Workshop G Clinical Immunology I

WorkshopG Clinical Immunology I

Rheumaklinik, 2357 Bad Bramstedt; Dept. of Rheumatology, University, 2400 Lubeck, Germany

G.1 Lysozyme: a new target antigen for anti-neutrophil cytoplasmic antibodies (ANCA) E. CSERNOK, W. L. SCHMITT, S. HAUSCHILD, A. RAUTMANN, and W. L. GROSS ANCA are found in vasculitic, rheumatological and renal disorders. In 674 pANCA (= perinuclear ANCA) positive sera we were able to identify the following target antigens for

ANCA: myeloperoxidase (MPO), lactoferrin (LF), cathepsin G (CG) and elastase (HLE). In 43 %, no target antigen was found. We studied whether lysozyme (LZ), a lysosomal protein of monocytes and PMN, could be one of the missing target antigens. We established an ELISA against LZ (Bottger, Berlin) and tested 1484 Sera from 1228 patients and 120 healthy blood donors for auto-antibodies (Abs) against LZ. In an inhibition assay, the specificity of positive sera was confirmed. In healthy controls, we did not find auto-Abs against LZ, in contrast to 142 Sera from 115 patients. In the routine ANCA-secreening, most of the sera showed a pANCA fluorescence, whereas only few had a cANCA (= classical ANCA). 89 Sera reacted with LZ only, whereas 53 sera were positive for other lysosomal proteins additionally (CG: 29 sera, LF: 25, PR3: 9, MPO: 7, HLE: 5). In total, 8 % of our 674 pANCA sera had Abs directed against LZ. The «gap" of not identified target antigens was reduced from 43 % to 37%. Clinically, anti-LZ-Abs were mainly associated with rheumatological and renal disorders such as SLE (14/77, 21 %), rheumatoid arthritis (14/87,16 %), Felty's and Still's syndrom (8 %) and rapidly progressive glomerulonephritis (8/84, 10 %). They were only rarely found in (cANCA positive) Wegener's granulomatosis (9/216, 4%). Additionally, we found anti-LZAbs in 3/10 patients with Crohn's disease and 6/32 cases of ulcerative colitis. Currently, we are testing the antibody titers in their relation to disease activity. In conclusion, Abs directed against lysozyme are new autoantibodies from the group of ANCA. They are associated with rheumatological and renal conditions but as pANCA in general, they are not specific for a certain disorder. Supported by BMFT, grant No. 01VM8622.

Rheumaklinik und Rheumaforschungsinstitut, 5100 Aachen, Germany

G.2 Clinical relevance of the autoantibody NOR-90 directed against the nucleolar organizer region T. DICK, K. FRANZ, R. MIERAU, E. WEINER, and E. GENTH NOR-90 autoantibodies directed against the nuclear organizer region located at the short arm of the chromosomes 13, 14, 15,21, and 22 were previously described as rare scleroderma associated antibodies (1). We studied the incidence of NOR-90 antibodies in patients with

88 . 23rd Meeting of the Society of Immunology systemic sclerosis (scleroderma) and describe their clinical associations. One hundred and eight sera from patients with systemic sclerosis were tested. In addition, more than 25,000 sera from patients with various rheumatic diseases were routinely screened for antinuclear antibodies by indirect immunofluorescent assay using HEp-2-cells. Sera with a typical pattern of tiny nucleolar sports were further tested in an immunoblot assay using Hela-S3 nucleolar extract electrophoretic ally separated on 5 to 20 % SDS gradient gels. A typical immunofluorescence pattern of NOR-90 antibodies confirmed by the detection of a double band of approximately 90 kD molecular weight in the immunoblot assay was found in one patient with systemic sclerosis (limited cutaneous scleroderma) and five other patients with various diagnoses (rheumatoid arthritis (2), atypical chronic polyarthritis, congestive heart failure, presumed fibromyalgia). The results confirm that NOR-90 antibodies are rare in systemic sclerosis. Furthermore they are not specific for this disorder. 1. RODRIGUEZ-SANCHEZ et al.:

J.

Immunol. 139: 2579-2584 (1987).

