J. Autoimmune Diseases

322 . Gesellschaft fur Immunologie - Societe Fran~aise d'Immunologie

lated rabbit anti-Fab IgG led to a 10 fold increase. The biotinylation was done in a controlled fashion to avoid changes in the binding properties of the substituted antibodies. In addition, the biotinylated antibodies were useful in immunoblotting procedures. In immunofluorescence studies such biotinylated antibodies and fragments were shown to be superior to standard assay systems when used with streptavidin-fluorochrome (FITC/RITC/Texas Red): they allow the detection of surface molecules present in only a small number of copies.

J. Autoimmune Diseases

Inserm U. 283, Hopital Cochin, 27, rue du Faubourg Saint-Jacques, 75674 Paris Cedex 14, France

J.1 Control of the proliferation and differentiation of the cloned human thyroid cells (GEJ)

Y. ABRAMOVICI, N. BOUCEKKINE, and J. CHARREIRE

Cloned human thyroid cells (GEJ) have been used to study the regulation of proliferation and the expression of differentiation. Proliferation has been evaluated by cell counting, 3H_ thymidine uptake and a rapid colorimetric assay for cellular growth using reduction of tetrazolium salt. Differentiation expression was based upon the evaluation of TSH receptors (TSH-R) detected on GEJ cells after specific binding of 125I-labeled TSH. Human thyrotropin, insulin, hydrocortisone, phorbol myristate acetate (PMA), dibutyryl cAMP (dbcAMP), epidermal growth factor (EGF) and platelet activating factor (PAF acether) were added at various concentrations during 1 to 7 days to quadruplicate of 5000 GEJ cells (per well) cultured in RPM I 1640 (Gibco, Paisley, Scotland) containing 5 % fetal calf serum. It appears that PMA (2 x 10-7M), insulin (800 ng/ml), db cAMP (5 x 1O-4M) and PAF acether (la-12M) significantly increase proliferation of GEJ cells of respectively 100,200,30 and 50 % on day 3 or 4 of culture. The expression of TSH -R was investigated after incubation of quadruplicate of 1 x 106 GEJ cells with PAF acether, a phospholipid supposed to act by binding to specific binding sites and activating a specific calcium and lipid dependent protein kinase, protein kinase C. This drug is able as soon as 30 min after incubation of the GEJ cells with 1O-12M PAF acether, the optimal time being one hour of incubation, to increase specific binding sites for TSH of approximately 150 %. Moreover, Scatchard's analysis similarly performed after incubation of GEJ cells with PAF acether in optimal conditions shows that PAF acether induced TSH-R exhibit a high affinity for TSH. The possible role of these other factors on TSH -R expression on GEJ cells as well as the detection of synergistic effects either on proliferation or differentiation are also studied.

Gesellschaft fiir Immunologie - Societe Fran~aise d'Immunologie . 323

Laboratoire d'Immunologie, Inserm U291 and Service d'Immuno-Rhumatologie, H6pital Saint-Eloi, Montpellier, France

J.2 Interleukin-2 (IL-2) abnormalities in rheumatoid arthritis

M. ANDARY, B. COMBE, B. KLEIN,]. SANY, and]. CLOT

In order to re-examine the participation of IL-2 in the immune response of patients with rheumatoid arthritis (RA) we have measured the percentage of IL-2 receptor-bearing cells with the CD25, B1.49.9. monoclonal antibody, the lymphoproliferative responses to exogenous IL-2 and to PHA, and the production of IL-2. These experiments were done with mononuclear cells isolated from synovial fluids (SFMC) and peripheral blood (PBMC) from 11 RA patients versus 11 age- and sex-matched controls. We report here that: 1) In RA patients the mean percentage of CD25 positive cells was not significantly higher among PBMC than in controls (2.2 ± 0.6 vs 1.1 ± 0.5), whereas it reached 4.9 ± 1.4 in SFMC. 2) Accordingly, the proliferative response of SFMC to exogenous IL-2 was increased as compared to RA and control PBMC; in contrast, their response to PHA was markedly reduced. 3) The production of IL-2 by RA SFMC and PBMC after PHA stimulation was very low. A three to four fold increase by either irradiation of suppressor cells, or indomethacin treatment, or phorbol myristate acetate was observed although the IL-2 production remained lower than in control PBMC.

University Hospital of Internal Medicine, 8700 Wiirzburg, FRG

J.3 Fine specificity of antimembrane antibodies in dilated cardiomyopathy

E. BAUER, B. MAISCH, and P. THOMETZEK

The etiology of dilated cardiomyopathy (DC) is by definition (WHO/ISCF task force) unresolved. Immunological reactions are also considered as pathogenetic factors. We therefore investigated sera of 35 patients with DC-active myocarditis being excluded by myocardial biopsy- for antimyolemmal (AM LA) and antisarcolemmal antibodies (ASA) and their fine specifities: the binding to isolated human adult atrial myocytes and to a highly enriched preparation of human sarcolemma by immunoblot analysis after SDS gel electrophoresis.

Results: ASAs were found in 27 our of 35 patients, AMLAs in 25 out of 35 patients of the homologous type. With immunoblot analysis it could be demonstrated that homologous AMLAs and ASAs are a microheterogeneity of antibodies with fix to bands of 220, 90, 78, 45, 42, 35 and 28 KD.

ASA% AM LA % (human)

KD pos neg pos neg 220 22 0 23 0 90 41 25 42 22 72 28 0 23 11 48 19 0 15 0 45 33 13 31 22 35 30 25 35 11

Conclusions: In DC ASAs and AMLAs can be demonstrated in substantial number of patients. Discrimination positive and negative sera can be attained best with antibodies that bind to the 220, 90 and 48 KD bands in the immunoblot. These data substantiate the hypothesis of a secondary imunopathogenesis of some forms of DC.

324 . Gesellschaft fur Immunologie - Societe Fran«aise d'Immunologie

lUnite d'Immunocytochimie, Institut Pasteur, 75015 Paris, France, and 2Laboratoire d'Immunologie, Faculte de Medecine de Tunis, Tunis, Tunisia

J.4 Natural autoantibody activity in sera of patients with systemic lupus erythematosus

F. BEN FADHELI, K. AYED2, and S. AVRAMEASI

We have tested, by ELISA, the sera of 52 patients with systemic lupus erythematosus and those of 4 patients with mixed connective tissue disease for their activity against a panel of antigens (DNA, actin, myosin, albumin, tubulin, TNP and a saline extract of calf thymus (ECT). The results were compared to those obtained by immunodiffusion with ECT, anti-Sm and anti-RNP as well as those obtained by immunofluorescence on Crithidia luciliae.

The results are as follows: - all sera examined showed an increased level of antibodies of the IgG and IgA isotypes against

all antigens, particularly a higher anti-DNA and anti-TNP activities. - the high anti-DNA activity found by ELISA is not correlated with the positivity obtained in

the Crithidia luciliae test. - there is a good correlation between the antibody activity with ECT detected by ELISA and

by immunodiffusion analysis. - in contrast, there is no correlation between the number of precipitating lines observed with

ECT, the identity with Sm and RNP and the anti-DNA activity detected by ELISA.

Lab. d'Immunologie, Faculte de Medecine, 13385 - Marseille, Cedex 5, France

J.5 Multiple Sclerosis: cell mediated immunity to human brain gangliosides

E. BERAUD, M. M. GOLSTEIN, B. SERLA, R. GALIBERT, F. VIALLET, R. KHALIL,]. L. GASTAUT, and D. BERNARD

Multiple Sclerosis (MS) is a demyelinating disease, the autoimmune nature of which is uncertain. A Delayed Type Hypersensitivity (DTH) to Myelin Basic Protein has been shown in MS patients, but inconstantly and not restricted to MS. Glycolipids are other components of myelin and, in MS, antibodies to gangliosides have been reported, as well as T lymphocytes «sensitized" to them by the «active E rosette» test (H. OFFNER et aI., J. Neurol. Sci. 1981. 52: 279). Furthermore, a MS-like disease has been induced in rabbits by immunization with gangliosides. For these reasons, we have performed Leucocyte Migration Inhibition Tests (LMIT) in MS, in the presence of highly purified human brain gangliosides and, as a control of cell mediated immunity, of candidin. 27 MS patients (19 being «definite», according to the classification of MacDonald and Halliday), 20 patients with Other Neurological Diseases (OND) and 25 healthy controls have been studied. MS group has been subdivided into MS with exacerbation, not treated (12), MS with exacerbation and treated (8) and MS without exacerbation (9). In the presence of gangliosides, the mean % inhibition of migration was 30.75 ± 3.43 in MS which exacerbation, not treated, 15.31 ± 4.48 in MS with exacerbation and treated, 12.33 ± 3.07 in MS without exacerbation, 8.41 ± 2.11 in OND and 15.44 ± 2.19 in

Gesellschaft fur Immunologie - Societe Franc;aise d'lmmunologie . 325

healthy controls. LMIT to gangliosides is therefore positive in MS with exacerbation (P < 0.001 vs. aND and healthy control groups), supporting the notion of a DTH to gangliosides. On the contrary, under treatment (mostly synacthene) and between exacerbations, MS patients show LMIT not significantly different from control groups. The possible pathogenic and diagnostic implications of these results will be discussed, as well as interesting individual data.

Depts of Ilnternal Medicine, University of Ulm, Steinhovelstra6e 9, D-7900 Ulm, FRG, and 2lmmunology, University Hospital, BP 824, F-29 285 Brest, France

J.6 Anticytoskeletal autoantibodies in systemic lupus erythematosus

M. BOHME\ J. JOUQUAN2, M. BLASCHEK2, and P. YOUINOU2

Sera from 38 patients with systemic lupus erythematosus and 63 normal controls were tested, using indirect immunofluorescence (IIF) techniques, for the presence of antibodies (ab's) to IF of the cytoskeleton of several cell lines (human hepatocytes and fibroblastes, and mouse fibroblastes). The antibody specificity was checked by an ELISA technique, using polystyrene plates coated with vimentin (V) or keratin (K). Class-specific antisera to human IgG and IgM were used throughout the study. Ab's to IF were found by IIF in 15, 12 and 10 sera, depending on the cell target. These ab's reacted with V in 4 cases, Kin 2 and both in 14, as shown by ELISA. Of 19 anti-V -positive sera, 3 had IgG, 9 IgM and 6 IgG plus IgM antibodies, and of 17 anti-K positive sera, I had IgG, 8 IgM and 7 IgG plus IgM antibodies. IgM-anti-V ab's correlated with IgM anti-K (p < 0.01) and anti-IgM anti-cardiolipin (p < 0.01) ab's, while there was no correlation between anti-V or anti-K on the one hand, and anti-dsDNA (Crithidia luciliae, ELISA and Farr assay) or anti-ssDNA (ELISA) on the other. The patient group was split into subgroups according to the clinical profile. Anti-Vas anti-K antibodies appeared to be significantly more frequent (p < 0.05) in patients with than in patients without joint involvement, but we could not show any relationships, so far, between these ab's and the presence or the absence of neurological or nephrological abnormalities. A longitudinal survey was therefore set up in our department, in order to evaluate the predictive value of these ab's.

Ilnserm U. 283 and 2Department of Pathology, H6pital Co chin, 27, rue du Fg Saint-Jacques, 75674 Paris Cedex 14, France

J.7 Progressive and chronic autoimmune polyarthritis induced with homologous type II collagen in mice

Immunization with heterologous type II collagen (CII) induces arthritis in DBA/l mice, a strain genetically susceptible to this disease. In order to develop an experimental model of autoimmunity more adequate for the study of human rheumatoid arthritis (RA), DBA/l mice were injected with 100 [lg of native CII that we purified from mouse xiphoideal cartilage. About 6 weeks later, the animals developed a chronic progressive polyarthritis involving the four paws but mainly confined to interphalangeal and metatarsophalangeal joints. The evolution of the disease involved fluctuating remissions and recrudescences. The evolution of


the disease involved fluctuating remissions and recrudescences. The initial lesions assessed by clinical observations and histologic examination were more severe when the disease occurred early than in the case of late onset. Interestingly, the incidence of arthritis was clearly preponderant in males and moreover the few females which developed arthritis had a mild disease with significantly lower arthritic score than the males. Varying levels of autoantibodies against mouse CII were found in the sera of immunized animals regardless of the development of arthritis. These data suggest that different immunogenic and arthritogenic epitopes are expressed on the CII molecule and that the injection of homologous arthritogenic determinants to mice causes a polyarthritis that is clinically and histologically closer to the human RA than the disease induced with heterologous CII.

