A, Tetanus Toxoid) as well. The monoclonal IgM antibody CB-03 was shown to realize different immune functions in vitro (Lipid A neutralization, anti-viral activity, tumor growth inhibition). Nucleotide sequencing of the respective VH gene showed homology to a germ line VH gene. However, 2 exchange mutations were seen both in the CDR-l (pos. 30: Gly....... Arg, 31:Tyr....... Ser) and in the CDR2 (pos. 48: Trp....... Arg, 60: Glu....... Gln). Obviously, one Arg residue located at the DHI]H junction was a result of N-sequence addition. Southern Blot hybridization of hybridoma DNA with a]H probe showed an identical IgH gene rearrangement in an independent hybridoma from the same autoimmune patient'S spleen. Data suggest an oligoclonal expansion of somatically mutated natural autoantibodies in autoimmune patients as a possible pre-requisite of pathogenetical autoantibody formation.
Div. of Immunobiology, Dept. of Medicine, University, 4000 Dusseldorf 1, Germany
G.5 Encapsulation in alginate does not protect rat Langerhans islets from lysis by activated macrophages in vitro K.-D. KRONCKE, F. WIEGAND, and V. KOLB-BACHOFEN
Type I (insulin dependent) diabetes is thought to be an autoimmune disease. In animal models many attempts have been made to transplant Langerhans islets into diabetic animals. For this purpose islets are usually encapsulated in alginate. In transplantation studies it has been shown that alginate encapsulated transplanted islets after several weeks are surrounded by pericapsular infiltrates, the majority being macrophages, granulocytes and lymphocytes. The viability of the islets at that time was greatly reduced which was postulated to be due to malnutrition. We recently showed that activated macrophages can lyse isolated rat islet cells in vitro via secretion of nitric oxide. We here report that activated but not resident macrophages are able to lyse alginate encapsulated rat islets in vitro within 24 h as determined by electron microscopy. Lysis of islets is dependent on the amount of macrophages and the concentration of Larginine. Lysis is inhibited by the nitric oxide synthase inhibitor N-methyl-L-arginine and is overcome by excess of L-arginine.
XXIInd Meeting of the Society of Immunology . 20S Div. of Immunology, Med. Institute of Environmental Hygiene, University, 4000 Diisseldorf 1, Germany
G.6 Murine systemic autoimmune disease induced by HgClz: Demonstration of Hg-specific T cells and of macrophages storing Hg in immunogenic form M. KUBICKA-MURANYI, O. BEHMER, M. UHRBERG, and E. GLEICH MANN
HgCh treatment of susceptible rats and mice induces a systemic autoimmune disease, and Th cells are required for its induction [1]. In the rat, HgCh treatment activates so-called autoreactive CD4+ T cells, i.e. cells that proliferate in response to class-II MHC molecules on syngeneic APC, and it was suggested that these T cells induce the autoimmune disease. The possible activation, however, of Th cells with specificity for Hg has not been studied [2]. Here, we show that HgCl2 treatment of susceptible H-2' mice activates Hg-specific Th cells. Since we were unable to induce an Hg-specific T cell response in vitro we used the adoptive transfer popliteal lymph node assay (PLNA). Splenic T cells of HgCh-treated or saline-treated BI0.S mice were injected into the footpad of syngeneic recipients. If the recipients received a small dose of HgCl2 in addition, the T cells of HgCh-treated donors responded in the form of an anamnestic response, as determined by increased cellularity and B cell activation in the draining PLN. An anamnestic response of the transferred T cells could also be induced when, instead of HgClb fragments of peritoneal macrophages obtained from HgClrtreated donor mice were injected into the footpad of recipients. Conclusions: 1) We assume that HgCh treatment renders self-proteins immunogenic and that these proteins are accumulated and stored in macrophages. 2) We propose that the activation of autoreactive T cells reported by PELLETIER et al. [2J is a byproduct of the activation of Hg-specific Th cells. 1. GOLDMAN, M., P. DRUET, E. GLEICHMANN: Immunol. Today 12, 7 (1990). 2. PELLETIER, L. et al.: J. Immunol. 140, 7S0 (1988).
Institute of Diabetes, 2201 Karlsburg, Germany, LEGENDO, K.U., 3000 Leuven, Belgium
G.7 Lack of autoimmune destruction of syngeneically grafted pancreatic islets in diabetic BB/Pfd rats B. KUTTLER, C. MATHIEU, R. BOUILLON, and H.
J.
HAHN
The BB rat is a well-known animal model for insulin-dependent diabetes which is autoimmune in nature. Normally, recurrence of the disease has been observed following syngeneic transplantation of pancreatic islets into diabetic BB rats due to the auto aggressive process. To prove this hypothesis we grafted 2000 syngeneic islets (donors 8-12 d of age) under the kidney capsules of diabetic rats (diabetes duration < 3 weeks) derived from the two BB rat strains BBI OK (n = 12) and BB/Pfd (n = 11). At transplantation pancreatic biopsies were taken to measure pancreatic insulin content and to characterize remaining islets by morphology (frozen sections). The glycaemia at transplantation was comparable (23.7 ± 2.2 mmolll vs. 2S.0 ± 1.0 mmolll) whereas the pancreatic insulin content was significantly lower in BB/Pfd rats (0.78 ± 0.14 pmol/mg vs. 0.29 ± 0.06 pmollmg, p < O.OS). Morphologically no insulin positive B-cells were found within the islets of BB/Pfd pancreases, whereas within BB/OK pancreases B-cells were still visible. In contrast to BB/OK rats no insulitis could be detected in BB/Pfd rats. In diabetic BB/OK rats graft destruction occurred in 10 of 12 rats (MST > 28.S ± 12.S d).
206 . XXIInd Meeting of the Society of Immunology Surprisingly, graft acceptance was observed in 10 of 11 grafted BB/Pfd recipients (MST> 111.6 ± 8.4 d). Despite the same origin of the two BB rat strains (Ottawa) we observed major differences in pancreatic morphology and no autoimmune destruction of syngeneically grafted islets in BBI Pfd rats possibly due to a lack of autoimmune memory.
Institute of Diabetes, 2201 Karlsburg, and lEUROIMMUN, 2400 Lubeck, Germany
G.8 Extraction and identification of glutamat decarboxylase from rat brain and its function as autoantigen in the type 1 diabetes F. LUHDER, W. STOCKERt, K. D. KOHNERT, and M. ZIEGLER
Sera of newly diagnosed type 1 diabetic patients are characterized by a high prevalence of antibodies reactive to islet cell antigens. One of the most prominent autoantigens is the 64 KD protein, recently identified by BAEKKESKOV et al. as the enzyme glutamate decarboxylase (GAD). Aim of this study was to extract GAD from rat brain and to detect autoantibodies against GAD in sera of type 1 diabetic patients. The extraction was made from rat brain by an ultracentrifugation at 100000 g. The supernatant was able to inhibit the binding of a stiff-man-syndrome (SMS) serum containing antibodies against GAD to the pancreas section by preincubation demonstrated by indirect immunofluorescence technique. In 3 out of 11 (27 %) sera of type 1 diabetic patients GAD autoantibodies were detectable by using this method. Furthermore the SMS-serum has labeled a band of 63/64 KD by Western blot analysis of the 100000 g supernatant. The results show the heterogeneity of the islet cell cytoplasmatic antibodies in the type 1 diabetes including autoantibodies against GAD.
lMax-Planck-Institut fur Psychiatrie, Abt. Neuroimmunologie, 8033 Martinsried, and 2Neurologische Universitatsklinik, 8000 Munchen, Germany
G.9 Multiple sclerosis: T cells specific for myelin basic protein (MBP) show extensive heterogeneity of epitope specificities and of T cell receptor (TCR) variable (V)~ region expression E. MEINLt, F. WEBER 1, G. SARUHAN 1, R. HOHLFELD 1,2
s.