2nd Med. University Clinic, 3200 Kiel; Dept. Internal Medicine, 2370 Rendsburg, Germany

G.3 Lymphocyte function during treatment-free remission in SLE patients H. H. EULER, B. EBERLlEN,

J.

D. HERR LINGER, E. TESKE, and J. O. SCHROEDER

Eight of 14 SLE patients treated according to an intensified regimen are currently in longterm, treatment-free remission (1 V2-5V2 years, mean: 2V, years). We investigated whether lymphocyte subclasses and function became normalized during the long-term course. Treatment consisted of a) cessation of previous immunosuppression, b) 3 plasmaphereses (60 mllkg) performed on consecutive days, c) subsequent pulse cyclophosphamide (Ctx) (36 mg/kg), d) prednisone (Prd) and peroral Ctx for 6 months, and e) withdrawal of all immunosuppressive drugs (including Prd) at month 6. This regimen aims at increased elimination of pathogenic clones during the period of compensatory activation following antibody elimination. Prior to therapy (Systemic Lupus Activity Measure/SLAM: 13-37) lymphocytopenia was noted, the T4/ T8 ratio was lowered, spontaneous immunoglobulin (IgG and IgM) secretion was elevated, and Pokeweed mitogen (PWM) stimulation was decreased. This constellation persisted at month 6 (SLAM: 2-13) and for up to 2 years. After 3-5 years the lymphocyte count, T4/T8 ratio, and spontaneous IgG and IgM secretion had normalized without SLE relapse (SLAM: 0-8). Elevated titers of anti-idiotypic antibodies were not found. Reduced PWM stimulation presisted in 7 of 8 patients; the single exception was the patient with the longest follow-up. Here, PWM stimulation normalized after 5V2 years without SLE recurrence (SLAM: 0). Thus, in long-term remission of SLE all of the parameters investigated became normalized. The data do not support the hypothesis that remissions in SLE are maintained by an enhancement of suppressive factors.

Rheumaklinik, 2357 Bad Bramstedt; Medical University, 2400 Lubeck, Germany

GA Standardization of solid-phase ELISAs for the detection of antineutrophil cytoplasmic autoantibodies found in Wegener's granulomatosis

B. K. FLESCH, E. CSERNOK, and W. L. GROSS Anti-neutrophil cytoplasmic autoantibodies exhibiting a cytoplasmic stammg pattern (cANCA) in indirect immunofluorescence (II F) on ethanol fixed polymorphonuclear

23rd Meeting of the Society of Immunology . 89 granulocytes (PMN) are closely related with Wegener's granulomatosis. Routine screening diagnosis is performed by lIF. However, a clear association to the target antigen is only possible by ELISA. In 97 % Wegener's auto antigen is identical to proteinase 3 (PR-3), a 29 kD neutral protease, located within the azurophil granules of PMN. We compared three different antigen preparations with respect to reliability and specificity for cANCA in ELISA. Preparation A «
2nd Med. University Clinic, 2300 Kiel, Germany

G.S G-CSF as an adjunct to i.v. pulse cyclophosphamide in autoimmune diseases P. HARTEN, E. TESKE,

J.

O. SCHROEDER, and H. H. EULER

In severe autoimmune diseases, e.g. vasculitis and SLE i.v. pulse cyclophosphamide (Ctx) is an alternative for established peroral Ctx protocols. A dosage-limiting factor is the risk of infection during the subsequent leukocytopenia. Recombinant granulocyte colony stimulating factor (G-CSF) is increasingly applied in aggressive oncological treatment protocols to limit the duration of cytopenia or exploit the increased scope for higher doses. Data on G-CSF following Ctx monotherapy in rheumatic diseases are not yet available. Three patients (f, 35-58 years) with severe SLE received high-dose pulse Ctx (45, 48, and 54 mg Ctx/kg body weight) over a period of 3 days. Beginning on day 4, 30 Mio U G-CSF s.c. were administered daily. The initial leukocyte count (WBC) on day 4 was 2100, 7300, and 13,200 WBC/mm 3 • Within 48 h after initiation of G-CSF an increase (probably due to distribution phenomena) in the WBC to 8000, 17,300, and 54,000/mm 3 was noted. The lower nadir of the subsequent leukocytopenia was reached on days 9, 10, and 11, respectively, with 200,100, and 1800 WBC/ mm 3 . By days 14, 15, and 16 the WBC exceeded 4000/mm 3 in all three patients and G-CSF was stopped. No effect on erythropoiesis and thrombopoiesis was observed. Infections did not occur. One patient reported mild influenza-like symptoms following the injections. There was no sign of SLE-activation. Compared with a retrospective control group of 33 patients treated with lower dose pulse Ctx (36 mg/kg), but without G-CSF, the leukocytopenia was significantly reduced. Based on these initial findings, G-CSF could represent a useful addition to the therapeutic repertoire of pulse Ctx treatment of SLE.