Diabetes Research Institute, University of Dusseldorf, FRG

J.8 Effect of interleukin 2 on autoimmune diabetes in BB rats

V. BURKART, J. ZIELASEK, U. KIESEL, and H. KOLB

We investigated the effect of exogenous Interleukin 2 (IL-2) on the development of autoimmune diabetes in three separate experiments with different sublines of BB rats. The animals were provided by Dr. A. A. LIKE, Worcester, USA. A total number of 138 animals received intraperitoneal injections of various doses of human recombinant IL-2 (Sandoz, Vienna, Austria). Controls received equivalent volumes of saline. The animals were checked daily for the development of diabetes. In the first experiment the animals (original breeding pairs RIOOC/RI07 A) received 2 x 20 I-tg IL-2/kg/day. This treatment accelerated the development of diabetes (mean age of diabetes manifestation 77 ± 4 versus 95 ± 5 days in the control group) and increased the diabetes incidence (53 %) compared to the control group (23 %). In the second experiment animals used had a higher spontaneous diabetes incidence (52 %) (original breeding pairs RIOOC/RI07 A). Injections of 1 x 20 I-tg IL-2/kg/day slightly accelerated diabetes development (mean age of diabetes manifestation 85 ± 5 versus 95 ± 6 days in the control group). 1 I-tg IL-2/kg/day delayed the development of diabetes (mean age of diabetes manifestation 110 ± 4 days). A dose of 5 I-tg IL-2/kg/day had almost no effect. At 150 days of age, the diabetes incidence in all groups was 43-55 %. In the third experiment, 73 % of the animals in the control group developed diabetes versus 38 % in the 2 x 20 I-tg IL-2/kg/day treated group (original breeding pairs I8BE 11 O/I8BE4 3). The mean age of diabetes manifestation was 85 ± 9 days in the IL-2 treated group versus 96 ± 5 days in the saline treated group. We conclude that exogenous IL-2 has a modulatory effect on the development of autoimmune diabetes in BB rats. But depending on the BB sub line we observed both enhancement and suppression of diabetes. We suggest that slight genetic differences between the sublines cause variability in the responsiveness towards exogenous IL-2. Research is in progress to determine whether these differences are subline dependent modulation of IL-2 receptor expression or of endogenous production of IL-2 or IL-2 inhibitor.


Max-Planck-Institut for Immunobiology, PO Box 1169, 7800 Freiburg, FRG, "·Clin. Res. Unit Multiple Sclerosis, 8900 Wiirzburg, FRG

J.9 Molecular biological analysis of rat T cell clones with specificity for myelin basic protein (MBP)

J. CHLUBA, H. SCHLUSENER"·, H. WEKERLE", and J. T. EpPLEN

T cells specific for auto-antigens have been developed into powerful tools to analyse auto aggressive diseases; e.g. a MBP-specific T cell-clone (Z85) from primed rats inadvertantly showed increased antigen sensitivity in comparison to the original clone (Z82) when tested after five years of continuous proliferation. An independently established T cell clone (LP1) specific for the MBP peptide C2 changed its specificity and suddenly recognized the peptide CI. The molecular mechanisms leading to increased specificity and/or to specificity switches are only poorly understood (1). Therefore, the rearrangements of the T cell receptor genes were analyzed by Southern blot and oligonucleotide hybridizations and compared to the germline configuration of the rat P chain locus. Respective germline phage clones had been isolated from an EMBL 3 genomic library. Furthermore, cDNA libraries were constructed to characterize the a and P T cell receptor genes. The results will be discussed with respect to their possible bearing on the pathogenesis of autoimmune disease.

1. J. T. EpPLEN, F. BARTELS, A. BECKER, G. NERZ, M. PRESTER, A. RINALDY, and M. M. SIMON (1986). Proc. Nat!. Acad. Sci. USA 83: 4441-4445.

CNRS UA 04-1159, 133, avenue de la Resistance, 92350 Le Plessis-Robinson, France

J .10 Lymphocyte activation status in myasthenia gravis

S. COHEN-KAMINSKY, and S. BERRIH-AKNIN

It is generally accepted that lymphocyte proliferation that involves the interaction between IL2 and its receptor depends upon activation of lymphocytes by mitogen or antigen. However, recent reports have clearly demonstrated that recombinant IL2 (rIL2) is able to induce lymphocyte proliferation without any previous stimulation, and regulates IL2 receptor expression. The aim of this study was to explore the lymphocyte activation state in Myasthenia Gravis (MG). Experiments performed with blood lymphocytes revealed that proliferation indexes in rIL2-treated cultures are generally higher in MG patients (values between 3 to 40) that in controls « 12). These findings have been obtained at different days of culture with rIL2 and at several rIL2 concentrations. Optimal conditions were 6 day treatment with 50U/ ml rIL2. Similar results were obtained with thymic cells isolated from hyperplastic MG thymuses. Indeed, after 6 days of culture with rILl, proliferation indexes of thymocytes from MG patients varied from 40 to 120, while those of controls were generally less than 35. This higher response of MG lymphocytes was correlated to an increased number of TAC-positive cells revealed by immunofluorescence with an anti-TAC monoclonal antibody. These data suggest that, in MG, a higher number of cells show functional signs of pre-activation. These cells could represent autoreactive cells involved in the pathogenesis of MG. However, we could not detect phenotypic signs of activated T cells (namely TAC antigen expression) before culture with rIL2. Our results raise the possibility that such lymphocytes from MG patients bear low density of IL2 receptors and/or have a higher capacity to express high affinity ILl receptors that are involved in the interaction with ILl and in the proliferative response.

328 . Gesellschaft fiir Immunologie - Societe Fran~aise d'lmmunologie

Universite Louis Pasteur, IDepartement d'lmmunologie et d-Immunopharmacologie moleculaires et cellulaires, BP 10, F-67048 Strasbourg Cedex, 2Clinique neurologique, 1 Place de I'h6pital, F-67091 Strasbourg Cedex, France

J .11 A new model of animal myasthenia gravis: effect of interferon on the evolution of the disease

J. CUEVA2, D. LOHSL2, P. ANDREI, S. BRAUNI, J. M. WARTER2, and P. POINDRON1

When Wistar rats were injected intramuscularly (1M) with a mixture of whole blood (from a patient with myasthenia gravis (MG)) and complete Freund's adjuvant, they developed a MGlike disease characterized by (1) clinical signs: muscular weakness prevailing in spinal and pelvic muscles, abnormal fatigability increased with exertion; (2) electrophysiological signs; decrementing electromyogram upon repetitive stimulation of the sciatic nerve, (3) pharmacological signs: transient improvement of the clinical status after administration of pyridostigmine bromide (an inhibitor of acetylcholinesterase) and transient correction of the electromyographic tracing after edrophonium chloride injection (Tensilon® test); aggravation (respiratory arrest) after intravenous injection of a curare; (4) immunological signs: presence of anti acetylcholine receptor antibodies at low but significant levels (0.6 to 1.5 nmole/l). The thymus of the diseased rats was not hyperplastic and no germinal centers could be seen upon histological examination. Control rats injected with a mixture of normal whole blood and complete Freund's adjuvant did not exhibit any sign of disease. 1M administration of purified rat interferon (2.5 to 3 X 106 U/mg protein, 10,000 U per injection, 20 injections at 3 day intervals) completely suppressed clinical, electrophysiological and pharmacological signs of the disease, but did not alter the anti acetylcholine receptor antibody content of the sera. These role played by the antibodies in the pathogenesis of the disease and the possible mode of action of IFN will be discussed.

Departments of Medical Microbiology and Immunology and "Internal Medicine, University ofUlm, FRG

J .12 Stimulation of eosinophilic granulopoiesis by T lymphocytes: clonal analysis in an idiopathic hypereosinophilic syndrome

B. FLEISCHER, S. FLEISCHER, and A.RAGHAVACHAR':·

The idiopathic hypereosinophilic syndrome (HES) comprises a heterogenous group of disorders with unknown pathogenesis characterized by persistent eosinophilia and eosinophilic infiltrates in skin and internal organs leading to severe dysfunctions. In the present study, T lymphocyte clones were randomly established from the blood of a patient with HES and propagated in culture with mitogen and IL-2.Whereas all clones were able to stimulate myeloid colony formation when cocultured with normal bone marrow cells in a double layer micro-agar culture system, one third of these clones stimulated preferentially pure eosinophil colonies (70--98 % of all colonies). This was due to the release of a lineagespecific colony stimulating factor (EoCSF) by these clones after appropiate stimulation. All these clones had the T4+g-phenotype and were capable of producing in addition IL-2 and IFN-y. Southern blot analysis of the T cell receptor ~-chain rearrangement of the EoCSF producing clones showed a different rearrangement pattern for each clone. These studies

Gesellschaft fur Immunologie - Societe Fran~aise d'Immunologie . 329

suggest a reactive T cell-mediated eosinophilia as pathogenetic mechanism in this case of HES and for the first time point to a biologic relevance of a lymphokine-induced stimulation of haemopoiesis.

L.I.R.1. Inserm U 269, e.H.U. Purpan, 31059 Toulouse Cedex, France, and "·I.B.M.e., CNRS, Strasbourg, France

J.13 Circulating DNA in lupus diseases and in various human and experimental situations

G. J. FOURNIE, F. LAVAL, J. LULE, V. GOSSELIN, M. A. MIGNON-CONTE, S. MULLER\ andJ. J. CONTE

In lupus diseases plasma (circulating, extracellular), DNA might playa role in the triggering of anti-DNA antibody and/or in the development of tissue lesions. Until now, technical problems have limited these studies. Plasma DNA levels were measured in various human and mouse situations including lupus diseases using a new method based on the «Nick Translation» labelling of DNA purified from 10--50 [!l of plasma.

In 54 lupus patients increased levels of plasma DNA were found more often in patients with active (clinicaly defined) forms of the disease (28/37 plasma samples) than in patients under remission (6/39 plasma samples). Increased levels of plasma DNA were also found in various other pathological situations including primary chronic glomerulonephritis, hemodialysis, thrombophlebitis, myocardial infarct, graft versus host reaction, malignancy, infections, surgery ...

In the mouse, increased levels of plasma DNA were found in NZB X NZW mice at the time when kidney lesions are rapidly worsening, in hairless mice 6- to 24 hours after ultra-violet exposure, in nude mice bearing solid human tumors, in C57BL/6 mice during endotoxinic shock, after surgery and after administration of cytotoxic drugs.

Molecular characterization of DNA using end labelling of purified DNA, polyacrylamide gel electrophoresis and autoradiography, showed that in most cases a major band of DNA ranging from 120--180 base pairs was present. Preliminary data suggested that circulating DNA might be precipitated in vitro by immunoglobulins with anti-histone activity.

Taken together these results strongly suggest that the presence of extracellular DNA in blood is the witness of cell death and the product of chromatin catabolism and that it circulates mainly as nucleosomes.

lInserm U93 H6pital St Louis, Paris, 2H6pital St Louis, Paris, 3H6pital Bichart, CHU. 46, rue Henri Huchard, Paris, France

J.14 HLA and type Ib diabetes with multi auto-immune manifestations

J. GONyl, A. MARCELLI-BARGE I, P. VEXIAu2, e. LHOMME3, M. SCHMID" J. e. POIRIERI, V.

BOCHUI, I. KALIDII, G. CATHELINEAU2, R. ASSANl, I. DESCHAMPS2, and J. HORS1

Seventy IDD patients (insulin-dependent diabetics) 48 female and 22 males, all adults at the onset of the diabetes, and suffering from, at least, one extrapancreatic Autoimmune Associated manifestation (AMM) (mainly Graves' disease or other thyroid dysfunction) were studied for HLA A, B, C, DR markers and Bf, C4 complement components.