SCHONBECK1.2, N. GOEBELS1, H. WEKERLE, and
In some mouse and rat strains encephalitogenic T cells specific for MBP are biased with respect to epitope specificity and TCR-V region usage. Both features have allowed specific immunotherapies. To determine the extent of heterogeneity of the human immune response against MBP we have established more than 200 long-term MBP specific T cell lines from 15 MS patients and 7 healthy donors and studied all three components of the trimolecular complex of T cell recognition. Using a panel of synthetic peptides of human MBP we found that MS patients recognize at least 15 different epitopes of MBP, individual patients up to 9 different epitopes. Some patients showed a clustering: Two DR2 patients had a cluster between aa86-105, two other patients between aa7-26 or 108-131. TCR-V~ usage was determined at
XXIInd Meeting of the Society of Immunology . 207 the protein level, allowing the identification of members of the families V~5, 6, 8, and 12. No dominance of any particular TCR-~ was evident. Individual patients used at least five different V~s for all epitopes and different TCR-V~s for the most frequently recognized peptide. The extent of heterogeneity of epitope specificity and TCR-V~ usage found in healthy donors was comparable to that in MS-patients. However, no clustering of epitopes was observed. Both in MS patients and in normal donors some MBP specific T cell lines were restricted by DR2b. Our results show that the T cell response against MBP is much more complex in humans than in rodents and that immunotherapy may be more difficult.
lMax-Planck-Institut fur Psychiatrie, Abt. Neuroimmunologie, 8033 Martinsried, and 2Neurologische Universitatsklinik, 8000 Munchen, Germany
G.10 Human myasthenia gravis thymus transplanted in SCID micea model of myasthenia gravis F. PADBERGl, S. SCHONBECK l ,2, H. WEKERLEl, and R. HOHLFELD l ,2
The thymus seems to be critical in the induction of myasthenia gravis (MG). To elucidate the events leading to anti-acetylcholine receptor antibody (AChR abs) production we transplanted pieces of MG thymus in SCID (severe combined immunodeficiency) mice beneath the kidney capsule. The thymus showed lympho-follicular hyperplasia. Every two weeks we determined plasma levels of human and mouse IgG and monitored anti-AChR «autoantibodies» using the a-bungarotoxin binding assay. After 2, 4, 6 or 8 weeks animals were sacrificed and cryosections of the transplant were immunohistochemically stained. At least up to 8 weeks after implantation human cells, positive for CD22, CD4, CD8, HLA-DR, cytokeratin peptide 13, desmin could be demonstrated within the graft area. Human immunoglobulines could be detected from 1 week after ingraftment and IgG titres up to 3 g/ml were maintained for more than two months. Mice were negative for murine IgG unless otherwise indicated. Early after implantation AChR abs began to rise in titer over at least 8 weeks. All the titres varied between individual mice from 0.5 to 5.07 nmol!l, levels typically found in MG patients. Deposits of human immunoglobulins could be stained at the muscle end-plate indicating binding of human antibodies to the murine AChR. Our findings show that grafts of human hyperplastic MG thymus can a) survive in SCID mice and b) initiate and sustain longterm AChR abs production. We currently use this model for studying human autosensitization in human MG.
lDiabetes Research Institute, University, 4000 Dusseldorf, 2FU Berlin, Universitatsklinikum Steglitz, Abt. fur Immunbiologie, 1000 Berlin, Germany, 3Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland
G.11 Aberrant TNFa production and deficient CD45R expression are genetically linked in the dp BB rat H. ROTHE\ A. SCHULLER2, G. RICHTER2, V. JUNGENEEL3, U. KIESELI, T. DIAMANTSTI·.IN 2 , T. BLANKENSTEIN2, and H. KOLE l
Type I diabetes is a T-cell and macrophage dependent disease in BB rats. Previous studies have shown an aberrant production of the macrophage product TNFa in diabetes prone (dp)
208 . XXIInd Meeting of the Society of Immunology BB rats. Classical Southern blot analysis could not detect a restnctlOn fragment length polymorphism (RFLP) in the TNFa locus in two control rat strains (Wistar rats and diabetes resistent (dr) BB rats) and dp BB rats. RFLP analysis of the TNFa locus in other models of autoimmunity also indicate that the TNFa locus is highly conserved. Further, the contiguous genetic arrangement of the lymphotoxin (L T) and the TNFa genes as described in mouse and man exist in the rat. In search of a polymorphic marker to detect a TNFa allele specific for the dp BB rat, we amplified a (CA)n:(GT)n microsatellite in the TNFa promotor region using polymerase chain reaction (PCR). We detected two alleles, a (CAh microsatellite shared by low TNFa producers (Wistar and dr BB rats) and the high TNFa producers (dp BB rats) and a (CA)}} in the Lewis.lA rats. Crosses between dp BB rats and Wistar, respectively Lewis.lA rats revealed that aberrant TNFa production of activated macrophages is largely recessive. Upregulated TNFa production of adherent in vivo (c. parvum) and in vitro (LPS) activated peritoneal macrophages was found in 7/19 (dp BB x Lew.1A)F2 rats and significantly correlated with lymphopenia (r=-0.77, P<0.001) and defective CD45R expression on lymphocytes (r=-0.8023, P<0.001), which is known to be deficient in dp BB rats. In all crosses the macrophage and T-cell defect was only found in animals carrying at least one RT1 u allele. So we conclude that a single linkage group, possible a single gene, is responsible for both: aberrant TNFa production and the defective T cell maturation in the BB rat.
Behringwerke AG, Research Laboratories, 3550 Marburg/Lahn, Germany
G.12 Curative effects of the anti-TCR and anti-CD3 monoclonal antibodies on the development of SLE-like disease in MRL/l autoimmune mice H. U. SCHORLEMMER, E.
J.
KANZY,
R. KURRLE, and F. R. SEILER
With the availability of the anti-mouse a/~-TCR (T cell receptor) monoclonal antibody (MAb) H57-597 (KUBO et aI., J. Immunol. 142,2736, 1989) and the anti-murine CD3-MAb 145-2.Cll (HIRSCH et aI., J. Immunol. 142, 737, 1989) we have been able to analyze the immunoregulatory capacity of these antibodies in experimental animal models. They have been investigated to their disease-modifying activity on the development of autoimmunity in MRLl1pr mice. These autoimmune mice spontaneously develop an SLE-like disease with clinical and serological characteristics that mimic not only human SLE but other autoimmune disorders such as rheumatoid arthritis (RA). The disorder of MRLl1pr mice is characterized by excessive T-lymphocyte proliferation and development of autoantibodies most prevalent directed against double stranded DNA (dsDNA) and type II collagen. These mice also have circulating rheumatoid factor (RF) and develop histological changes in their joints characterized by pannus formation, cartilage and bone erosions. Treatment of MRLllpr mice with the MAbs resulted in an improved survival rate, lowered the percentage of animals with swollen lymph nodes and splenomegaly, reduced the amount of autoantibodies and inhibited proteinuria of the developing glomerulonephritis. Even in the established disease the MAbs could inhibit the proteinuria and returned urine-protein values and renal functions (serum urea and creatinine) to normal levels. Also circulating dsDNA autoantibodies and RF were reduced and the increase in joint swelling (polyarthritis) was inhibited. But even more important also F(ab)z-fragments of these MAbs significantly prolonged the survival time in this model. Thus these types of MAbs should be useful drugs in the treatment of chronic degenerative and autoimmune disease, i.e. an illness resembling human SLE or RA.