90 . 23rd Meeting of the Society of Immunology Div. of Immunology, Dept. of Medicine, Medical School, 3000 Hannover, Germany

G.6 Why does the FcyRIIIb-deficiency of neutrophils from patients with paroxysmal nocturnal hemoglobinuria not cause an immunodeficiency? M. HUNDT, M. ZIELINSKA-SKOWRONEK,

J.

SCHUBERT, and R. E. SCHMIDT

Human neutrophils express constitutively two low-affinity Fcy receptors, FcyRII (CDw32) and FcyRIIIb (CD16), for IgG immune complex interactions. FcyRII is a transmembranous protein and FcyRIIIb is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. In paroxysmal nocturnal hemoglobinuria (PNH) the abnormal cells are characterized by the lack of GPI-linked proteins on their cell surface. Therefore, PNH neutrophils express FcyRII in normal and FcyRIIIb in very low density. In normal neutrophils it has been demonstrated that IgG 1x cryoglobulin complex induced calcium release and H 2 0 2 production was predominantly mediated via FcyRIIIb. In addition, neutrophils treated with PI-PLC to cleave GPI-linked FcyRIIIb in vitro demonstrated significantly reduced calcium mobilization that was mediated by FcyRII and FcyRIIIb. Consequently a significant defect in IgG immune complex activation of PNH neutrophils was to be expected. But PNH neutrophils with a lower expression of FcyRIIIb than PI-PLC treated neutrophils with a lower expression of FcyRIIIb than PI-PLC treated neutrophils released calcium and produced H 2 0 2 only slightly reduced in comparison to neutrophils from healthy donors. In contrast to normal neutrophils, this activation was mediated via FcyRII and not via FcyRIIIb. Therefore, we propose that the ability of FcyRII to take over functions of FcyRIIIb prevents an expected immunodeficiency in PNH patients.

1 Institute for General and Exp. Pathology, 2 Dept. of Surgery and 3 Neurology, University Medical School, 6020 Innsbruck; 4 Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck, Austria

G.7 Heat shock protein 65 is expressed in human atherosclerotic lesions R. KLEINDlENST\

Q. xU., F. R. WALDENBERGER2 , J. WILLEIT3 , and G. WICK 1•4

Previous work in our laboratory revealed the presence of a great number of activated Tlymphocytes in early human atherosclerotic lesions and showed the atherogenic property of Mycobacterium tuberculosis heat shock protein 65 (hsp65) in normocholesterolemic rabbits when immunizing them with this stress protein. To demonstrate endogeneous hsp 65 expression and infiltration of T-lymphocytes expressing the y/o T-cell receptor (TCR) in human atherosclerotic lesions, we examined specimens of aorta, carotid-, and internal mammary arteries and of internal mammary-, and saphenous veins obtained from 25 patients aged 33-80 years, by means of immunohistochemical- and immunofluorescence techniques on serial frozen tissue sections. Whereas venous samples of small diameter as well as internal mammary arteries served as a reference for normal intima and showed neither hsp 65 expression nor mononuclear infiltration, expression of this stress protein could be detected on endothelial-, smooth muscle-, and mononuclear cells of almost all aortic and carotid specimens, correlating with severity of inflammatory signs and progression of atherosclerosis. We found 9.7 % of Tcells bearing y/o TCR in the transition zone from normal intima to fatty streak, decreasing to 6.6 % and 4.3 % in fatty streak and atherosclerotic plaque, respectively. We conclude, that the intensity of hsp 65 expression positively correlates with severity of atherosclerosis and may be responsible for a specific immunological induction of the inflammatory process in this disease. Infiltrating y/o TCR positive cells constitute a considerable population in early lesions, but later on the process seems to be mediated by a/~ TCR bearing cells. Supported by the Austrian Research Council (project no. 8925).