330 . Gesellschaft fur Immunologie - Societe Fran\aise d'Immunologie

Comparisons were made with a series of 261 «type Ia» IDD patients. The well known increase in HLA-B8 among IDD + AAM patients (36 %) is confirmed, compared to the frequency in Ia diabetics (22 %) (p < 0.02). There was a decrease in DR4 among diabetics with AMM (34%), leading to a low frequency of the allelic combination DR3/4 (13%): the corresponding frequency in Ia IDD patients were 62 % and 34 %, respectively (p < 10-7

,

10-3). These results confirm the heterogeneity of the disease based on immuno-genetic

markers and support the originality of a variety of IDD with AMM characterized also by a late onset and a higher incidence among females.

Laboratoire d'Immunologie. "·Clinique Endocrinologique. Universite Nancy I, Nancy, France

J .15 Clinical, biological and immunological signs of mucosal pathology in auto-immune diseases of the thyroid

v. GUERIN, M. C. BENE, C. SERIOT"", J. LECLERE"", and G. FAURE

Auto-immune pathologies of the thyroid can be associated with other systemic diseases, often involving mucosal areas, such as Sjogren's syndrome or pernicious anemia. Besides, IgAproducing plasma cells are frequently observed in thyroid glands involved with Hashimoto's or Graves' diseases. We investigated, in 40 patients suffering from one of these conditions, the possible clinical and/or biological involvment of the Mucosal Associated Lymphoid Tissue (MALT). Clinical examination was systematically completed by an assessment of the frequency and type of past or present mucosal pathologies and a Schirmer test. A sugar test was performed in 10 patients. Serum was obtained in all cases for the detection of anti-parotid and antithyroid antibodies. Anti-duct cells antibodies were assayed in indirect immunofluorescence (IFI) using frozen-cut human parotid sections. The levels and isotypes of anti-microsomal and anti-thyroglobulin (TG) antibodies were established by IFI and E.L.I.S.A. Clinical symptoms of mucosal infectious or atopic involvment were frequently observed (30 %). The Schirmer test detected in 56 % of the patients a diminution of the lacrymal secretion. Hyposialosis was detected by the sugar test in 8 out of 10 patients. Anti-parotid antibodies were positive in 64 % of the sera, with a mean titer of 64. Anti-TG antibodies of IgA isotype were present in most of the cases with a higher frequency in patients with Hashimoto thyroiditis, and/or anti-parotid antibodies. In the latter subgroup, the light chain of anti-TG antibodies was more often of lambda class. These results establish clinical and biological involvment of the MALT in autoimmune diseases of the thyroid.

Centre de Genetique Moleculaire, CNRS, Gif sur Yvette, France, U. 108 Inserm, Hopital Saint Louis, Paris, France

J .16 Autoantibodies to nuclear lamin B in a patient with thrombopenia

M. N. GUILLY, F. DANON, J. C. BROUET, M. BORNENS, and J. C. COURVALIN

We report the characterization of autoantibodies of original nuclear specificity in the serum of a patient with systemic lupus erythematosus. On rat liver tissue section, the immunofluorescent staining of the nucleus was homogeneous, speckled and brightly perinuclear up to a dilution of a 1/1000. Immunofluorescent studies in cycling cells and absorption experiments

Gesellschaft fur Immunologie - Societe Fran<;:aise d'Immunologie . 331

showed that the antigen was localized in the nuclear envelope. Bidimensional analysis of immunoprecipitated nuclear proteins revealed a unique acidic protein (pI 5.4) of 68 kDa molecular mass. This component was identified as lamin B, one of the three major polypeptides of the vertebrate nuclear envelope. Antibodies, shown to be polyclonal immunoglobulins G, were directed against conserved determinant( s) of the protein. They were not related to other autoantibodies present in the serum (anti-DNA and anti-platelets). This new nuclear specificity is an additional example of the antigenicity of proteins related to intermediate filament proteins family in patients with autoimmune disorders.

Institute of Clinical Immunology and Rheumatology, University of Erlangen, Department of Orthopedics, Center for Rheumatic Disease, Bad Abbach, FRG

J .17 Growth frequency and stimulation of T cell clones and peripheral blood lymphocytes by supernatants of cultured synovial lining cells and synovial fluid

N. HAIN, G. R. BURMESTER, B. JAHN, J. ZACHER, and J. R. KALDEN

In vivo activated Il-2 receptor positive T cells in peripheral blood, synovial fluid and synovium are characteristic of the inflammatory process in rheumatoid arthritis (RA). The frequency of T cells responding to Il-2 in peripheral blood and synovial tissue was determined by limiting dilution. Patients with RA showed higher frequencies in comparison with healthy controls which was in accordance with an increased percentage of Il-2 receptor bearing cells. T cell clones which in the vast majority were CD4 + were generated by limiting dilution technique and by the addition of Il-2 alone. These clones were incubated with supernatants from cultured synovial lining cells (SLC-SN) and cultured chondrocytes (CC-SN). One population of clones could be stimulated both by autologous and allogeneic SLC-SN whereas CC-SN proved to be inhibitory. Incubation of native peripheral blood lymphocytes and synovial tissue T cells with synovial fluid (SF) from RA patients showed an augmentation of T cell proliferation in almost every donor in contrast to SLC-SN enhancing the proliferation in only few cases. Significant stimulation was always dependent on the presence of exogenous Il-2. Thus synovial fluid and supernatants of cultured synovial lining cells presumably contain activating as well as inhibitory mediators which will have to be characterized by biochemical analysis.

Institute for General and Experimental Pathology, University of Innsbruck, Medical School, Innsbruck, Fritz-Pregl-StraBe 3, A-6020 Innsbruck, Austria

J .18 Genetic aspects of spontaneous autoimmune thyroiditis of chicken: a model for hashimoto thyroiditis

K. HALA, and G. WICK

For the analysis of the genetic background of thyroiditis we used Obese strain (OS) chickens which develop a spontaneous autoimmune thyroiditis (SAT). Practically all animals from this strain develop severe lymphoid infiltration of the thyroid gland and circulating autoantibodies

332 . Gesellschaft fi.ir Immunologie - Societe Fran~aise d'Immunologie

against thyroglobulin (TgAAb) within a few weeks of hatching (1). Of the 3 haplotypes (B\ BU

, B15) that are present in the as, B15/B 15 birds were used for genetic analysis. The syngeneic

line CB BIZ / stZ was selected as a second line for cross-breeding experiments. This inbred line was established in 1933 and is kept by the brother x sister mating. All animals from this line are healthy, without thyroid infiltration orTgAAb (2).

From the results obtained withFI and Fz hybrids and with backcrosses between FI with both parental lines (aS or CB) we draw the following conclusion: There are two gene families that are important for the disease. The first family of genes is regulating the hyperreactivity of the immune system against self antigens, and at least one gene is responsible for the specificity of the hyperreactivity against thyroglobulin. The second family contains a minimum of 3 genes; one of these is recessive and determines the target organ (thyroid) susceptibility to the autoimmune attack. In addition to these genes with major importance, minor genes were also found. Some of them are associatedlidentical with the genes of the major histocompability complex (MHC) or with non-MHC genes controlling the immune response. Sex hormones playa modulatory role. The full development of the disease is only possible when an individual possesses respective genes from both families.

1. G. WICK, J. MOST, K. SCHAUENSTEIN, G. KROMER, H. DIETRICH, A. ZIEMIECKI, R. FASSLER, N. NED, and K. HALA (1986). Immunology Today 6: 359.

2. N. NED, K. HALA, H. DIETRICH, and G. WICK (1986). Int. Archs. Allergy App!. Immuno!. 80: 168.

Supported by the Austrian Research Council (project Nr. S-41105) and the Austrian Ministry of Science and Research.

Department of Neurology, University of Di.isseldorf, Moorenstr. 5, 4000 Di.isseldorf, FRG

J .19 The role of inflammatory mediators in the pathogenesis of experimental allergic neuritis

H. P. HARTUNG, B. SCHAFER, B. BLOMENKAMP, and K. V. TOYKA

Experimental allergic neuritis (EAN) is an animal model of the acute Guillain-Barre syndrome, a monophasic inflammatory polyradiculoneuropathy. EAN can be induced in rats by immunization with purified peripheral nerve myelin. Pathologically, the hallmark of both the experimental model and the human condition is the infiltration of nerves and spinal roots by macrophages. Their pathogenic function is considered to be due to the phagocytosis of myelin. As a result the denuded axon cannot propagate nerve impulses properly. However, since macrophages can synthesize and release a multitude of inflammatory mediators it is also conceivable that other mechanisms are operative in imparting tissue damage. Hence we investigated whether in vivo administration of antiinflammatory pharmacologic compounds with known sites of action would influence the course of the disease. Treatment of immunized Lewis rats with steroids, cyclooxygenase, lipoxygenase, joint arachidonate conversion blockers, oxygen radical scavengers, or the organoselenic drug ebselen was initiated on day 7, 13, or 17 post immunization, and continued until sacrifice of animals be perfusion on day 21. The course of the disease was followed clinically, and more sensitively by monitoring nerve conduction properties applying electrophysiologic methods (1, 2). After perfusion plastic sections were prepared for morphologic evaluation of nerves and roots. Further, ex vivo generation of eicosanoids and reactive oxygen species by macrophages was examined (3). Most of the agents employed attenuated or prevented the disease albeit to a different extent. Ex vivo macrophage studies corroborated predicted sites of actions of the tested compounds. We

Gesellschaft fur Immunologie - Societe Fran«aise d'Immunologie . 333

conclude that macrophage-derived inflammatory mediators such as arachidonic acid metabolites and toxic oxygen intermediates contribute to the damage inflicted upon the nerve sheath in EAN.

1. HEININGER et al. (1986). Ann. Neurol. 19: 44. 2. HARTUNG et al. (1986). Neurology 36 (Suppll): 185. 3. HARTUNG et al. (1986). J. Immunol. 136: 3856.

Supported by DFG, SFB 200, B5; and Hertie Stiftung.

Klinik fur Innere Medizin der Medizinischen Universitat zu Lubeck, FRG

J .20 Autoantibody-profiles in disorders of the pancreas. The autoimmune reactions against exocrine pancreas in pancreatitis differ significantly from those in Crohn's disease

D. HELLWIG, M. OTTE, U. REDDIG, D. STRUVE, and W. STOCKER

Introduction: Sera of patients with pancreatitis only sometimes contain autoantibodies (Aab) against pancreatic acini (Pab; 1,2). On the contrary, Pab are common in Crohn's disease (CD; 3). Are there immunological similarities between pancreatitis and CD regarding Pab and other Aab?

Methods: Aab-profiles were established by the indirect immunofluorescent technique in the sera of 26 patients with acute and 51 patients with chronic pancreatitis, of 22 patients with carcinoma of the pancreas and of 100 healthy subjects. In all patients the simultaneous presence of CD was excluded. The Titerplane-technique was employed (4), using frozen sections of 17 different human organ-tissues as antigenic substrates. When Pab were detected in the sera, their immunoglobulin-class was identified and their ability to fix complement was tested. The capacity of Pab to neutralize the CD-related autoantigen (isolated from human pancreatic juice) was controlled according to (5).

Results: In patients with disorders of the pancreas significant concentrations of Pab (titers 1 :10 or higher) were recorded only in 5 % (acute pancreatitis: 3/26, chronic pancreatitis: 2/51, carcinoma of the pancreas: 0122; healthy subjects: 1/100). Titers did not exceed 1 :32. Pab of the immunoglobulin-class IgG could not be observed. Pab in pancreatitis did not fix complement and they were not able to neutralize the CD-related autoantigen. In patients with pancreatitis, Aab against cell nuclei (usually IgM) and against organ-specific and other autoantigens of 17 human tissues were present with the same frequency as in healthy subjects.