XXIInd Meeting of the Society of Immunology . 209 2nd Med. Dept., University, 2300 Kiel 1, Germany
G.13 High dose immunoglobulins in systemic Lupus erythematosus (5LE)
J. o. SCHRODER, A.
H. HEER, H. H. EULER, and H. LOFFLER
We investigated the effect of high dose intravenous immunoglobulins in systemic Lupus erythematosus (SLE). 12 patients (£.) with active SLE were treated with 3 x 40 g ivIg (Venimmun®) on 4 consecutive days. In the majority of cases the same treatment was repeated after three weeks. 4/12 patients presented with severe disease; since, however, cytotoxic drug treatment could not be applied because of prior surgery andlor severe infection, treatment with plasmapheresis and corticosteroids was initiated. Additionally, ivIg were given to prevent plasmapheresis induced disease exacerbation. In all cases (4/4) control of symptoms could be achieved. However, the individual effect of ivIg could not be assessed. In 8/12 further patients receiving only ivIg as a new measure, pretreatment, consisting of corticosteroids (7/8), azathioprine (2/8) or hydroxychloroquine (1/8), was not changed during and at least 6 weeks after ivIg-treatment. In 4/8 cases treatment was followed by clinical improvement, particularly by a rise in platelet cound and hemoglobin and by a mitigation of erythema and arthritis. In 118 patients clinical benefit was regarded as probable, in 3/8 patients no improvement was seen. Similar results were found for serologic parameters: 5/13 treatment cycles were followed by a decline in ANA-titer (at least two dilutions) and by decreased levels of antibodies to ds-DNA. 2/13 treatment cycles were followed by an increase of these parameters. 6/13 treatment cycles were not followed by a change in the serological status. Complement proteins were not influenced at all. - These data seem to confirm other studies showing that iv Ig are of clinical value in some patients with SLE. Thus, ivIg may represent a treatment modality in case other options are not available. However, to define the role of ivIg in SLE more precisely, controlled studies seem to be necessary.
lInstitute of Immunology and Molecular Genetics, 7500 Karlsruhe, and 2Dept. of Medicine and Immunology and Bloodbank, University, 3000 Hannover, Germany
G.14 Antibodies to Ro (55-A) and La (55-B) detected by ELI5A with recombinant Ro and La fusion protein H. P. SEELIG \ H. EHRFELDl, M. RENZI, K. HARTUNG 2, R. COLDEWEy2, H. DEICHER2, and the Members of the SLE Study Group Autoantibodies to Ro (SS-A) antigens frequently occurring in Sjogrens syndrome and systemic lupus erythematosus (SLE) react with at least four antigenically distinct forms of human Ro (60, 54, 52, 48 kD proteins). Anti-La antibodies (SS-B) react with a 50 kD protein antigen of a ribonucleoprotein complex. cDNAs enconding the 60 kD Ro and 50 kD La were obtained by polymerase chain reaction and ligated into pEx34b expression vector. Recombinant proteins were purified by SDS-PAGE. 2-3 ttg/ml protein were coated to micro titer plates. Sera were analyzed at 1:300 dilutions using polyvalent anti-human IgGI AIM and OPD as substrate. Results were calculated using standard curves prepared from anti-Ro and anti-La positive sera assigning 400 Ulml to the 1:300 dilution. 866 sera of SLE patients (N = 377), sera of their relatives (N = 386) and 640 sera of patients with suspected or manifest rheumatic
210 . XXIInd Meeting of the Society of Immunology disease were analyzed. In sera of 25 healthy persons anti-Ro was 12.5 ± 4.1 U/ml, anti-La 9.6±3.6 U/ml. Anti-60 kD Ro was found in 16% of SLE patients, anti-La in 14%, both antibodies in 7.7 % of these patients. Relatives showed anti-Ro in 1.5 %, anti-La in 4 %, both antibodies in 1 %. In patients with suspected or manifest rheumatic disease anti-Ro was found in 27 %, anti-La in 15.1 %, both antibodies in 7.6 %. In SLE patients 2 to 3 sera could be tested in most cases of which 96 % showed congruent results for anti-60 kD Ro, 98 % for anti-La. Antibody activities up to several thousand U could be detected. In the patient group with suspected or manifest rheumatic diseases the proportion of lower anti-Ro and anti-La activities « 40 U/ml) was with 15 % (anti-Ro) or 8 % (anti-La) higher than in the SLE group (5 % antiRo, 4 % anti-La). The ELISA revealed significant differences in anti-60 kD Ro and anti-La antibody frequencies of healthy persons, SLE patients and their relatives. Our figure of 16 % for anti-Ro is lower than that of 37 % reported from an immunoblot analysis with natural antigens. Compared to other studies using ELISA with natural or recombinant La-antigens our figures in SLE patients are also lower but in the routine sera group (N = 640) 26 % anti-60 kD Ro positive tests were obtained, figures which are in the magnitude of antibody frequencies obtained with our natural antigens. In parallel tests with natural La-antigen the concordance was 88 %.
Chirurgische Klinik u. Institut fur Med. Immunologie, Charite, Humboldt-Universitat, 1040 Berlin, Germany
G.1S Natural autoantibodies in the fetal human B cell repertoire U. SETTMACHER, G. GROTZ, K. WINKLER, A. LUKOWSKY, H. WOLFF, R. VON BAEHR, and S.JAHN
Human hybridomas were generated from fetal lymphocytes (15-36 week of gestation) hybridized with the heteromyeloma line CB-F7. Independently of the gestational age, 7-10 % of the IgM-producing hybridomas were found to secrete autoantibodies reacting with ssDNA, keratin or thrombocytes. Some of the monoclonal antibodies, however, were polyspecific, and reacted with foreign antigen material (Lipid A, Tetanus Toxin) as well. The nucleotide sequences of the respective VH and VL genes have been determined. The usage of unmutated germline genes belonging both to the VHI and VHIII families was shown. The DNA derived from 10 independent hybridomas (6 fetuses) were hybridized with a JH probe. No clonal relationship between the maternal B lymphocytes producing natural autoantibodies could be detected. We discuss the frequent occurrence of autoimmune B cells in the fetal immune system as an important aspect of natural repertoire generation, including anti-bacterial specificities which may guarantee a first line defense in the newborn organism.
XXIInd Meeting of the Society of Immunology· 211 Augenklinik der Universitat, Mathildenstr. 8, 8000 Miinchen 2, Germany, and National Eye Institute, NIH, Bethesda, MD 20892, USA
G.t6 Oral suppression with IRBP in the acute and chronic relapsing mouse models of experimental autoimmune uveitis (EAU) S. R. THURAu,
c.-c. CHAN, E. SUH, B. WIGGERT, R. B. NUSSENBLATT, and R. R. CASPI
Uveitis is an inflammatory autoimmune disorder in man, which affects ocular tissues (uvea and retina) and can cause blindness in severe therapy refractive cases. Mouse EAU serves as a model for human uveitis. EAU is a T cell-dependent autoimmune disease, which is experimentally induced by footpad immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP) emulsified in complete Freund's adjuvant. In the following study we investigated the potential of antigen feeding to downregulate acute as well as chronic relapsing EAU in B10.A mice. In the first set of experiments mice were fed 4 times with IRBP or with Keyhole Limpet Hemocyanin (KLH) prior to the active immunization with 100 [.lg IRBP, which induces an acute uveitis. Three weeks later histology revealed significantly reduced damage of the retina in the IRBP fed mice. In vitro proliferative responses to IRBP were also reduced in treated mice. In the second set of experiments chronic relapsing EAU was induced with a low dose immunization scheme and feeding was started 8 weeks later after mice had recovered from their first attack of uveitis as determined by fundoscopy. For the following 5 weeks animals received IRBP or KLH. On fundoscopy control mice developed a relapse of uveitis 3 weeks after the first attack, while the relapse was inhibited in mice fed with IRBP. Again, histology showed significantly reduced retinal destruction in treated mice. Interestingly, specific antibody titers were not reduced, while cellular responses to IRBP did not always correlate with the clinical responses in the various groups.