23rd Meeting of the Society of Immunology . 91 Cell- and Immunbiol. Lab., Dept. of Internal Medicine, Medical University, 2400 Lubeck

G.8 Autoantibody profiles in needle biopsies from autoimmune human thyroid glands by means of isoelectrofocusing (IE F) and reverse immunoblotting (rIB) with defined antigens T. KLUGMANN, S. GRAMMERSTORF, and B. E .. WENZEL We developed a method whereby in homogenized thyroid tissue from needle biopsies of patients with autoimmune thyroid diseases (AITD) Ig-classes and subclasses were separated by IEF. Bands obtained in IEF were transfered to nitrocellulose membranes which subsequently were incubated with biotinylated antigens. The immunocomplexes were developed by Streptavidin-alkaline-phosphatase staining. In AITD (n = 10) antibodies against thyroglobulin (Tg), thyroid peroxidase (TPO), heats hock protein 72 (HSP 72) and Yersinia virulence antigens (YOP) were detected. These antibodies were absent in non-immune thyroid tissues. YOP and HSP 72 antibodies were found at different pIs in comparison to corresponding antibodies in serum from the same patients, while the pIs of Tg and TPO antibodies were identical in both tissue ad serum. HSP 72 antibodies in tissues were IgA class, whilst YOP antibodies were classed as IgG and IgM. These findings support our hypothesis that HSP 72 and YOPs are involved in situ autoimmune processes. Supported by BMFT 01-Ki 8903/1.

1. Med. Klinik, Universitat, 6500 Mainz, Germany; ! Dept. of Radiobiology and Immunology, Primate Centre, TNO, Rijswijk, Netherlands

G.9 Induction of an anti-vaccine response by T cell vaccination in primates and humans A. W. LOHSE, N. BAKKER!, E. HERMANN, T. PORALLA, M. JONKER!, and K.-H. MEYER ZUM BOSCHENFELDE Experimental and spontaneous autoimmune disease in animals can effectively be prevented and treated by application of the pathogenic autoreactive T cells in an attenuated form. This approach has become known as T cell vaccination. T cell vaccination exploits specifically the ability of the immune system to regulate its autoreactive T cells by mechanisms of network control. The success of T cell vaccination in a variety of rodent animal models has raised hopes for it as an effective and specific therapy in human autoimmune disease. The aim of this study was to induce an anti-T cell response by T cell vaccination in humans and primates as a preclinical study into the feasibility and toxicity of T cell vaccination. Using bulk cultures of T cells from the peripheral blood or an inflamed joint it was possible to induce a T cell response specific for the injected vaccine and its activation state both in rhesus monkeys and in two patients with active rheumatoid arthritis. In one of the patients there was already a spontaneous T cell response against a mitogen driven T cell line from the peripheral blood, but not against a control T cell line specific for tetanus toxoid, suggesting that regulatory T cell networks are operative in patients with autoimmune disease. Significant clinical effects of sideeffects were not observed. The results suggest that T cell vaccination in humans is feasible and non-toxic. It is likely to influence an already ongoing regulatory process. Conditions of making T cell vaccination an effective therapy need still to be worked out by futher studies both in primates and in less complex human immune processes.