Discussion: There are fundamental differences between pancreatitis and Crohn's disease with respect to the autoimmune reactions against exocrine pancreas: Pab were rare in pancreatitis and, when present, their concentration was low. On the contrary, Pab in CD could be detected in 39 % of the cases, titers of 1: 1 00 or higher occurred in 29 %. Pab in CD consisted predominantly of IgG and IgA and sometimes they fixed complement (3). And only in CD Pab could be neutralized with the CD-related auto antigen (5). Furthermore, a pancreatitis-specific autoimmunity against antigens of cell nuclei cannot be supported by the present investigation.

1. LENDRUM, R., and G. WALKER (1986). Gut 16: 365-371. 2. LANKISCH, P. G., H. Koop, R. SEELIG, and H. P. SEELIG (1981). Diguestion 21: 65-68. 3. STOCKER, W., M. OTTE, S. ULRICH, D. NORMANN, K. STOCKER, and G. JANTSCHEK (1984).

Dtsch. med. Wschr. 109: 1963-1969. 4. STOCKER, W. (1985). Acta histochem. (Suppl.) Jena 31: 269-281. 5. FINKBEINER, H., S. BOCK, U. BURMESTER, D. GRAGE, U. REDDIG, D. STRUVE, M. OTTE,

and W. STOCKER (1985). Immunobiol. 170: 20-21.

334 . Gesellschaft fiir Immunologie - Societe Franc;aise d'Immunologie

Institute for Clinical Immunology and Rheumatology, University of Erlangen-Niirnberg, 8520 Erlangen, FRG

J .21 Antibody fixing high molecular weight DNA and RNA in SLE patients' plasmas

M. HERRMANN, F. KRAPF, and J. R. KALDEN

15 patients with severe systemic lupus erythematosus and several controls - 3 patients with M. W ALDENSTROM, 2 patients suffering from rheumatoid arthritis and 3 healthy controls -underwent plasmapheresis. Two liters of plasma were precipitated using polyethylene glycol 6.000 with a final concentration of 5 %. Thus, excluding precipitation of protein-free double stranded DNA and monomeric immunoglobulin. The precipitate was divided in two aliquots, the first one was used for the preparation of nucleic acids, the second one for the purification of autoantibodies.

The nucleic acids were prepared using standard methods including ion exchange chromatography, proteinase K- and RNAse digest. Nevertheless, not only DNA of high molecular weight - up to 20 kb - could be demonstrated in these preparations but also RNA of at least 20 b, whereas not detectable amounts of nucleic acids were found in the control groups. Caesiumchloride buoyant gradients showed inhomogenous molecules, excluding pure viral or bacterial origin. The RNA contents contained 30 up to 80 % riboguanosine proven by nucleosid analysis. Similarities in hyperchrome shifting due to increasing temperature -indicating a transition in confirmation and secondary structure - between the nucleosid preparations from the patients compared to pure synthetic polyriboguanosine indicated high amounts of polyriboguanosine in the patients' plasma.

In rabbits, antibody production could be induced by using the nucleic acid preparations and by synthetic polyriboguanosine, not, however, by double stranded DNA from salmon testis. These rabbit heteronantisera reacted with synthetic polyriboguanosine and with double stranded DNA as well.

The second aliquot used for immunoglobulin preparations was purified to 95 up to 98 % by repeated ammonium sulfate precipitation and affinity chromatography using matrix-bound double stranded DNA. The immunoglobulin preparation contained IgG and small amounts of IgM. The autoantibodies from patients' plasmas reacted, similar to those drawn from the rabbits, with double stranded DNA, the nucleic acid preparations from patients with SLE and pure polyriboguanosine.

!Inserm U93, H6pital St Louis, Paris, 2Inserm U290, H6pital Herold, Paris, France

J .22 Five risk factors for the development of insulin-dependent diabetes in siblings of diabetic patients

J. HORS!, I. DESCHAMPS2, A. MARCELLI-BARGE!, J. C. POIRIERt, M. SCHMIDt, H. LESTRADET2,

and D. COHEN!

In 51 diabetic multiple case families one secondary affected child from each family - the proband being excluded from the study - have been compared to the 265 non affected siblings. Comparisons have been made using relative risk (R. R.) calculation for five parameters: age at onset, birth order in the sibship, sex, HLA DR3 and/or DR4 alleles and complement markers including BfF1 and C4BQo. The highest R. R. (comprised between 4.3 and 12, p < 0.001) were observed in case of DR3 and/or DR4 phenotype, BfF1, DR3 orC4BQo, DR3 haplotype, 2

Gesellschaft fur Immunologie - Societe Fran"aise d'Immunologie . 335

haplotypes shared with the proband, and a young age « 10 years old) at the onset of disease in the index case. Siblings born after the proband were also at greater risk (R. R. 3.4, P < 0.01). On the opposite, low R. R. values were observed among siblings bearing neither DR3 nor DR4, or sharing no HLA haplotype with the index case (R.R.: 0.1, p < 0.01). Evidence emerges from the study that both class II and III alleles of the MHC possibly in conjunction, and also age related factors, are able to increase the penetration of the disease in diabetic families. These parameters are important to be taken into consideration for future possible preventive attitude.

IHopital Henri Mondor, Creteil, 2Institut Pasteur, Paris, France

J .23 Presence of natural autoantibodies against cytoskeletal proteins after allogeneic bone marrow transplantation. Relationship with chronic GVHD?

L. INTRATORt, C. CORDONNIERl, P. TORTEVOYE2, J. P. VERNANTt, and G. DIGHIERd

22 patients (acute myeloid leukemia: 13, acute lymphoid leukemia: 5, chronic myeloid leukemia: 4) with a mean age of 25 years (range 8 to 36 y.), had received allogeneic bone marrow transplantation (BMT) from an HLA identical sibling. The follow-up of these patients after BMT was 4 to 65 months. All patients were given Cytoxan and total body irradiation and received Methotrexate in order to prevent graft versus host disease (GVHD). 11122 patients exhibited chronic graft versus host disease (C.GVHD) (extensive in 5, limited in 6 at the time of the study) assessed on classical and histological parameters. Serum samples were collected from these patients every two or three months. Sequential studies of auto-antibodies against cytoskeletal proteins (actin, tubulin, myosin) and against ds and ss DNA were performed by an Elisa method. Simultaneously immunoelectrophoresis and measurement of complement fractions C3, C4 were performed on each sample. Auto-antibodies against cytoskeletal proteins were found in 10/11 patients with C.GVHD and were absent in 11111 patients without C.GVHD; none of them exhibited anti DNA activity. At the same time, C4 levels were decreased in 7 of these patients with C.GVHD. Monoclonal immunoglobulins IgG and IgM (2-15 gil) were found in 8/11, but the antibody activity was never found to be located within the M component. These results indicate a clear relationship between the presence of these auto-antibodies and occurence of C.GVHD but the predictive value of these autoantibodies could not be assessed in this retrospective study. These auto-antibodies could constitute an immunological marker of C.GVHD.

Depts of 'Immunology and 2Internal Medicine, University Hospital, Brest, France, and 3Dept of Medicine, Medical School University of Ioannina, Greece

J .24 T- and B cell activation in patients with primary Sjogren's syndrome

J. JOUQUANl, Y. PENNEd, H. M. MOUTSOPOULOS3, and P. YOUINOUl

Twelve patients with a biopsy-confirmed diagnosis of primary Sjogren's syndrome (12 females ranging in age from 30 to 74 years) and 12 sex- and agematched normal controls were entered in the study. Peripheral blood mononuclear cells were partially depleted of monocytes by incubating them for 1 hour at 37°C on Petri dish and separated into T - and B-cell-enriched populations by rosetting with neuraminidase-treated sheep red blood cells. Rosetting cells (regarded as «T cells») were double stained, first with monoclonal antibodies (Mab) to DR


framework, interleukin 2 receptor (anti-Tac Mab) (both conjugated with FITC) or transferrin receptor (OKT9 Mab) revealed by FITC-F(ab')2 anti-mouse IgG) and then with biotinlabelled Leu 3a or leu 2a Mabs (revealed by TRITC-avidin). The proportions of DR, OKT9 and Tac-positive T cells in the patients were 28.4, 1.7 and 11.4 %, respectively, as opposed to 4.8, 0.7 and 1.9 %, respectively, in the controls. Non-rosetting cells (regarded as «B cells,,) were double-stained, first with Mabs to kappa or lambda chains (revealed by TRITC-F(ab')2 anti-mouse IgG) and then with FITC-Mab to transferrin receptors. The proportions of kappa and lambda-positive B cells with transferrin receptors in the patients were 2.2 and 1.1 %, respectively, as opposed to 0.7 and 0.1 %, in the controls. Homogeneous protein bands were found in the serum of patients with high proportion of activated «B cells}}, be means of immuno-electrophoresis and/or immunofixation.

Institute for Clinical Immunology and Rheumatology, "'Neurological Clinic, University of Erlangen-Nurnberg, 8520 Erlangen, FRG

J .25 Inhibition of Con A stimulated peripheral blood mononuclear cells by autologous suppressor cells generated by acetylcholine receptor activation in myasthenia gravis patients and healthy donors

H. KACHELRIES, 1. KALIES, J. R. KALDEN, and K.-F. DRUSCHKY

The leading feature of the autoimmune disease Myasthenia gravis (MG) is the destruction of the postsynaptic membrane caused by autoantibodies to the acetylcholine receptor (ACHR). This autoantibody production might be due to a functional loss or decrease of T suppressor cells directed against auto reactive lymphocytes. Analysis of cell surface phenotypes, however, did not reveal any significant difference in the suppressor cell number (T8) between MG patients and healthy controls. A new approach to investigate suppressor activity is the introduction of specific activated suppressing cells in a concanavalin A (Con A)-induced suppressor test. Ficoll-separated PBMC from MC patients and healthy donors were treated with high doses of either Con A or purified ACHR from Torpedo c. for 48 hours. Activated cells were then blocked by mitomycin and washed three times with 30 mM a-methyl-Dmannoside. Suppressing potential of these cells was measured by the capacity to inhibit Con A-activated transformation of autologous responder PBMC. The Con A-induced suppressor test showed a reduction of proliferation due to Con A or ACHR-incuded suppressing cells of 47 % or 66 % respectively on average. In MG patients, however, the inhibitory effect of ACHR-induced suppressing cells was only 17%, while Con A-induced suppressing cells showed normal inhibition of 32 % in the Con A-induced suppressor test. These findings suggest that specific suppressing cells can be induced with high mitogen or antigen concentrations. Furthermore, our results might indicate a malfunction of a cell population involved in antigen-specific suppression in MG patients.

Supported by DFG Ka 325/9.

University of Wurzburg, Medical School, FRG

J .26 Cardiac involvement in chronic polyarthritis and ankylosing spondylitis - an echo cardiographic and immunologic study

D. KLOPF, B. MAISCH, B. RAAB, 1. O. AUER, G. SPROTIE, and K. KOCHSIEK

Cardiac involvement in chronic polyarthritis and ankylosing spondylitis widely varies in the few studies that have been undertaken. It was the purpose of this investigation to revisit cardiac


involvement in rheumatic and collagen diseases by ECG, TM and twodimensional echocardiography and to find out, if antimyolemmal antibodies (AM LAs) directed against the cardiac membrane may be of additional help in establishing the probability of cardiac manifestation. Cardiac abnormalities were graded positive when either AV-block (2nd-3rd degree), pericardial effusion or thickening (> 7 mm), valvular vegetations, cardiomegaly, atrial dilatation or mitral valve prolapse were present. Immunoserological findings including antimyolemmal (AMLA), antinuclear (ANA) antibodies and HLA-B 27 were calculated for the total of all patients (p) and for those with cardiac involvement (c. inv.).

ankylosing chronic IllClplent spondylitis polyarthritis chronic polyarthritis

all.p. LIllV. all.p. LIllV. all.p. C.IllV.