Dept. of Immunology, Medical School, University, 2300 Kiel, Germany
G.t7 A clinically successful protocol to suppress autoantibody production in SLE patients is analyzed for its efficacy to inhibit natural xenophile antibodies (NXA) K. ULRICHS, H. H. EULER, and W. MULLER-RuCHHOLTZ Plasmapheresis in conjunction with a defined, time-related course of CY injections has been shown to be very effective in treating patients who suffer from severe forms of systemic lupus erythematodes (SLE). It was the aim of our study to analyze the effect of this established clinical protocol on the kinetics of natural xenophile antibodies (NXA) against porcine pancreatic islet cells, which are known to be responsible for acute and hyperacute rejection of islet xenografts. For this purpose, sera from 5 SLE patients were additionally tested for NXA against porcine pancreatic cells by quantitative immunofluorescence (a) before, (n) immediately after, and (c) long-term after treatment. Results: (1) In contrast to healthy individuals, SLE patients are strongly positive for disease-related anti-nucleic antibodies (ANA), but negative for NXA. (2) Immediately after treatment, both ANA and NXA are negative. (3) Six to twelve months after treatment, only a marginal reoccurrence of ANA is observed. Reoccurrence of NXA reaches titers, considered normal. (4) ANA and NXA are of IgG rather than IgM isotype. Conclusions: (I) Plasmapheresis combined with a short-term course of the alkylating agent CY appears to be very effective in eliminating activated B cell clones but rather
212 . XXIInd Meeting of the Society of Immunology ineffective in eliminating «resting» clones, such as NXA clones. (II) In the context of xenotransplantation, this clinical protocol could be particularly effective in eliminating those antibodies which may be produced by xenograft-activated B cells.
Dept. of Chemistry, University, 6750 Kaiserslautern, Germany
G.18 Antigen-specific therapy of experimental autoimmune myasthenia gravis with acetylcholine receptor-gelonin conjugates
in vivo I. L. URBATSCH, R. K. M. STERZ, K. PEPER, and W. E. TROMMER The major pathophysiological deficiency in myasthenia gravis as well as in its animal model, experimental autoimmune myasthenia gravis (EAMG) is the T cell-dependent production of autoantibodies against acetylcholine receptors (AChR) in the muscle membrane by specific B cells. This leads to an accelerated degradation of functional AChR and associated ionic endplate channels giving rise to the clinical symptoms of impaired neuromuscular transmission (i.e., weakness and various degree of paralysis). In order to eliminate these specific B cell clones, rats with EAMG were injected with antigen toxin conjugates constructed by specific coupling of purified AChR with the plant toxin gelonin. This led to a marked improvement of clinical symptoms and an increase in the number of ionic endplate channel density compared to untreated animals with EAMG. The immune response to irrelevant control antigens was not altered by this treatment. Injections of equivalent amounts (molar basis) of gelonin or AChR alone had no therapeutic effect.
!Dept. of Immunology, Dept. of Internal Medicine, Medical University, 3000 Hannover 61, 2Fraunhofer-Institut, 3000 Hannover, Germany
G .19 Increased number of macrophages and monocyte precursor cells in organs of autoimmune male BXSB mice G. VIETEN!, K. BEHRENS2, A. EMMENDORFFER2, K. HARTUNG!, and H. DEICHER!
BXSB mice spontaneously develop progressive fatal autoimmune disease which resembles human systemic lupus erythematosus (SLE). During their lifetime male BXSB mice develop a high level of serum immunoglobulins, including anti-nuclear antibodies (ANA), a progressive diffuse proliferative glomerulonephritis and an increasing monocytosis in the peripheral blood which is unique among lupus mice. As we could show previously there is a strain-specific high number of myeloid precursor cells in the bone marrow especially monocyte precursor cells (M-CFC). The number of M-CFC is especially high in male BXSB mice and increases with progressing disease. Our latest data show that there is an extramedullary appearance of MCFC in spleen and liver of male BXSB mice which increases with progressing disease. In addition to a morphologic alteration of the organs there is a change in the number and distribution of organ resident macrophages. The alteration of the macrophage population may be one reason for the rapid progression of the autoimmune disease in male BXSB mice. Supported by DFVLR 01 VM 8606.
III. Medizinische u. Poliklinik, Universitat, 6300 Giessen, Germany
G.t Organ specific autoimmune markers in type I diabetes mellitus F. BECKER, K. KRAUTER, S. SCHERER, H. SAUER, and K. FEDERLIN
The use of islet-cell antibodies (ICA) and insulin antibodies (IAA) has made the recognition of the long pre-diabetic period in Type I diabetes mellitus possible. Testing for these antibody specificities in susceptible individuals appears to make prediction of future diabetes possible. In the present study, more than 750 healthy first degree relatives of Type I diabetic patients were followed prospectively for almost 6 years and more than 8000 serum samples screened for the presence of ICA and IAA. The overall prevalence for parents and siblings are 5.1 and 2.8 % for ICA and 7.8 resp. 4.9 % for IAA in this group. While fluctuation of ICA was only observed in two cases with very low titre « 5 JDF-units) ICA, this could be seen in 7 out of 15 IAA-positive patients tested over the whole observation period. The titres of these fluctuating IAA tended to be lower than average. Only one seroconversion was observed with respect to both ICA and IAA each. There was a strong correlation between high titre ICA and the presence of IAA. So far, 4 individuals from the study population have turned diabetic with both tests identifying 3 of these 4 persons. Metabolic testing revealed a faster decline of insulin secretion in individuals with high titre ICA and those with both antibody specificities. Though there is a strong link between the two tests, testing for both ICA and IAA enlarges the probability to identify subjects at risk to develop IDDM.
Institute of Diabetes, 2201 Karlsburg, Institute of Med. Immunology, Humboldt-Universitat, 1040 Berlin, and Institute of Immunology, Free University, 1000 Berlin, Germany
G.2 Tolerance induction after anti-CD25 therapy of diabetic BB/OK rats H.
J. HAHN, B.
KUTILER, A. DUNGER, H. D. YOLK, and T. DIAMANTSTEIN
The autoaggressive disease «insulin-dependent diabetes» of BB/OK rats can be cured by a short-term (10 d) anti-CD25/CyclosporinA (CS-A) therapy, since the majority (> 70 %) of treated rats maintained normoglycaemia for life. In the experiments to be presented we investigated the induction of a possible tolerance 30, 120 or 240 days after treatment. Diabetic BB/OK rats were syngeneically grafted with BB/OK rat islets and treated for 10 d with 1.0 mg ART-18/kg b.w. and 1.5 mg CS-A/kg b.w. 30,120 and 240d later the first graft was removed
XXIInd Meeting of the Society of Immunology . 203 and after relapse into hyperglycaemia a second syngeneic islet graft was given without any immunotreatment into the BB/O K rats. In a second series of experiments the spleen cells of grafted normoglycaemic BB/OK rats were prepared 30,120 or 240d after treatment and given to a diabetic BB/OK rat with a syngeneic islet graft. Neither the recipients (age of diabetes diagnosis, hyperglycaemia, pancreatic insulin content) nor the grafts (islet number and insulin content) differed significantly. Whereas 30 d after the primary islet transplantation only 9 of 14 recipients (64 %) accepted a secondary graft, the secondary graft acceptance increased to 100 % in the other animals investigated. The lymphocytes transfused (no differences in the phenotypically characterized subsets) prevented the graft destruction in 64 %, 66 % or 50 % of the animals. The results let us assume that a lifelong tolerance induction can be achieved within 30d.