92 . 23rd Meeting of the Society of Immunology I. Medical Dept., University, 6500 Mainz, Germany

G.IO Antibodies to proteinase 3 increase adhesion of neutrophils to human endothelial cells W.-]. MAYET, E. HERMANN, B. FLEISCHER, and K.-H. MEYER ZUM BOSCHENFELDE Adhesion of neutrophils to endothelial cells (EC) represents an early and requisite event in acute inflammation. Neutrophil-mediated EC injury is discussed to be important in the pathogenesis of Wegener's granulomatosis (WG) and related vasculitides. Antibodies (Ab) directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in WG. The aim of this study was to investigate the effect of affinity purified PR-3-Ab on the adhesion of neutrophils to cultured human EC. EC were isolated by collagenase digestion of human umbilical veins and cultured in gelatine coated flasks using the standard procedure of JAFFE et al. (1973) with minor modifications. EC of several donors were pooled to exclude the influence of ABH-antigens. Purity of culture was confirmed by reaction with Ab to v. Willebrand factor and Ulex lectin. Neutrophils were sedimented under pyrogen-free conditions and adhesion was measured in a Bengal-Red release assay after incubation with PR-3-Ab (IgG- and F(ab'lz fractions) and a human monoclonal PR-3-Ab. Incubation of EC with PR-3Ab led to an marked increase of neutrophil dehesion with a peak after 4 h and could be inhibited by preincubation of the Ab with purified PR-3 or an Ab against ELAM-l. Furthermore, incubation of EC with PR-3-Ab similar to TNF-alpha led to an increase of endothelial ELAM-1-expression as measured in a cyto-ELISA and by flow cytometry. Our data demonstrate a direct effect of PR-3-Ab on neutrophil-endothelial interactions. The enhanced neutrophil adhesion occurs via induction of ELAM-1 and PR-3-Ab seem to have TNF-alpha-like effects. PR-3-Ab induced neutrophil adhesion to vascular endothelium may play an important role in the pathogenesis of WG and other ANCA-related vasculitides.

Institute of Immunology, Medical Academy, 8080 Dresden; «Arztliche Gutachtergemeinschaft», 9154 Niederdorf, Germany

G.ll Non-organ specific autoantibodies as a prognostic sign of a possible development of a progressive systemic sclerosis in quartz dust exposed miners K. CONRAD,]. MEHLHORN, A. JACOBI, U. GAWEHN, and K.-H. FRANK Workers with an intensive quartz dust exposition take a higher risk to develop a progressive systemic sclerosis (PSS) than non-exposed persons. Because anticentromere antibodies (ACA) may be detected several years before disease manifestation we examined sera of 751 dust exposed uranic miners and 1004 non-exposed blood donors for the expression of ACA and other non-organ specific autoantibodies (AAb) by indirect immunofluorescence on tumor cell monolayer, immunodiffusion, immunoblotting and enzyme immunoassays. The detected AAb specificities (8 ACA, 7 anti-Scl-70, 6 anti-U1-RNP, 28 anti-SSA/-SSB, 1 anti-dsDNA and 5 antinucleolar antibodies) are characteristic for connective tissue diseases and were not seen in sera of blood donors. In a group of 20 persons with symptoms indicative of a developing PSS (Raynaud, arthralgy, pulmonary interstitial fibrosis) 14 were positive for such AAbs. In 7 of them PSS according to the ARA criteria manifested 2-5 years after the first detection of AAb (2 ACA, 3 anti-Scl-70, 1 anti-SSA, 1 antinucleolar AAb). One quartz dust exposed miner without any signs of connective tissue disease carried anti-Scl-70 at least 5 years

23rd Meeting of the Society of Immunology . 93 until the onset of PSS. The observation of the clinical course of the other AAb positive persons will show, if these AAb specificities are prognostic signs of a developing connective tissue disease. Besides that, these results may be the basis for further immunological and molecularbiological studies of the pathogenesis of systemic autoimmune diseases.

Institute of Clinical Immunology, ! Dept. of Internal Medicine, University, 7010 Leipzig, Germany