9 8 8 5 9 4 ALMA (human) 6 6 3 4 5 4 ALMA (rat) 5 5 6 4 5 3 ANA 3 3 5 4 3 3 HLA-B 27 7 7 0 0 0 0

SLE scleroderma no pos all.p. LIllV. all.p. C.IllV. all p. = all patients

C.inv. = cardiac involvement

AM LA (human) AM LA (rat) 0 0 ANA 1 1 0 0 HLA-B 27 0 0 0 0

Conclusions: Patients with ankylosing spondylitis showed a higher incidence of cardiac involvement than other patients with rheumatic and collagen diseases. Antibodies against human cardiac membranes are of additional help in the diagnosis of cardiac manifestations in either of the diseases since they are closely associated to cardiac involvement in rheumatic and collagen diseases.

Klinik fiir Innere Medizin und Klinik fiir Psychosomatik und Psychotherapie der Medizinischen Universitat zu Liibeck, FRG

J .27 Family members of patients with Crohn's disease and with ulcerative colitis do not exhibit serum-antibodies against exocrine pancreas and against intestinal goblet cells

T. H. KOSEGARTEN, M. DOSCHER, G. jANTSCHEK, U. BURMESTER, S. SCHMIDT, M. OTIE, and W. STOCKER

Introduction: In patients with Crohn's disease (CD), autoantibodies against exocrine pancreas (Pab) are common (39 %), high titers occur in 29 % (CU: 0 %, healthy subjects: 0 %; 1). A possible role of Pab in the pathogenesis of CD was discussed (2). In ulcerative colitis (UC) autoantibodies against intestinal goblet cells (Gab) can be demonstrated in 28 % of the patients (CD 0 %, healthy subjects: 0 %; 1). Family members of patients with CD and UC carry an elevated risk for developing chronic inflammatory bowel disease (3). In this study, the prevalence of Pab and Gab in the sera of healthy appearing relatives was determined.

Methode: First degree family members of patients with inflammatory bowel disease were interviewed, and their state of health was recorded (71 family members of 52 patients with CD,

338 . Gesellschaft fUr Immunologie - Societe Franc;aise d'Immunologie

114 family members of 46 patients with UC). Diagnoses of the patients were confirmed both by endoscopy and histology. A collective of 36 families comprising 115 healthy persons served for control. Blood was drawn from all subjects for determination of Pab and Gab by the indirect imunofluorescent technique, using frozen sections of human adult pancreas (blood group 0) and human fetal ileum as antigenic substrates.

Results: 114 relatives of UC-patients exhibited chronic inflammatory bowel disease in 4 %, 71 relatives of CD-patients in 13 %. Of the 52 patients with CD, 20 demonstrated Pab (39 %). Eight sera of the 46 patients with UC contained Gab (17%). None of the patients family members and none of the control persons exhibited Pab or Gab in significant concentrations (titers 1 :10 or higher).

Discussion: This investigation was conducted to find out, if subjects at high risk for developing CD or UC can be identified by determination of Pab and Gab. But these autoantibodies could not be detected in the sera of the family members, so Pab and Gab seem to be disease-specific, and they do not yet indicate a disposition for CD or Uc.

1. STOCKER, W., M. OTRTE, S. ULRICH, D. NORMANN, K. STOCKER, and G. JANTSCHEK (1984). Dtsch. med. Wschr. 109: 1963-1969.

2. STOCKER, W., M. OTTE, and P. C. SCRIBA (1984). Dtsch. med. Wschr. 109: 1984-1986. 3. KOSTER, W., and W. LENZ (1984). Erg. Inn. Med. Kinderheilk. 53: 103-132.

Institute for General and Experimental Pathology, University of Innsbruck, Medical School, Fritz-Pregl-Straile 3, A-6020 Innsbruck, Austria

J .28 T cell hyperreactivity in obese strain (OS) chickens: divergent mechanisms in spleen and peripheral blood

G. KROMER, K. SCHAUENSTEIN, and G. WICK

Spontaneous autoimmune thyroiditis (SAT) of Obese strain (OS) chickens is associated with a marked T cell hyperreactivity (Con A hyperresponsiveness, IL 2 hypersecretion) detectable in spleen, peripheral blood, and - most extensively - in the lymphocyte population which infiltrates the thyroid gland. We have shown earlier that splenic T cell hyperfunction of the OS is exclusively due to a dominantly inherited deficiency in nonspecific inhibitors of IL 2 function and action. In the present study, the cellular substrate of this phenomenon is demonstrated to be an autochthonous functional defect of the OS macrophage: By pretreatment of normal (NWL) splenocytes with ph ago cytotropic agents (carbonyl iron, silica) or depletion of nylon wool adherent cells, we were able to mimick the OS defect in vitro. Moreover, i.v. injection of silica particles into NWL chicks induced a defect in low molecular weight IL 2 antagonists in the serum similar to that of the OS, whereas neonatal thymectomy or bursectomy had no effect. The defect in IL 2 antagonists of the OS was excluded to be a phenomenon secondary to known alterations of the immunoendocrine system (hypothyroidism, decreased corticosterone tonus, thymic defect) as substitution of the respective hormones or injection of MHC compatible normal thymocytes did not enhance the immunosuppressive capacity of the OS macrophage, the absolute and relative number of which is not different from normal controls. In contrast to the sessile macrophage, circulating monocytes from peripheral blood of OS produce normal levels of IL 2 antagonist. Peripheral blood lymphocytes (PBL) - but not spleen cells - of the OS were shown to contain an increased frequency of T lymphocytes capable to express IL 2 receptors upon Con A stimulation. Hence, the mechanism of Con A hyperresponsiveness is totally different in the two peripheral lymphoid organs of the OS chicken. This conclusion is further supported by secondary abnormalities of 2-3 day

Gesellschaft fur Immunologie - Societe Fran~aise d'lmmunologie . 339

cultivated as lymphocytes, which are found in the spleen, but not in peripheral blood: enhanced density of IL 2 receptors, elevated IL 2 response, and a decreased susceptibility to suppressive regulation by IL 2 antagonists.

Supported by the Austrian Research Council (grants No. S-41105 and 4679).

ILaboratoire d'immunopathologie du diabete, Inserm U. 211, and 2Clinique medicale B-Faculte de Medecine, 1 rue Gaston Veil, 44035 Nantes, France

J .29 Insulin-dependent diabetes (IDD): obtainment of human T-cell lines by initial rosette formation with insulin-producing pancreatic cells

F. LANGI, D. MAUGENDREI, 2, E. HOUSSAINTI, B. CHARBONNEL2, and P. SAil

Human T-lymphocyte lines sensitized against insulin-producing cells (~cells) would help to understand the autoimmune pathogenesis and lead to specific immunotherapies of IDD, We have found that lymphocytes forming rosettes with normal or tumoral murine ~ cells were more numerous (P < 0,001) in recent-onset diabetics (n = 20) than in control subjects (n = 21), We have documented the ~ cell specificity, the phenotype, and the kinetics of these <,diabetic-rosettes» that may represent a useful marker of anti-~ cell autoimmunity. The activated T-lymphocytes forming rosettes with ~ cells were then separated, cultured with IL2 for more than 6 months, and restimulated every 15 days with ~ cells and autologous mononuclear cells. Three T-cell lines, with a predominant «helper» phenotype, displayed a 250 % specific proliferation (thymidine uptake) when stimulated with ~ cells and autologous mononuclear cells. Two other lines, with a «cytotoxic/suppressor» phenotype, displayed (at a target/effector ratio of 1 :2) a 20 % specific cytotoxicity for ~ cells in a IICr release assay, These lines have also NK-like activity against K562. These data suggest that: 1) the specific rosette formation of IDD lymphocytes with ~ cells is a marker of cellular autoimmunity; 2) the culture of these «diabetic-rosettes» can generate anti ~ cell human cell lines.

Laboratory of Immunology and Department of Neurology, C.H.U. Saint-Etienne, France

J .30 Band T cells differentiation antigens in myasthenia gravis thymus

].c. LE PETIT, ].c. ANTOINE, G. POMIER, S. BOUCHERON, F. FREYCON, and D. MICHEL

In seven thymus of myasthenia gravis (MG) patients, histological pattern was correlated with lymphocyte differentiation markers identified by an immunofluorescence technique using monoclonal antibodies. T Lymphocyte markers tested were: CD2 (TIl), CDI (T6), CD3 (T3); B cells markers were CD19 (B4), CD21 (lOBI and 10Bla). CD19 and lOBI are known to be two different B cells antigens, 1 DB 1 a is restricted to a subpopulation of B cells homed in germinal center of secondary lymphnodes follicules.


Results: cortical T cells were positive for CD2 and CDl and negative for CD3 according to the T cells normal maturation process. In 4/5 patients with follicular hyperplasia, we found a very strong 10Bla fluorescent pattern of medullar germinal centers of lymphoid follicules. Only three of them weakly reacted with the other B antisera (one with CD19 and two with 10Bl). Two patients (one involuted thymus, one thymoma) showed no B cells markers.

These data confirmed the B cells origin of follicular hyperplasia in M. G. Thymus. 10Bla antigen might be a marker of the involuted B cells of the autoimmune process of M. G.

1. Medizinische Universitatsklinik, SchittenhelmstraBe 12,2300 Kiel, FRG

J .31 ELISA for autoantibodies in Wegener's granulomatosis

J. LUDEMANN, B. UTECHT, G. LUDEMANN, and W. L. GROSS

Recently IgG antibodies directed against cytoplasmic antigens of neutrophils (ACPA) have been described as a disease specific marker in Wegener's Granulomatosis (WG) that correlates strongly with the disease activity (1). We confirmed these findings using an indirect immunofluorescence technique (2) and succeeded now in developing a sensitive ELISA technique to detect the ACPA. An immunoadsorbent column was prepared with cyanogen bromide activated Sepharose 4B coupled with IgG extracted from a serum positive for ACPA. A homogenate of neutrophils, obtained by ultrasonification, was centrifuged (80000 g, 30 min) and the supernatant was passed over this column. After extensive washing the bound material was eluated with 3M guanidine. The eluate was diafiltrated and used as antigen in an ELISA. First results indicate that this rapid, sensitive ELISA is very useful to detect the ACPA and therefore of importance to monitor the disease activity and therapy in WG. Investigations to determine the molecular weight and the nature of the purified antigen are in progress.

1. VAN DER WOUDE, F. J. et al. (1985). Autoantibodies against neutrophils and monocytes: Tool for diagnosis and marker of disease activity in Wegener's Granulomatosis. Lancet, i, 425.

2. GROSS, W. L. et al. (1986). Anticytoplasmic antibodies in Wegener's Granulomatosis. Lancet, i, 806.

Supported by the Deutsche Forschungsgemeinschaft (grant Gr 609/4-2).

'Unite d'Immunocytochimie, Institut Pasteur, 75015 Paris, and 2Groupe de Recherches sur la Pathologie Renale et Vasculaire, Inserm U28, H6pital Broussais, 75014 Paris, France

J .32 Specificity of the antibodies eluted from renal biopsies of patients with systemic lupus associated nephritis

PEGGY MATSIOTA', P.DRUET2, P. BOSQUET2

, B. GUILBERT" and S. AVRAMEASI

We have tested, by ELISA, the sera of 25 patients with lupus associated nephritis as well as the antibodies eluted from 9 renal biopsies, against a panel of 6 antigens (DNA, TNP, actin, tubulin, myosin and albumin).

We have found that: - the sera exhibit a high level of IgG and IgA with anti-DNA and anti-TNP activities, and a

slightly increased level of antibodies against the panel of antigens. - the anti-TNP antibodies isolated from a lupus serum using immuno-adsorption on a TNP-


Sepharose column and subsequent elution with DNP-Iysine are able to react with TNP as well as with DNA.

- the majority of the antibodies eluted from renal biopsies when tested on the panel of antigens reacts only with DNA, however some react with DNA as well as with all the antigens of the panel. These results demonstrate that in lupus sera, there are several populations of anti-DNA

antibodies, one strictly anti-DNA, one recognizing DNA and TNP as well, and one polyspecific. At least 2 populations are found in renal biopsies and although the presence, in majority, of specific anti-DNA antibody is in favour of the pathogenic role of this anti-DNA antibody population, one cannot exclude the role of the other anti-DNA antibody population.