Institute of Clinical Immunology and Rheumatology, Dept. of Medicine III, University Erlangen-Niirnberg, 8520 Erlangen, Germany
G.3 Analysis of retroviral genes and transcripts in patients with autoimmune diseases M. HERRMANN, F. E. KRAPF, and J. R. KALDEN
Although the etiology of SLE is unknown, the detection of lentivirus interferon and of antibodies directed against retroviral gene products suggest an involvement of retroviruses in the etiopathogenesis. Recently, GARRY et al. described the detection of a human intracisternal A-type retroviral particle which is antigenic ally related to HIV I in patients with Sjogren's syndrome [1]. We investigated ubiquitous retroviral genes and their transcripts in patients with autoimmune diseases. PCR primers (DONEHOWER et al. [2]) suitable to amplify pol-genes of most known infectious and of many endogenous retroviruses were used to generate pol fragment libraries from lymphocytes from patients with SLE and normal healthy controls. Sequence analysis of a limited number of clones revealed homology to retroviral pol genes in most cases. Moreover, open reading frames were present in 50 % of the clones. To select clones for Northern and Southern Blot analyses the retroviral pol fragment library was screened by reverse Dot Blot hybridization. PCR amplified (universal pol primers) extrachromosomal DNA was used as a probe to detect potential retroviral replication intermediates. Retroviral transcripts were detected by PCR amplified cDNA. To exclude false positive reactions by contaminating genomic DNA in the cDNA preparations, a modified PCR protocol was used for the generation of probes. A pol gene specific upstream primer in addition to a cDNA specific downstream primer was employed. Northern and Southern Blot analysis of selected clones will be presented. 1. GARRY, R. F. et al.: Science 250, 1127-1129 (1990). 2. DONE HOWER, L. A. et al.: J. Virol. Meth. 28, 33-46 (1990). Supported by the Deutsche Forschungsgemeinschaft SFB 263.
204 . XXIInd Meeting of the Society of Immunology Institut fur Med. Immunologie, Charite, Humboldt-Universitat, 1040 Berlin, Germany
G.4 Natural autoantibodies and autoimmune disease S. ]AHN, B. NIEMANN, G. KOTINER, S. T. KIESSIG, and R. VON BAEHR
The healthy organism may possess autoantibody producing B cells. Natural (physiological) autoantibodies encoded by germline genes were often found to be polyspecific, i.e. react with different non-self and autoantigens. From the immortalization of splenic B cells from a patient with idiopathic thrombocytopenia hybridomas were obtained producing polyspecific antibodies recognizing autoantigens (ssDNA, thrombocytes) and non-self material (Lipid A, Tetanus Toxoid) as well. The monoclonal IgM antibody CB-03 was shown to realize different immune functions in vitro (Lipid A neutralization, anti-viral activity, tumor growth inhibition). Nucleotide sequencing of the respective VH gene showed homology to a germ line VH gene. However, 2 exchange mutations were seen both in the CDR-l (pos. 30: Gly....... Arg, 31:Tyr....... Ser) and in the CDR2 (pos. 48: Trp....... Arg, 60: Glu....... Gln). Obviously, one Arg residue located at the DHI]H junction was a result of N-sequence addition. Southern Blot hybridization of hybridoma DNA with a]H probe showed an identical IgH gene rearrangement in an independent hybridoma from the same autoimmune patient'S spleen. Data suggest an oligoclonal expansion of somatically mutated natural autoantibodies in autoimmune patients as a possible pre-requisite of pathogenetical autoantibody formation.
Div. of Immunobiology, Dept. of Medicine, University, 4000 Dusseldorf 1, Germany
G.5 Encapsulation in alginate does not protect rat Langerhans islets from lysis by activated macrophages in vitro K.-D. KRONCKE, F. WIEGAND, and V. KOLB-BACHOFEN
Type I (insulin dependent) diabetes is thought to be an autoimmune disease. In animal models many attempts have been made to transplant Langerhans islets into diabetic animals. For this purpose islets are usually encapsulated in alginate. In transplantation studies it has been shown that alginate encapsulated transplanted islets after several weeks are surrounded by pericapsular infiltrates, the majority being macrophages, granulocytes and lymphocytes. The viability of the islets at that time was greatly reduced which was postulated to be due to malnutrition. We recently showed that activated macrophages can lyse isolated rat islet cells in vitro via secretion of nitric oxide. We here report that activated but not resident macrophages are able to lyse alginate encapsulated rat islets in vitro within 24 h as determined by electron microscopy. Lysis of islets is dependent on the amount of macrophages and the concentration of Larginine. Lysis is inhibited by the nitric oxide synthase inhibitor N-methyl-L-arginine and is overcome by excess of L-arginine.
XXIInd Meeting of the Society of Immunology . 20S Div. of Immunology, Med. Institute of Environmental Hygiene, University, 4000 Diisseldorf 1, Germany
G.6 Murine systemic autoimmune disease induced by HgClz: Demonstration of Hg-specific T cells and of macrophages storing Hg in immunogenic form M. KUBICKA-MURANYI, O. BEHMER, M. UHRBERG, and E. GLEICH MANN
HgCh treatment of susceptible rats and mice induces a systemic autoimmune disease, and Th cells are required for its induction [1]. In the rat, HgCh treatment activates so-called autoreactive CD4+ T cells, i.e. cells that proliferate in response to class-II MHC molecules on syngeneic APC, and it was suggested that these T cells induce the autoimmune disease. The possible activation, however, of Th cells with specificity for Hg has not been studied [2]. Here, we show that HgCl2 treatment of susceptible H-2' mice activates Hg-specific Th cells. Since we were unable to induce an Hg-specific T cell response in vitro we used the adoptive transfer popliteal lymph node assay (PLNA). Splenic T cells of HgCh-treated or saline-treated BI0.S mice were injected into the footpad of syngeneic recipients. If the recipients received a small dose of HgCl2 in addition, the T cells of HgCh-treated donors responded in the form of an anamnestic response, as determined by increased cellularity and B cell activation in the draining PLN. An anamnestic response of the transferred T cells could also be induced when, instead of HgClb fragments of peritoneal macrophages obtained from HgClrtreated donor mice were injected into the footpad of recipients. Conclusions: 1) We assume that HgCh treatment renders self-proteins immunogenic and that these proteins are accumulated and stored in macrophages. 2) We propose that the activation of autoreactive T cells reported by PELLETIER et al. [2J is a byproduct of the activation of Hg-specific Th cells. 1. GOLDMAN, M., P. DRUET, E. GLEICHMANN: Immunol. Today 12, 7 (1990). 2. PELLETIER, L. et al.: J. Immunol. 140, 7S0 (1988).
Institute of Diabetes, 2201 Karlsburg, Germany, LEGENDO, K.U., 3000 Leuven, Belgium
G.7 Lack of autoimmune destruction of syngeneically grafted pancreatic islets in diabetic BB/Pfd rats B. KUTTLER, C. MATHIEU, R. BOUILLON, and H.
J.