G.12 Immunological aspects of fibrinolytic therapy of acute myocardial infarction by recombinant plasmin ogene activator (rt-PA) H. SCHADLICH, I. BLECHSCHMIDT, U. SACK, K. DROSSLER, A. BLUHM!, L. ENGELMANN!, and G. METZNER Reperfusion injury and myocardial stunning after acute myocardial infarction are accompanied by a cytokine-mediated activation of immune processes. We examined polymorph nuclear neutrophils, monocytes, and lymphocyte sub populations (T cells, activated T cells, CD4+ and CD8+ T cells, CD4/CD8 ratio, NK cells, B cells), and HLA-DR+ monocytes in right ventricular blood of patients with myocardial infarction with and without rt-PA therapy on 0, 1, 2, 3, 4, 8, 12, and 24 h after admission to the hospital and then daily for 10 days in FACS analysis. C reactive protein (CRP) and Interleukin-6 (IL-6) were detected using a commercial ELISA. IL-6 arises some hours after infarction. This is followed by an increase of CRP for three days. On first day, we find an enhanced content of NK cells and a diminished number of T cells, especially of CD4+ lymphocytes. CD4/CD8 ratio is also diminished. Lymphocyte populations normalize up to the 10th day. This findings rather are due to the reperfusion of myocardial tissue than to a non specific acute phase reaction after infarction.

! Clinical Research Unit on Rheumatology, 2 Dept. of Rheumatology and Clinical Immunology, University Medical Center, 7800 Freiburg, Germany

G.13 Specificity of monoclonal rheumatoid factors derived from patients with rheumatoid arthritis M. SCHLEDZ!, A. HOHLBAUM!, H. H. PETER2, U. RUDOLPH!!, and I. MELCHERS! Two systems were used to analyze B cells producing rheumatoid factors (RF) under clonal conditions: the activation system developed by WEN et al. (Eur. J. Immunol. 17,887, (1987)), which generates short term clones with high efficiency (system A), and the establishment of hybridomas, resulting in stable clones (system B). Using system A under limiting dilution conditions we were able to distinguish between clones producing monospecific RF and clones producing multireactive RF by testing individual culture supernatants on a variety of (auto-) antigens. In patients with rheumatoid arthritis the frequency of monoreactive clones usually extends the frequency of multireactive clones, whereas the contrary is the case in healthy control persons. Using system B, hybridomas were established, which represent typical populations defined in system A. In detail, the features of IgA-RF derived from peripheral blood B cells and IgG-RF derived from synovial tissue B cells will be described. Supported by DFG grant Pe 151/10.

94 . 23rd Meeting of the Society of Immunology 2nd Med. University Clinic, 2300 Kiel, Germany

G.14 Treatment of SLE with high-dose i.v. immunoglobulins

J. o.

SCHROEDER, A. H. HEER, E. TESKE, and H. H. EULER

We investigated the effect of high-dose intravenous immunoglobulins (ivIg) in Systemic Lupus Erythematosus (SLE). To date 12 patients with a mean age of 28 years (range: 16-55) have been enrolled in a pilot trial. The patients fulfilled a mean of 5 (range: 4-8) of the 1982 ACR diagnostic criteria. The patients showed a mild mean disease activity of 7.9 (range: 3-15) as determined by the Systemic Lupus Activity Measure (SLAM). The predominant symptoms were thrombocytopenia (n=5), anemia (n=4), arthritis (n=4), Raynaud's phenomenon (n = 4), facial erythema (n = 3), and lupus nephritis (n= 1). Treatment consisted of infusion of 30 g ivIg on days 1 to 4 and a repetition of this regimen on days 21 to 24. Previous therapy consisting of prednisone (n= 10), azathioprine (n =2), or hydroxychloroquine (n = 1) was not changed for at least 6 weeks. In the majority of patients (7/12) a positive clinical effect was noted. The mean thrombocyte count rose from 49/nl prior to therapy to 103/nl after one week and 112/nl after 6 weeks. In addition, improvement was noted in the degree of arthritis, Raynaud's phenomenon, and erythema. The mean SLAM decreased from 7.9 to 6 in week 4. Whereas ANA titers and complement proteins (C3 and C4) remained constant in the majority of patients, a slight decline in antibodies against ds-DNS occurred. Thus, high-dose ivIg was effective in at least a portion of patients and could be useful in treatment of SLE when immunosuppression by cytotoxic agents is contraindicated.