ICentre de Transfusion Sanguine et d'Immunopathologie, 2Laboratoire de Biochimie A, and 3d'Histologie Embryologie, CHU de Grenoble, 38700 La Tronche, France

J .33 A monoclonal IgMK from macroglobulinaemia expresses autoantibody activity against basement membranes laminin and cross reactions against some cellular nuclei and mitotic spindle apparatus

C. MICOUINt, B. ROUSSELl, E. HUDRyl, J. C. RENVERSEZ2, E. POTHAINI, P. LORIMIER>,

F. CHENAISt, B. SCHWEIZERt, and J. C. BENSAI

A monoclonal IgMK (IgM-Rod) was isolated froma patient with macroglobulinaemia. It had an autoantibody activity - without complement activation - against the laminin component of basement membranes. In vivo binding was demonstrated by immunohistologic examination of a patient's skin biopsy and of kidney sections obtained from a rat previously injected with IgM-Rod. In vitro binding was demonstrated by indirect immunofluorescence on various frozen tissues sections and by immunoelectron microscopy on rat kidney sections, using the IgM-Rod and its Fab (It) fragments. Specificity of the antibody was investigated by immunoblotting and ELISA using purified laminin (purchased from B.R.L.). Cross reactions were also observed between this IgM and nuclei and mitotic spindle apparatus, but Fab fragments were not tested against these structures whose precise nature remains unknown. Using a mouse monoclonal antibody specifically directed to the IgM-Rod (anti-idiotype obtained in our laboratories by M. C. JACOB, and F. BERNARD-GUELLE) we observecd a total inhibition of this IgM against laminin and a partial inhibition against intracellular structures. All these data highly suggest - as it is well known today - that this natural monoclonal autoantibody binds to various substances.

Dept. of Internal Medicine, University of New Mexico, Albuquerque, U.S.A. and Laboratory of Immunology, H6pital Saint-Eloi, Montpellier, France

J .34 Studies of possible anti-idiotypic control mechanisms in Graves', disease

M. MOYNIER, and R. C. WILLIAMS jr.

The present report describes studies directed at definition of possible idiotype-antiidiotypic mechanisms involved in modulation of host reactivity in thyrotoxicosis. Using ELISA, we


studied the presence of anti-F(ab')2 antibodies in the serum of patients with Graves' disease. Active and inactive Graves' patients showed higher levels of IgG anti-F(ab')2 antibodies than normal controls, but no significant difference was found when active Graves' patients were compared to those with inactive disease. In the case of IgM anti-F(ab')2 antibody, highest levels were noted in active Graves' patients in comparison to similar levels among inactive or normal controls. This pattern of anti-F(ab')2 reactivity was distinctly different from that previously documented in systemic lupus erythematosus where anti-F(ab')2 was low during active disease and highest during disease in remission (F. SILVESTRIS et aI., Arthritis Rheum., 1984, 27: 1387). We also studied the presence of antibodies directed against highly defined murine monoclonal antibodies against epitopes of thyroid stimulating hormone (TSH) and TSH-receptor. When dilutions of whole serum were studied both active and inactive patients showed a slightly higher reactivity with the monoclonal anti-TSH than normal controls. A significantly but slightly higher reactivity with two anti-TSH receptor monoclonal antibodies was noted in samples from inactive Graves' disease patients in comparison to sera from either active Graves' or normal controls. Using anti-F(ab')2 eluates from Sepharose-F(ab')2, a completely different pattern of reactivity was apparent. These data and preliminary results obtained from the study of EB virus stimulated cell lines supernates could not provide an absolute evidence of an idiotypic-anti-idiotypic control mechanism of the disease. It seems possible that if such a regulatory mechanism plays an important role either in pathogenesis or control of Graves' diesease, such reactions may be occuring primarily within the gland itself and may not be readily detectable within serum at least using our technical approach.

'Centre Hospitalier Regional, Amiens, France, and 2Inserm U. 283, H6pital Co chin, 27, rue du Faubourg St-Jacques, 75674 Paris Cedex 14, France

J .35 Thyroid stimulating immunoglobulins synthesis by pokeweed mitogen stimulated peripheral blood lymphocytes from Graves' disease patients

B. PAUTARD', and J. CHARREIRE2

Lymphocytes from freshly diagnosed untreated Graves' disease patients can be induced to produce in vitro immunoglobulins, and more precisely thyroid stimulating immunoglobulins in the presence of pokeweed mitogen, a T cell dependent polyclonal activator of B cells. Thyroid stimulating immunoglobulins were measured from day 8 to 41 of culture, using a functional cAMP production assay, after the deposition of culture supernatants on human thyroid epithelial cells. Spontaneous or pokeweed mitogen induced immunoglobulins synthesis was optimal on day 28 of culture for Graves' disease mononuclear cells, with amounts of immunoglobulins in pokeweed mitogen stimulated Graves' disease culture supernatants twice those found in unstimulated Graves' disease mononuclear cells. Moreover only pokeweed mitogen stimulated Graves' disease mononuclear cells produce immunoglobulins which induce cAMP production by cultured human thyroid epithelial cells, with a significant correlation between in vitro immunoglobulins and cAMP productions.

Gesellschaft fur Immunologie - Societe Fran"aise d'lmmunologie . 343

University of Vienna, Austria, and Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France

J .36 Antibodies to his tones : novel markers of primary biliary cirrhosis

E. PENNER, S. MULLER, D. ZIMMERMANN, and M. H. V. VAN REGENMORTEL

Liver disease sera were tested for antibodies to histones. Histones are small, basic, evolutionary highly conserved proteins, involved in the packing of the deoxyribonucleic acid molecule into chromatin. Sera were obtained from patients with alcoholic liver disease (ALD), hepatitis B virus-associated disorderes (HBV), primary biliary cirrhosis (PBC), and healthy individuals (N). Histones were extracted from calf thymus nuclei, and separated into the individual histones HI, H2A, H2B, H3 and H4 by gel exclusion chromatography; their purity was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An ELISA was used in tests for histone antibodies. The results are given below.

% Antibodies to:

Disease No. HI H2A H2B H3 H4

ALD 16 0 0 0 0 0 HBV 15 0 0 0 0 0 PBC 53 74 6 13 13 6 N 15 0 0 0 0 0

Clinically, there was no difference between PBC sera positive or negative for histone antibodies. Our data show that antibodies to histones exclusively occur in PBC; a preferential immune response is mounted towards histone HI, the protein associated with the internuc1eosomallinker region. This antibody seems to constitute a novel serologic marker for PBC.

Inserm U. 283, H6pital Cochin, 27, rue du Fg Saint-Jacques, 75674 Paris Cedex 14, France

J .37 Biochemical studies of thyrotropin receptor from the cloned human thyroid cells (GEJ)

J. J. REMY, J. SALAMERO, and J. CHARREIRE

Purification of the binding sites for thyrotropin of cloned human thyroid cells (GEJ) was performed after biosynthetic labeling of the celis, affinity chromatography on human thyrotropin sepharose column and polyacrylamide gel electrophoresis in sodium dodecylsulfate (PAGE-SDS). The apparent molecular weight of the thyrotropin receptor of the GEJ cells was approcimately 48 Kd. This molecular weight was confirmed using cross-linking of 1251_ thyrotropin to GEJ binding sites with two homobifunctional agents: dimethylsuberimidate and disuccinimidylsuberate. We found that the cross-linked complexes have a 78 Kd molecular weight still yielding a thyrotropin receptor size of 48 Kd, by subtraction of the 30 Kd molecular weight of the thyrotropin, assuming the cross-linking of one thyrotropin molecule per binding site. Moreover, the lack of effect of dithiothreitol demonstrated that the binding sites for thyrotropin of the GEJ cells is formed by a single chain without any disulfide bond. Lastly the effect of trypsin on thyrotropinlthyrotropin receptor complexes from GEJ cells detects two tryptic sites.

344 . Gesellschaft fur Immunologie - Societe Fran<;aise d'Immunologie

Inserm U28, H6pital Broussais and Lab. Hemostase, H6pital Cochin, Paris, France

J .38 Recovery from anti-VIII autoimmune disease is dependent on generation of anti-idiotypes against anti-VIII autoantibodies

F. ROSSI, Y. SULTAN, and M. D. KAZATCHKINE

Plasma samples obtained from a patient 6 weeks, 6 months and 4 years after recovery from anti-VIII:C (anti-hemophilic factor) autoimmune disease, were found to contain antibodies which inhibited anti-VIII:C activity in patient'S prerecovery plasma and in the plasma of two other patients with anti-VIII:C autoantibodies. F(ab')2 fragments from post-recovery IgG suppressed anti-VIII:C activity in F(ab')2 fragments from prerecovery IgG within a narrow range of molar ratios. Anti-VIII:C activity in F(ab')2 autoantibodies was also inhibited by F(ab')z fragments from polyspecific therapeutic immunoglobulins prepared from a large pool of normal donors (IVlg). IgG from pre recovery plasma bound to F(ab')2 from postrecovery IgG and to F(ab')2 from IVIg, as assessed by ELISA. Affinity chromatography experiments demonstrated that F(ab')2 from postrecovery IgG preferentially bound anti-VIII:C antibodies among F(ab')2 fragments from prerecovery plasma containing anti-VIII:C autoantibodies. F(ab')z from pre recovery plasma bound in higher amounts to post-recovery F(ab')z than to IVlg. Insolubilized F(ab')z fragments from postrecovery plasma also bound F(ab')2 fragments prepared from the plasma of another patient with anti-VIII:C autoimmune disease, although in lesser amounts than patient's own prerecovery anti-VIII:C F(ab')2 antibodies. These observations suggest that human anti-VIII:C autoantibodies share idiotypic determinants and that spontaneous recovery from anti-VIII:C autoimmune disease occurs through idiotypic suppression of autoantibodies. Both in patients who recover from autoimmune disease and in patients in whom autoantibodies have been suppressed by infusions of IVlg, anti-idiotypic antibodies, possibly by providing internal images of the antigen, may have shifted the immune system towards the steady state equilibrium that prevents autoimmunity in normal individuals.

Inserm U. 283, H6pital Cochin, 27, rue du Fg Saint-Jacques, 75674 Paris Cedex 14, France

J .39 Determination of the thyroglobulin epitope responsible for syngeneic T cell reactivity

J. SALAMERO, J. J. REMY, D. ZUCMAN, and J. CHARREIRE

Monoclonal autoantibodies obtained using the native thyroglobulin molecule for immunization were shown to react against a defined epitope of the thyroglobulin molecule. Moreover, previous studies have shown that this epitope, expressed on cultured thyroid epithelial cells, is responsible for in vitro primary syngeneic sensitization of T lymphocytes. Its biochemical characterization was undertaken, using papain or trypsin enzymatic treatments of porcine thyroglobulin, followed by gel filtration, affinity chromatography, SDS-PAGE electrophoresis and HPLC analysis. Immunoreactivity tested by immunoblotting with appropriate monoclonal anti-Tg antibody allowed the detection of two peptidic fragments, of 20 Kd and 5-10 Kd, after limited proteolysis with respectively papain and trypsin. From these experiments, it can be postulated that both these peptidic fragments, which react specifically with blocking monoclonal anti-Tg antibodies, contain the peptidic amino-acid sequence responsible for the in vitro proliferative response of the T cells which induce thyroiditis when injected into syngeneic recipients.