HAHN
The BB rat is a well-known animal model for insulin-dependent diabetes which is autoimmune in nature. Normally, recurrence of the disease has been observed following syngeneic transplantation of pancreatic islets into diabetic BB rats due to the auto aggressive process. To prove this hypothesis we grafted 2000 syngeneic islets (donors 8-12 d of age) under the kidney capsules of diabetic rats (diabetes duration < 3 weeks) derived from the two BB rat strains BBI OK (n = 12) and BB/Pfd (n = 11). At transplantation pancreatic biopsies were taken to measure pancreatic insulin content and to characterize remaining islets by morphology (frozen sections). The glycaemia at transplantation was comparable (23.7 ± 2.2 mmolll vs. 2S.0 ± 1.0 mmolll) whereas the pancreatic insulin content was significantly lower in BB/Pfd rats (0.78 ± 0.14 pmol/mg vs. 0.29 ± 0.06 pmollmg, p < O.OS). Morphologically no insulin positive B-cells were found within the islets of BB/Pfd pancreases, whereas within BB/OK pancreases B-cells were still visible. In contrast to BB/OK rats no insulitis could be detected in BB/Pfd rats. In diabetic BB/OK rats graft destruction occurred in 10 of 12 rats (MST > 28.S ± 12.S d).
206 . XXIInd Meeting of the Society of Immunology Surprisingly, graft acceptance was observed in 10 of 11 grafted BB/Pfd recipients (MST> 111.6 ± 8.4 d). Despite the same origin of the two BB rat strains (Ottawa) we observed major differences in pancreatic morphology and no autoimmune destruction of syngeneically grafted islets in BBI Pfd rats possibly due to a lack of autoimmune memory.
Institute of Diabetes, 2201 Karlsburg, and lEUROIMMUN, 2400 Lubeck, Germany
G.8 Extraction and identification of glutamat decarboxylase from rat brain and its function as autoantigen in the type 1 diabetes F. LUHDER, W. STOCKERt, K. D. KOHNERT, and M. ZIEGLER
Sera of newly diagnosed type 1 diabetic patients are characterized by a high prevalence of antibodies reactive to islet cell antigens. One of the most prominent autoantigens is the 64 KD protein, recently identified by BAEKKESKOV et al. as the enzyme glutamate decarboxylase (GAD). Aim of this study was to extract GAD from rat brain and to detect autoantibodies against GAD in sera of type 1 diabetic patients. The extraction was made from rat brain by an ultracentrifugation at 100000 g. The supernatant was able to inhibit the binding of a stiff-man-syndrome (SMS) serum containing antibodies against GAD to the pancreas section by preincubation demonstrated by indirect immunofluorescence technique. In 3 out of 11 (27 %) sera of type 1 diabetic patients GAD autoantibodies were detectable by using this method. Furthermore the SMS-serum has labeled a band of 63/64 KD by Western blot analysis of the 100000 g supernatant. The results show the heterogeneity of the islet cell cytoplasmatic antibodies in the type 1 diabetes including autoantibodies against GAD.
lMax-Planck-Institut fur Psychiatrie, Abt. Neuroimmunologie, 8033 Martinsried, and 2Neurologische Universitatsklinik, 8000 Munchen, Germany
G.9 Multiple sclerosis: T cells specific for myelin basic protein (MBP) show extensive heterogeneity of epitope specificities and of T cell receptor (TCR) variable (V)~ region expression E. MEINLt, F. WEBER 1, G. SARUHAN 1, R. HOHLFELD 1,2
s.
SCHONBECK1.2, N. GOEBELS1, H. WEKERLE, and
In some mouse and rat strains encephalitogenic T cells specific for MBP are biased with respect to epitope specificity and TCR-V region usage. Both features have allowed specific immunotherapies. To determine the extent of heterogeneity of the human immune response against MBP we have established more than 200 long-term MBP specific T cell lines from 15 MS patients and 7 healthy donors and studied all three components of the trimolecular complex of T cell recognition. Using a panel of synthetic peptides of human MBP we found that MS patients recognize at least 15 different epitopes of MBP, individual patients up to 9 different epitopes. Some patients showed a clustering: Two DR2 patients had a cluster between aa86-105, two other patients between aa7-26 or 108-131. TCR-V~ usage was determined at
XXIInd Meeting of the Society of Immunology . 207 the protein level, allowing the identification of members of the families V~5, 6, 8, and 12. No dominance of any particular TCR-~ was evident. Individual patients used at least five different V~s for all epitopes and different TCR-V~s for the most frequently recognized peptide. The extent of heterogeneity of epitope specificity and TCR-V~ usage found in healthy donors was comparable to that in MS-patients. However, no clustering of epitopes was observed. Both in MS patients and in normal donors some MBP specific T cell lines were restricted by DR2b. Our results show that the T cell response against MBP is much more complex in humans than in rodents and that immunotherapy may be more difficult.
lMax-Planck-Institut fur Psychiatrie, Abt. Neuroimmunologie, 8033 Martinsried, and 2Neurologische Universitatsklinik, 8000 Munchen, Germany
G.10 Human myasthenia gravis thymus transplanted in SCID micea model of myasthenia gravis F. PADBERGl, S. SCHONBECK l ,2, H. WEKERLEl, and R. HOHLFELD l ,2
The thymus seems to be critical in the induction of myasthenia gravis (MG). To elucidate the events leading to anti-acetylcholine receptor antibody (AChR abs) production we transplanted pieces of MG thymus in SCID (severe combined immunodeficiency) mice beneath the kidney capsule. The thymus showed lympho-follicular hyperplasia. Every two weeks we determined plasma levels of human and mouse IgG and monitored anti-AChR «autoantibodies» using the a-bungarotoxin binding assay. After 2, 4, 6 or 8 weeks animals were sacrificed and cryosections of the transplant were immunohistochemically stained. At least up to 8 weeks after implantation human cells, positive for CD22, CD4, CD8, HLA-DR, cytokeratin peptide 13, desmin could be demonstrated within the graft area. Human immunoglobulines could be detected from 1 week after ingraftment and IgG titres up to 3 g/ml were maintained for more than two months. Mice were negative for murine IgG unless otherwise indicated. Early after implantation AChR abs began to rise in titer over at least 8 weeks. All the titres varied between individual mice from 0.5 to 5.07 nmol!l, levels typically found in MG patients. Deposits of human immunoglobulins could be stained at the muscle end-plate indicating binding of human antibodies to the murine AChR. Our findings show that grafts of human hyperplastic MG thymus can a) survive in SCID mice and b) initiate and sustain longterm AChR abs production. We currently use this model for studying human autosensitization in human MG.
lDiabetes Research Institute, University, 4000 Dusseldorf, 2FU Berlin, Universitatsklinikum Steglitz, Abt. fur Immunbiologie, 1000 Berlin, Germany, 3Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland
G.11 Aberrant TNFa production and deficient CD45R expression are genetically linked in the dp BB rat H. ROTHE\ A. SCHULLER2, G. RICHTER2, V. JUNGENEEL3, U. KIESELI, T. DIAMANTSTI·.IN 2 , T. BLANKENSTEIN2, and H. KOLE l
Type I diabetes is a T-cell and macrophage dependent disease in BB rats. Previous studies have shown an aberrant production of the macrophage product TNFa in diabetes prone (dp)
208 . XXIInd Meeting of the Society of Immunology BB rats. Classical Southern blot analysis could not detect a restnctlOn fragment length polymorphism (RFLP) in the TNFa locus in two control rat strains (Wistar rats and diabetes resistent (dr) BB rats) and dp BB rats. RFLP analysis of the TNFa locus in other models of autoimmunity also indicate that the TNFa locus is highly conserved. Further, the contiguous genetic arrangement of the lymphotoxin (L T) and the TNFa genes as described in mouse and man exist in the rat. In search of a polymorphic marker to detect a TNFa allele specific for the dp BB rat, we amplified a (CA)n:(GT)n microsatellite in the TNFa promotor region using polymerase chain reaction (PCR). We detected two alleles, a (CAh microsatellite shared by low TNFa producers (Wistar and dr BB rats) and the high TNFa producers (dp BB rats) and a (CA)}} in the Lewis.lA rats. Crosses between dp BB rats and Wistar, respectively Lewis.lA rats revealed that aberrant TNFa production of activated macrophages is largely recessive. Upregulated TNFa production of adherent in vivo (c. parvum) and in vitro (LPS) activated peritoneal macrophages was found in 7/19 (dp BB x Lew.1A)F2 rats and significantly correlated with lymphopenia (r=-0.77, P<0.001) and defective CD45R expression on lymphocytes (r=-0.8023, P<0.001), which is known to be deficient in dp BB rats. In all crosses the macrophage and T-cell defect was only found in animals carrying at least one RT1 u allele. So we conclude that a single linkage group, possible a single gene, is responsible for both: aberrant TNFa production and the defective T cell maturation in the BB rat.
Behringwerke AG, Research Laboratories, 3550 Marburg/Lahn, Germany
G.12 Curative effects of the anti-TCR and anti-CD3 monoclonal antibodies on the development of SLE-like disease in MRL/l autoimmune mice H. U. SCHORLEMMER, E.
J.
KANZY,
R. KURRLE, and F. R. SEILER
With the availability of the anti-mouse a/~-TCR (T cell receptor) monoclonal antibody (MAb) H57-597 (KUBO et aI., J. Immunol. 142,2736, 1989) and the anti-murine CD3-MAb 145-2.Cll (HIRSCH et aI., J. Immunol. 142, 737, 1989) we have been able to analyze the immunoregulatory capacity of these antibodies in experimental animal models. They have been investigated to their disease-modifying activity on the development of autoimmunity in MRLl1pr mice. These autoimmune mice spontaneously develop an SLE-like disease with clinical and serological characteristics that mimic not only human SLE but other autoimmune disorders such as rheumatoid arthritis (RA). The disorder of MRLl1pr mice is characterized by excessive T-lymphocyte proliferation and development of autoantibodies most prevalent directed against double stranded DNA (dsDNA) and type II collagen. These mice also have circulating rheumatoid factor (RF) and develop histological changes in their joints characterized by pannus formation, cartilage and bone erosions. Treatment of MRLllpr mice with the MAbs resulted in an improved survival rate, lowered the percentage of animals with swollen lymph nodes and splenomegaly, reduced the amount of autoantibodies and inhibited proteinuria of the developing glomerulonephritis. Even in the established disease the MAbs could inhibit the proteinuria and returned urine-protein values and renal functions (serum urea and creatinine) to normal levels. Also circulating dsDNA autoantibodies and RF were reduced and the increase in joint swelling (polyarthritis) was inhibited. But even more important also F(ab)z-fragments of these MAbs significantly prolonged the survival time in this model. Thus these types of MAbs should be useful drugs in the treatment of chronic degenerative and autoimmune disease, i.e. an illness resembling human SLE or RA.
XXIInd Meeting of the Society of Immunology . 209 2nd Med. Dept., University, 2300 Kiel 1, Germany
G.13 High dose immunoglobulins in systemic Lupus erythematosus (5LE)
J. o. SCHRODER, A.
H. HEER, H. H. EULER, and H. LOFFLER
We investigated the effect of high dose intravenous immunoglobulins in systemic Lupus erythematosus (SLE). 12 patients (£.) with active SLE were treated with 3 x 40 g ivIg (Venimmun®) on 4 consecutive days. In the majority of cases the same treatment was repeated after three weeks. 4/12 patients presented with severe disease; since, however, cytotoxic drug treatment could not be applied because of prior surgery andlor severe infection, treatment with plasmapheresis and corticosteroids was initiated. Additionally, ivIg were given to prevent plasmapheresis induced disease exacerbation. In all cases (4/4) control of symptoms could be achieved. However, the individual effect of ivIg could not be assessed. In 8/12 further patients receiving only ivIg as a new measure, pretreatment, consisting of corticosteroids (7/8), azathioprine (2/8) or hydroxychloroquine (1/8), was not changed during and at least 6 weeks after ivIg-treatment. In 4/8 cases treatment was followed by clinical improvement, particularly by a rise in platelet cound and hemoglobin and by a mitigation of erythema and arthritis. In 118 patients clinical benefit was regarded as probable, in 3/8 patients no improvement was seen. Similar results were found for serologic parameters: 5/13 treatment cycles were followed by a decline in ANA-titer (at least two dilutions) and by decreased levels of antibodies to ds-DNA. 2/13 treatment cycles were followed by an increase of these parameters. 6/13 treatment cycles were not followed by a change in the serological status. Complement proteins were not influenced at all. - These data seem to confirm other studies showing that iv Ig are of clinical value in some patients with SLE. Thus, ivIg may represent a treatment modality in case other options are not available. However, to define the role of ivIg in SLE more precisely, controlled studies seem to be necessary.
lInstitute of Immunology and Molecular Genetics, 7500 Karlsruhe, and 2Dept. of Medicine and Immunology and Bloodbank, University, 3000 Hannover, Germany
G.14 Antibodies to Ro (55-A) and La (55-B) detected by ELI5A with recombinant Ro and La fusion protein H. P. SEELIG \ H. EHRFELDl, M. RENZI, K. HARTUNG 2, R. COLDEWEy2, H. DEICHER2, and the Members of the SLE Study Group Autoantibodies to Ro (SS-A) antigens frequently occurring in Sjogrens syndrome and systemic lupus erythematosus (SLE) react with at least four antigenically distinct forms of human Ro (60, 54, 52, 48 kD proteins). Anti-La antibodies (SS-B) react with a 50 kD protein antigen of a ribonucleoprotein complex. cDNAs enconding the 60 kD Ro and 50 kD La were obtained by polymerase chain reaction and ligated into pEx34b expression vector. Recombinant proteins were purified by SDS-PAGE. 2-3 ttg/ml protein were coated to micro titer plates. Sera were analyzed at 1:300 dilutions using polyvalent anti-human IgGI AIM and OPD as substrate. Results were calculated using standard curves prepared from anti-Ro and anti-La positive sera assigning 400 Ulml to the 1:300 dilution. 866 sera of SLE patients (N = 377), sera of their relatives (N = 386) and 640 sera of patients with suspected or manifest rheumatic
210 . XXIInd Meeting of the Society of Immunology disease were analyzed. In sera of 25 healthy persons anti-Ro was 12.5 ± 4.1 U/ml, anti-La 9.6±3.6 U/ml. Anti-60 kD Ro was found in 16% of SLE patients, anti-La in 14%, both antibodies in 7.7 % of these patients. Relatives showed anti-Ro in 1.5 %, anti-La in 4 %, both antibodies in 1 %. In patients with suspected or manifest rheumatic disease anti-Ro was found in 27 %, anti-La in 15.1 %, both antibodies in 7.6 %. In SLE patients 2 to 3 sera could be tested in most cases of which 96 % showed congruent results for anti-60 kD Ro, 98 % for anti-La. Antibody activities up to several thousand U could be detected. In the patient group with suspected or manifest rheumatic diseases the proportion of lower anti-Ro and anti-La activities « 40 U/ml) was with 15 % (anti-Ro) or 8 % (anti-La) higher than in the SLE group (5 % antiRo, 4 % anti-La). The ELISA revealed significant differences in anti-60 kD Ro and anti-La antibody frequencies of healthy persons, SLE patients and their relatives. Our figure of 16 % for anti-Ro is lower than that of 37 % reported from an immunoblot analysis with natural antigens. Compared to other studies using ELISA with natural or recombinant La-antigens our figures in SLE patients are also lower but in the routine sera group (N = 640) 26 % anti-60 kD Ro positive tests were obtained, figures which are in the magnitude of antibody frequencies obtained with our natural antigens. In parallel tests with natural La-antigen the concordance was 88 %.