Abt. Rheumatologie und Klinische Immunologie der Medizinischen Universitatsklinik, Hugstetter StraGe 55,7800 Freiburg, und "Max-Planck-Institut fur Immunbiologie, Stubeweg 51, 7800 Freiburg, FRG

J.40 Functional and molecular characterization of an auoreactive T cell clone derived from a patient with reactive arthritis

M. SCHLESIER, C. RAMB-LINDHAUER, A. E. HINKKANEN, J. T. EpPLEN, and H. H. PETER

From the synovial fluid of a patient with reactive arthritis after rubella infection several T cell clones were established by limiting dilution. One of these clones (UA-S2) expressing the phenotype T3+ T4+ T8- exhibited a strong proliferative response to autologous irradiated mononuclear cells but not to a panel of allogeneic cells. This autoproliferative response was MHC class II-restricted since it was strongly inhibited by two monoclonal antibodies detecting common, monomorphic determinants encoded by the HLA-DR- and DP-regions, but not by monoclonal antibody W6132 which recognizes a monomorphic determinant on MHC class I molecules. Analysis of the family members revealed that the auto reactive T cell clone UA-S2 recognized a determinant of the MHC haplotype HLA-A2 or 32; B27; Cw1; DRw11 which was contributed by the patient's mother. Cells from the patient'S father, which also typed HLA-DRw11 serologically, were unable to stimulate UA-S2. Thus, the MHC determinant recognized by this clone seems not to be simply associated with Drw11. Molecular characterization of UA-S2 included the analysis of T cell receptor gene rearrangements and expression of alpha and beta chain gene products. The genes coding for the T cell receptor as well as for the alpha and beta subunits of HLA-DRw11 are currently isolated from eDNA and genomic gene libraries. These results will be discussed with respect to pathogenesis of rheumatic disease.

J.41 C. SCHMITT et al. (see Addendum, p. 499)

Medical Policlinic, University of Heidelberg, and Institute of Virus Research, German Cancer Research Center, Heidelberg, FRG

J.42 Enhanced lymphoproliferation to an antigen derived from mycoplasma arthritidis in patients with ankylosing spondylitis

M. SEITZ, W. HUNSTEIN, and H. KIRCHNER

The group of Cole et al and subsequently KIRCHNER et al isolated a mitogen for T lymphocytes from the supernatant of cultured mycoplasma arthritidis organisms (subsequently called MAS). In recent experiments we observed a marked antigenic stimulation of T lymphocytes of patients with ankylosing spondylitis by purified MAS. The lymphoproliferative response to purified MAS was strictly monocyte dependent and inhibited by a monoclonal antibody to the HLA-DR determinants of the cell surface.

Lymphocytes of healthy individuals and of patients with a variety of other rheumatic disorders did not respond to this antigen with lymphoproliferation. Our findings indicate that

346 . Gesellschaft fur Immunologie - Societe Fran~aise d'lmmunologie

supernatants of cultured mycoplasma arthritidis organisms not only contain a nonspecific mitogen for T cells but also a specific antigen for T lymphocystes from patients with ankylosing spondylitis. This may suggest a mycoplasma-related pathogenesis for ankylosing spondylitis or at least an immunogenitically determined predisposition to mycoplasmal infection in patients with this rheumatic disorder.

Inserm U.25 and L.A. 122, H6pital Necker, 149 rue de Sevres, 75743 Paris Cedex 15, France

J.43 Monoclonal (M.) autoantibodies (Ab.) inhibiting insulin-secretion in animal models of insulin-dependent diabetes mellitus (IDDM)

J. TIMSIT, C. BOITARD, C. BE COURT, A. BENDELAC, M. DEBRAY-SACHS, and J. F. BACH

Anti-islet cell autoantibodies (Ab) have been described in IDDM in man as well as in animal models such as the Bio-Breeding (BB) rat and the Non-Obese-Diabetic (NOD) mouse. Some of these Ab. are directed against islet-cell surface antigens and might be directly involved in the pathogenesis of IDDM by exerting either cytotoxicity or a functionnal inhibitory effect against the insulin-secreting islet cell. We investigated this latter hypothesis by fusing spleen cells from diabetic BB rats and NOD with a non secreting mouse myeloma (P.A.I.) in order to obtain anti-islet cell M. Ab. Hybridoma supernatants were first screened for islet cell surface (ICS) Ab. using a binding radio-immuno-assay on living rat insulin om a cells (RIN 5F). ICS Ab. positive supernatants were then studied for their ability to inhibit the secretion of insulin by RIN 5F cells. Insulinoma cells were pre-cultured for 24 hours in 96 wells microplates. Culture medium was then replaced by hybridoma supernatant for 2 hours. After washing the cells 4 wells were incubated for 1 hour at 37° with basal medium (minimum essential medium containing 10 % fetal calf serum) and 4 with stimulatory medium (basal medium plus theophylline 5 mMolil and arginine 20 mMolil). For control experiments cells were incubated either with culture medium or with supernatants containing unrelated M. antibodies. A significant increase of insulin secretion was observed in control experiments (basal insulin secretion: 2.62 ± 0.31 f.\UIlO' cells/h, stimulated secretion: 7.53 ± 0.58, P < 0.001). One ICS Ab. positive supernatant (F 16-9) was shown to inhibit enhancement of insulin secretion after stimulation (basal: 2.39 ± 0.23, stimulated: 2.96 ± 0.53), while another one (F 20-53) inhibited both basal (0.96 ± 0.12) and stimulated (1.44 ± 0.19) secretions. This demonstrate that Ab. inhibiting insulin secretion are produced at the onset of overt diabetes in animal models of IDDM. Further studies of these «blocking" M. Ab., and more specially characterization of the corresponding antigen( s), are needed for the understanding of the role of such a phenomenon in the pathogenesis of IDDM, but also of the physiology of insulin secretion.

1st Dept. of Int. Med. and Institute of Pathology, University of Mainz, 6500 Mainz, FRG

J.44 Antiglomerular basement membrane antibodies recognize similar target antigens in basement membranes prepared from human glomeruli, lung, and placenta

M. WEBER, H. KOHLER, M. MANNS, H.-P. BAUM, and K.-H. MEYER ZUM BOSCHENFELDE

Goodpasture's syndrome is characterized serologically by autoantibodies to glomerular (anti-GMB AB) and alveolar basement membranes. Several laboratories including our own (WEBER et aI., Proc. EDTA-ERA 22: 746-751, 1985) partially characterized the presumed

Gesellschaft fUr Immunologie - Societe Fran\aise d'Immunologie . 347

target antigens in glomerular basement membrane (GBM). It was the aim of this investigation to identify the target antigens of anti-GBM AB in alveolar (ABM) and placenta (PBM) basement membrane. GBM was prepared according to WESTBERG and MICHAEL (1970). ABM and PBM were prepared as described by MEEZAN et al. (1976). Collagenase-digests of the basement membrane (BM) preparations were separated by gel filtration columns and the fractions rich of Goodpasture(GP)-antigens were identified by an antibody-inhibition enzymimmunoassay. SDS-polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membranes was performed with the antigen-rich pool of each basement membrane preparation and IgG bound to the nitrocellulose membranes was detected by immunoperoxidase staining. Sera from patients with floride GP-syndrome and anti-GBM AB (4) as well as sera from these patients in clinical remission and without anti-GBM AB were investigated. In addition, sera from patients with systemic lupus erythematosus (5), autoimmune chronic active hapatitis (5), rapidly progressive glomerulonephritis (4), and sera from healthy blood donors (10) were investigated. Sera with anti GBM AB identified target antigens with molecular weights (MW) of 24, 26, 44 and 50 KD in the GBM and of 44 and 50 KD in the ABM and PBM. No corresponding reactivity was observed using GP-sera in clinical remission or the other sera. The antigens were sensitive to reduction. We conclude, that antigens of similar MW can be identified in basement membranes of human glomeruli, lung and placenta as well. Further investigations on basement membranes of other organs should clarify, whether the GPantigens may be common to all basement membranes.

lKlinik f. Innere Medizin, Med. Universitatsklinik Lubeck, and 2Chirurgische Klinik, Allgemeines Krankenhaus, Hamburg-Harburg, FRG

J.45 Autologous mixed lymphocyte reaction (MLR) induced in cocultures of T cells and iodine preincubated thyroid epithelial cell cultures (TEC) from patients with Graves' disease (GD)

An association between highly iodized diet and autoimmune thyroiditis has been demonstrated with genetically susceptible chicken or with BB rats. Since clinical observations have suggested a coincidence of high iodine administration and the prevalence of thyroiditis, we were interested in the effect iodine preincubation of TECs expressing class-I! antigen would have in co-cultures with autologous T cells. Human TECs were established from thyroid tissue obtained at operation of GD-patients. After plating, class-I! antigen was induced with interferon y (IFN y), phytohemagglutinin (PHA) or thyrotrophin (TSH) in serum free medium. Simultaneously with Class-I! induction, TECs were incubated with or without sodium iodide (NaJ, 0.001-0.1 mM), sodium perchlorate (NaCl04, < 0.1 mM) and/or methimazol (MMI, < 0.1 mM). Thereafter, autologous peripheral or intrathyroidal T cells and TECs were co-cultured for 6 days with or without interleukin 2 (IL-2, - 20 %), the 3H-thymidine uptake was measured and the stimulation indices (SI = ± Il-2) were calculated. Preincubation with NaJ (> «.01 mM) toglether with Class-II antigen expression of TECs induced a proliferative response of peripheral T cells (SI = 5.2 ± 1.2, n = 9, P < 0.01) in contrast to intrathyroidal T cells (SI = 0.8 ± 1.1, n = 5, P < 0.1). Iodine pretreatment ofTECs without class-II induction increased the thymidin uptake only slightly (SI = 2.1 ± 0.8, n = 5, P < 0.1), whilst class-II antigen alone on TECs had no effect. NaCl04 could not induce a proliferative T cell response, whereas MMI abolished it in iodine pretreated TECs. Our findings suggest that iodine induced autoantigen together with class-I! expression of TECs from genetically autoimmune-susceptible individuals (GD-patients) activates peripheral memory lymphocytes, which might be related to early cellular events in thyroid autoimmunity.

Supported by Deutsche Forschungsgemeinschaft, SFB 232/C4.

348 . Gesellschaft fiir Immunologie - Societe Fran~aise d'Immunologie

!Service de Rhumatologie, and 2Laboratoire d'Immunologie, CHU de Nancy, Vandoeuvre, France

J.46 Phenotypic characterization of polykaryons present in the rheumatoid synovial membrane (case report)

G. WERYHAl, M. c. BENE2, G. FAuRE2, and J. POUREL!

Giant multinucleate cells in rheumatoid synovial membrane have been reported morphologically for many years. But their precise lineage remains disputable. We determined the phenotypic characteristics of polykaryons found in the synovium of one patient with seronegative rheumatoid arthritis. Frozen tissue sections were stained by enzyme-histochemistry techniques: alkaline and acid phosphatases, non specific esterase, peroxidase. Immunofluorescence methods were used to evidence lymphocyte antigens (CD3, CD4, CD8, CD19), HLA class II antigens (DP, DQ, DR), mononuclear phagocyte antigens (CDll, CDw14), NK cell antigens. Multinucleate giant cells exhibited enzymatic and immunologic phenotype of mononuclear phagocytes as described in other studies: they stained for lysozomal enzymes and expressed strongly HLA class II antigens. Synovial polykaryons can be considered as mononuclear phagocyte derived cells. Absence of peroxidatic activity and of CD11 antigen expression sustain that these cells represent tissue macrophages at a late stage of differentiation.

Institute for clin. Immunol. Rheumatol., Univ. Erlangen-Niirnberg, 8520 Erlangen, FRG, and "·Institute for clin. expo Tumorresearch, Tiefenauspital, Univ. Bern, Switzerland

J.47 IgG subclass distribution of anti-doublestranded DNA antibodies in sera of patients with systemic lupus erythematosus

T. H. G. WINKLER, T. W. A. HENSCHEL, 1. KALIES, F. SKVARIL", H.-W. BAENKLER, andJ. R. KALDEN

Antibodies of the IgG class against double stranded DNA (dsDNA) are regarded as marker antibodies in systemic lupus erythematosus (SLE), but the insufficient correlation between antibody titer and disease manifestation leads to the question about their pathogenetic importance. Because of the known differences in biological functions the distribution of the Ig G subclasses among autoantibodies may have relevance for this divergence. Therefore we developed a subclass specific ELISA system for a-dsDNA antibodies. Sl nuclease treated calf thymus DNA was used for coating microtiter plates, doublestrandedness of DNA was proven by HPLC on hydroxylappatit. After incubation with sera, subclass specific monoclonal antibodies in appropriate dilutions were added. As a second antibody, biotinylated a-mouse IgG from sheep was used. Finally, streptavidin-biotin-peroxidase compexes and OPD as chromogenic substrate were added. Subclass-specific antibodies differ in avidity for their antigen. To obtain comparable results, standard dilution curves with myeloma proteins of the respective subclass were run on every ELISA plate. Results from more than 100 a-dsDNA positive sera showed specific antibodies within the first three subclasses. IgG 4 was not detected in significant amounts. Subclass distribution differed from patient to patient in a wide range. In longitudinal studies (1-2 years) the subclass pattern of each patient remained constant. Our findings do not confirm former results showing a restriction to the IgG 1 and IgG3 subclass. This could be due to the recently developed a-IgG2 antibody HP 6014 which


binds with tenfold higher affinity than the formerly used GOM 1 and GOM 2. A correlation between disease manifestation and subclass pattern of the a-dsDNA antibodies could not be established up to now.