Chirurgische Klinik u. Institut fur Med. Immunologie, Charite, Humboldt-Universitat, 1040 Berlin, Germany
G.1S Natural autoantibodies in the fetal human B cell repertoire U. SETTMACHER, G. GROTZ, K. WINKLER, A. LUKOWSKY, H. WOLFF, R. VON BAEHR, and S.JAHN
Human hybridomas were generated from fetal lymphocytes (15-36 week of gestation) hybridized with the heteromyeloma line CB-F7. Independently of the gestational age, 7-10 % of the IgM-producing hybridomas were found to secrete autoantibodies reacting with ssDNA, keratin or thrombocytes. Some of the monoclonal antibodies, however, were polyspecific, and reacted with foreign antigen material (Lipid A, Tetanus Toxin) as well. The nucleotide sequences of the respective VH and VL genes have been determined. The usage of unmutated germline genes belonging both to the VHI and VHIII families was shown. The DNA derived from 10 independent hybridomas (6 fetuses) were hybridized with a JH probe. No clonal relationship between the maternal B lymphocytes producing natural autoantibodies could be detected. We discuss the frequent occurrence of autoimmune B cells in the fetal immune system as an important aspect of natural repertoire generation, including anti-bacterial specificities which may guarantee a first line defense in the newborn organism.
XXIInd Meeting of the Society of Immunology· 211 Augenklinik der Universitat, Mathildenstr. 8, 8000 Miinchen 2, Germany, and National Eye Institute, NIH, Bethesda, MD 20892, USA
G.t6 Oral suppression with IRBP in the acute and chronic relapsing mouse models of experimental autoimmune uveitis (EAU) S. R. THURAu,
c.-c. CHAN, E. SUH, B. WIGGERT, R. B. NUSSENBLATT, and R. R. CASPI
Uveitis is an inflammatory autoimmune disorder in man, which affects ocular tissues (uvea and retina) and can cause blindness in severe therapy refractive cases. Mouse EAU serves as a model for human uveitis. EAU is a T cell-dependent autoimmune disease, which is experimentally induced by footpad immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP) emulsified in complete Freund's adjuvant. In the following study we investigated the potential of antigen feeding to downregulate acute as well as chronic relapsing EAU in B10.A mice. In the first set of experiments mice were fed 4 times with IRBP or with Keyhole Limpet Hemocyanin (KLH) prior to the active immunization with 100 [.lg IRBP, which induces an acute uveitis. Three weeks later histology revealed significantly reduced damage of the retina in the IRBP fed mice. In vitro proliferative responses to IRBP were also reduced in treated mice. In the second set of experiments chronic relapsing EAU was induced with a low dose immunization scheme and feeding was started 8 weeks later after mice had recovered from their first attack of uveitis as determined by fundoscopy. For the following 5 weeks animals received IRBP or KLH. On fundoscopy control mice developed a relapse of uveitis 3 weeks after the first attack, while the relapse was inhibited in mice fed with IRBP. Again, histology showed significantly reduced retinal destruction in treated mice. Interestingly, specific antibody titers were not reduced, while cellular responses to IRBP did not always correlate with the clinical responses in the various groups.
Dept. of Immunology, Medical School, University, 2300 Kiel, Germany
G.t7 A clinically successful protocol to suppress autoantibody production in SLE patients is analyzed for its efficacy to inhibit natural xenophile antibodies (NXA) K. ULRICHS, H. H. EULER, and W. MULLER-RuCHHOLTZ Plasmapheresis in conjunction with a defined, time-related course of CY injections has been shown to be very effective in treating patients who suffer from severe forms of systemic lupus erythematodes (SLE). It was the aim of our study to analyze the effect of this established clinical protocol on the kinetics of natural xenophile antibodies (NXA) against porcine pancreatic islet cells, which are known to be responsible for acute and hyperacute rejection of islet xenografts. For this purpose, sera from 5 SLE patients were additionally tested for NXA against porcine pancreatic cells by quantitative immunofluorescence (a) before, (n) immediately after, and (c) long-term after treatment. Results: (1) In contrast to healthy individuals, SLE patients are strongly positive for disease-related anti-nucleic antibodies (ANA), but negative for NXA. (2) Immediately after treatment, both ANA and NXA are negative. (3) Six to twelve months after treatment, only a marginal reoccurrence of ANA is observed. Reoccurrence of NXA reaches titers, considered normal. (4) ANA and NXA are of IgG rather than IgM isotype. Conclusions: (I) Plasmapheresis combined with a short-term course of the alkylating agent CY appears to be very effective in eliminating activated B cell clones but rather
212 . XXIInd Meeting of the Society of Immunology ineffective in eliminating «resting» clones, such as NXA clones. (II) In the context of xenotransplantation, this clinical protocol could be particularly effective in eliminating those antibodies which may be produced by xenograft-activated B cells.
Dept. of Chemistry, University, 6750 Kaiserslautern, Germany
G.18 Antigen-specific therapy of experimental autoimmune myasthenia gravis with acetylcholine receptor-gelonin conjugates
in vivo I. L. URBATSCH, R. K. M. STERZ, K. PEPER, and W. E. TROMMER The major pathophysiological deficiency in myasthenia gravis as well as in its animal model, experimental autoimmune myasthenia gravis (EAMG) is the T cell-dependent production of autoantibodies against acetylcholine receptors (AChR) in the muscle membrane by specific B cells. This leads to an accelerated degradation of functional AChR and associated ionic endplate channels giving rise to the clinical symptoms of impaired neuromuscular transmission (i.e., weakness and various degree of paralysis). In order to eliminate these specific B cell clones, rats with EAMG were injected with antigen toxin conjugates constructed by specific coupling of purified AChR with the plant toxin gelonin. This led to a marked improvement of clinical symptoms and an increase in the number of ionic endplate channel density compared to untreated animals with EAMG. The immune response to irrelevant control antigens was not altered by this treatment. Injections of equivalent amounts (molar basis) of gelonin or AChR alone had no therapeutic effect.
!Dept. of Immunology, Dept. of Internal Medicine, Medical University, 3000 Hannover 61, 2Fraunhofer-Institut, 3000 Hannover, Germany
G .19 Increased number of macrophages and monocyte precursor cells in organs of autoimmune male BXSB mice G. VIETEN!, K. BEHRENS2, A. EMMENDORFFER2, K. HARTUNG!, and H. DEICHER!
BXSB mice spontaneously develop progressive fatal autoimmune disease which resembles human systemic lupus erythematosus (SLE). During their lifetime male BXSB mice develop a high level of serum immunoglobulins, including anti-nuclear antibodies (ANA), a progressive diffuse proliferative glomerulonephritis and an increasing monocytosis in the peripheral blood which is unique among lupus mice. As we could show previously there is a strain-specific high number of myeloid precursor cells in the bone marrow especially monocyte precursor cells (M-CFC). The number of M-CFC is especially high in male BXSB mice and increases with progressing disease. Our latest data show that there is an extramedullary appearance of MCFC in spleen and liver of male BXSB mice which increases with progressing disease. In addition to a morphologic alteration of the organs there is a change in the number and distribution of organ resident macrophages. The alteration of the macrophage population may be one reason for the rapid progression of the autoimmune disease in male BXSB mice. Supported by DFVLR 01 VM 8606.