Supported by BMFT.

ILaboratoire d'Immunologie, Centre Hospitalier Regional, Nice, and 2Inserm U 210, Faculte de Medecine (Pasteur), Nice, France

J.48 Antibasal keratinocyte activity restricted to IgG 1 lambda in a patient with no evidence of myeloma

J. P. ZAPPITELUI, D. MONDINI\ C. BELLET\ s. ALTARE\ and P. SOUBIRAN1, 2

A 70-year-old woman with post radiotherapy pulmonary fibrosis and no skin lesions showed the presence inher serum of an anti epidermis antibody as detected by indirect immunofluorescence on human skin, monkey oesophagus and rabbit lip. This auto-antibody reacted only with the first layer of epithelia exhibiting an «intercellular type» pattern. Using monoclonal antibodies specific of Ig subclasses, the basal keratinocytes activity was found to be restricted to IgG 1 Lambda. Immunoelectrophoresis did not show any evidence of IgG Lambda myeloma. These findings indicate that this auto-antibody is: a) directed towards a membrane antigen that might be a marker of keratinocyte differentiation or cell proliferation and b) is issued from an oligo or monoclonal B cell proliferation. Biochemical caracteristics of the antigen are under current study.

Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis, Wurzburg, FRG, "'Institut Pasteur Hellenique, Athens, Greece

J.49 AChR specific B-T lymphocyte interactions: privileged presentation of AChR by anti-a chain specific B hybridomas

Y. ZHANG, S. TZARTOS"', und H. WEKERLE

In preliminary report, we suggested that B cells can upregulate activation of acetylcoline receptor (AChR) specific T line cells by two different ways: either directly by presentation of the antigen AChR, or indirectly by enhancing the immunogenicity of AChR via secreted antiAChR antibodies (1). We have now screened a total of 23 AChR specific B hybrid om as differing in their Ig isotype, AChR epitope specificity and membrane phenotype for their capacity of presenting AChR to specific Lewis rat T line cells. As compared to standard antigen presenting cells (APC), 10 hybrid om as showed 3-4 fold enhanced, «privileged» presentation; 10 others had similar or lower efficiency. These twenty hybridomas presented other antigens to relevant T cells like standard APC. Three hybridomas presented neither AChR nor any other antigen. Privileged presentation did not correlate with an elevated density of membrane Ia on hybrids. The isotype of the secreted Ig was not relevant, but then was a close dependence on the target epitope of the hybrid om a antibody. Only hybrids specific for the AChR a-chain, particularly the Major Immunogenic Region (MIR), showed privileged


presentation. Our data lend further support to the concept that: 1) B cell can actively modulate T cell function, and 2) the a-chain, in particular its MIR, plays a dominant role in the pathogenesis of myasthenia gravis.

1. ZHANG, Y. et al. Ann. NY Acad. Sci. (in press).

Inserm U. 283, Hopital Co chin, 27, rue du Fg Saint-Jacques, 75674 Paris Cedex 14, France

J .50 Obtainment of thyroglobulin (T g) specific human T cell lines from Graves' disease (GD) thyroid glands

D. ZUCMAN, and J. CHARREIRE

The HLA-DR positive thyroid glands of patients with Graves' disease contain infiltrating activated T lymphocytes which were separated and selected using the classical method of sub cloning in the presence of recombinant IL-2 (x). After successive stimulations by autologous thyroid epithelial cells (TEC), two T cell lines were obtained from two different GD thyroid glands. These cell lines which show helper phenotypes (OKT4 = 90 and 92 %, DR = 65 and 70 %) were generated from wells seeded with 100 lymphocytes two months earlier.

Functional studies demonstrated that each T cell line could be stimulated by only autologous TEC, allogeneic TEC or EBV cell lines giving only background proliferative responses. Moreover when anti-DR antibodies were added during the autologous coculture on TEC as stimulators, the thymidine uptake of T cell line was strongly reduced. Lastly",'preliminary experiments of blocking of proliferative response by addition of monoclonal arlti-T g antibodies during the cocultures demonstrated that these T cell lines are specific for Tg.

(x) Roussel-Uclaf, Romainville, France.

Inserm U. 283, Hopital Cochin, 27, rue du Fg Saint-Jacques, 75674 Paris Cedex 14, France

J .51 Partial characterization of the epitopes of human thyroglobulin recognized by autoimmune sera

D. ZUCMAN, J. SALAMERO, J. J. REMY, and J. CHARREIRE

Thyroglobulin (Tg) is a large polypeptide of 660 kDa. Antibodies directed against Tg are usually detected in Graves disease and Hashimoto's thyroiditis sera; however, the precise epitopes recognized by these antibodies are still poorly known. To characterize these immunogenic epitopes of the Tg molecule, we trypsinized human thyroglobulin and study their reactivity towards ten human sera containing high anti-Tg antibody titers (> 1/1000 in hemagglutination). Thyroglobulin was extracted from 100,000 g supernatants obtained from different human thyroid glands. Then gel filtration on a Sepharose 4B column was performed and the thyroglobulin obtained (10 mg/ml in NH4 HCO, pH 8.2) was digested during 4 h with trypsin TPeK (1/25 w/w) before SDS-PAGE electrophoresis, electrotransfert on nitrocellulose and incubation with sera diluted 11100 in PBS-I0 % FCS-0.2 % Triton X100. After washings the antibodies were revealed by anti-human peroxidase conjugated antibodies.


Regularly three major bands were found, located respectively at 60 kDa, 28 kDa and 22 kDa. These three bands were detected in each of the sera tested except one which only recognize the 60 kDa one. The corresponding three pep tides were purified by electro-elution and HPLC before testing in ELISA. The ELISA showed that trypsinized Tg was much less immunoreactive than native Tg as assessed by inhibition studies. We also demonstrated that the 22 and 28 kDa peptides could share very closely related epitopes whatever the test used mutual inhibition in ELISA. immunoblot with monoclonal anti-human Tg antibodies (kindly given by M. SCHLUMBERGER, Villejuif) or anti-mouse thyroglobulin (3B8G9).

Whereas the 60 kDa peptide exhibits minimal interaction with other trypsinized Tg peptides. This study evidenced both the immunological role of conformational epitopes on Tg molecules and the existence of two different regions recognized by human autoimmune sera. Purification, production and sequencing of the 22 kDa peptide of the Tg molecule are now under investigation.

K. T-Cell Receptors, Markers and Subsets

IINSERM U93, H6pital Saint-Louis, 75010 Paris, 2Inst. G. Roussy, Villejuif, France, 3INR sui cancro Genova, Italy

K.1 Comparison and structural homologies of the three CD1 (THY, gp 43-49, 12) molecules

M. AMIOT!, H. DASTOT!, M. FABBI3, A. BERNARD2, and L. BOUMSELL1

The similarities of the CD1 molecules, in terms of their tissue distribution and association with ~2 microglobulin, prompted us to look for structural homology of the three heavy chains at the level of their peptides by 2 D-peptide maps. We observed that the T cell line HD-Mar carried high density of the CD1 (43 Kd) and (49 Kd) molecules, whereas the anti-CD1 (45 Kd) monoclonal antibodies intensively stained HPB-All cells. 2D peptide maps were thus performed with molecules isolated from 1251 surface labelled cells from two aforementioned T cell lines. We show that the three CDt heavy chains share only one tyrosil peptide, while the 49 and 43 Kd heavy chains or the 49 and 45 Kd heavy chains have another peptide in common, and finally the 49 Kd and 45 Kd heavy chains have eight peptides in common. We also observed that the CDt (49 Kd) heavy chain has twelve unique peptides, the CDt (45 Kd) has eight unique peptides and the CD1 (43 Kd) has ten unique peptides. Our results indicate that the three CDt molecules are related but confirms that they are clearly different molecules. However the extent of the differences does not permit to conclude whether the CDt molecules are the products of different genes. We also looked for the existence of a polymorphism of these three molecules on normal human thymus cells and showed that it is very limited.


Laboratoire d'Immunogenetique, Chu Pitie Salpetriere, Paris, France

K.2 Phenotypic and functional analysis of T cell precursors in a T -CFU assay

B. AUTRAN, M. C. COUTY, and P. DEBRE

The definition of the T-CFU assay has always been controversial since T-CFC can be driven from T depleted (E-) bone-marrow (BM) mononuclear cells (MNC) as well as from unfractionated PBL or BM-MNC but not from E- PBL. We have analyzed, with IF techniques, the cell surface markers of these E- and E+ derived T-cell colonies. 90 % of these cells displayed a phenotype of mature T cells (CD1-, CD22+, CD3+, Ti+, CD4 or CD8+, CDS+, CD6+, CDr, CDw18+) and were partially activated (MHC class II antigens positive, Tac+, OKT9+, OKT10+). These cells were functional in the allogeneic MLR and had a capacity of self renewal in the same T-CFC assay. However T cell depletion of these primary (I) T cell colonies, (by complement dependent cytotoxicity using a cocktail of CD2, CD3 and CDS moAbs), did not abrogate the development of mature T-colonies from the residual E- cells. We then analyzed the phenotypic and functional properties of thelse E - precursor cells of II -T -CFC derived from PBL or BM-MNC. These cells displayed the same absence of surface expression of the TCR and of all the T -differentiation antigens, except for the CD7 antigen which is consistently present on PB but not on BM derived E- cells. The E- cells of both origin did not express any marker of the B, NK or K cells nor of the myelomonocytic lineage and did not express the LFA1 antigen. Most of them were positive for the MHC-class II and Tac antigens and were OKT10+ but OKT9-. Kinetics studies showed that both the PB and BM derived E- cells were able of in vitro maturating in CD2+, CD3 +, Ti+ cells in 48 H and could proliferate in the allogeneic and autologous MLR. These data suggest that 1) immature T cells phenotypically different in PB and BM can generate mature T-colonies, 2) the T-CFU assay provides an in vitro model for the T cell differentiation analysis.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK, and "-Permanent address: Laboratoire d'Immunogenetique, Universite des Sciences et Techniques du Languedoc, Montpellier, France

K.3 Organisation of the T cell receptor a-chain gene and rearrangement in human T cellleukaemias

R. BAER, M.-P. LEFRANC", A. FORSTER, M. A. STINSON, and T. H. RABBITTS

Three different genes undergo DNA rearrangement during the course of thymic differentiation. Two of these (a and ~) appear to encode the TCR receptor. Analysis of the sequence of the a eDNA suggests the presence of V, J and C (1, 2, 3). The Ja have been shown recently to occupy a large segment of genomic DNA. We have therefore analysed the genomic organisation and rearrangement of the human a-chain locus. Analysis of a-chain rearrangements in the DNA of 53 various cell lines and primary T cell tumours indicates that at least 6 distinct Ja segments occur within a region of about 19 kb upstream of the unique Ca gene (4). A cluster of rearrangements (10 alleles out of 106) was observed to a J a arbitrarily called J aSP, located about 4 kb upstream from Ca and which seems to be the first active J a. Since we find that only about 28 % of the T cells examined are rearranged to this area further J a segments must occur

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