Abt. Immunologie, Med. Hochschule, 3000 Hannover 61, Germany; Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA

G.15 Impaired activation of GPI-deficient lymphocytes from PNHpatients

J. SCHUBERT!, M. LEDWON!, M. ZIELINSKA-SKOWRONEK!, M. E. MEDOF2 , and R. E. SCHMIDT! Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder in which parts of peripheral blood cells deficient in glycoinositol phospholipid (GPI)-anchored surface proteins are present in the circulation. A series of these molecules have recently been described being able to activate lymphocytes after crosslinking. In these studies the role of GPI-anchored proteins in T cell activation was analyzed using fresh T cells and T cell lines from PNH patients. For control GPI-molecule expressing T cells from the same patients were used. Unstimulated GPI-deficient T cells exhibited a significantly reduced surface expression of the activation antigen CD45RO. In addition, measuring proliferation, cytokine production and second messengers, a decreased response of CD48- compared to CD48+ T cells was observed after stimulation with PHA but not when using CD3 monoclonal antibodies. Biochemical analysis revealed that all deficient T cell lines are able to produce the first 2 intermediates as well as Dol-P-Man but fail to produce further mannosylated intermediates of the biosynthetic pathway. We conclude that GPI-anchored surface molecules play an important role in T lymphocyte activation. We are currently investigating the construction of «high mannose» oligo saccharides by the deficient cells since this selective activation defect also suggests an Nlinked glycosylation defect of proteins. Supported by DFG Schm 596/3-2, DFG Schu 713/1-2 and NIH 23598 PO 10K38181.

23rd Meeting of the Society of Immunology . 95 Private Institute of Immunology and Molecular Genetics, 7500 Karlsruhe, Germany

G.t6 Autoantibodies directed against Golgi antigens in patients with rheumatic diseases H. P. SEELIG, C. WIEMANN and M. RENZ Antibodies against antigens at the Golgi apparatus (GA) have been described in a few patients with Lupus erythematosus or non-infectious hepatitis. In two patients with rheumatic diseases with suspicion of sclerodermia or Sjogren syndrome we found antibodies which showed the characteristic fluorescence pattern of these rarely occurring anti-«GA» antibodies. Immunohistological studies with various culture cell lines and tissue sections confirmed the affinity of these antibodies to components of the «GA» (juxtanuclear localization, fragmentation of «GA" after mitomycin treatment and during mitosis). To identify the nature of the antigen(s) about 106 plaques of a human cell line (HeLa) AZAPII eDNA expression library (Stratagene) were screened with the patients' sera. Positive colonies were purified by repeated screenings yielding several phagemids which contained overlapping eDNA inserts. After insert excision of the phagemid carrying the largest insert (3.2 kb) and recloning in an bacterial expression vector the patients' sera were affinity purified with the recombinant antigen. The affinity purified antibodies reacted in immunohistological experiments exclusively with structures of the Golgi apparatus. In addition the recombinant antigen was used to develop a sensitive and specific Elisa for antibody detection in patient sera. Since the so far sequenced cDNAs (about 900 nt) showed no significant similarity « 60 %) to any of the sequences deposited in the GenBank/EMBL data library this Golgi protein is presumed to be new. Whether this seldom occurring antibody will gain any pathogenetic or diagnostic significance needs to be evaluated. The biological function of the antigenic protein might be elucidated by sequence comparison with known proteins after completion of the sequence of the entire coding eDNA as well as by electron microscopic studies.

Institute of Immunology, Dept. of Medicine, University, 2500 Rostock; Clinical Research Unit MPG> University, 8520 Erlangen, Germany

G.t7 Immunological follow-up after monoclonal antibody therapy in chronic inflammatory bowel diseases M. SEYFARTH, S. BUCHWALD, U. SCHULZ, M. NAUSCH, J. EMMRICH, and F. EMMRICH Conventional treatment protocols often do not lead to continued remission in inflammatory bowel diseases. Recently, successful pilot trials with a monoclonal antibody (mab) to CD4 have reported in rheumatoid arthritis. 3 pat. with Crohn's disease and 4 pat. with ulcerative colitis were treated for 7 days with an anti-CD4 mAb (MAX 16H5). Immunological monitoring revealed a transient, selective depletion of CD4+ cells after the first infusion. The proliferative response of blood leukocytes to mitogens was diminished after antibody treatment. The addition of anti-IL6 to the PWM culture led to the neutralization of de-novo IL6 and influenced the immunoglobulin production in vitro. 2 pat. reached a complete remission lasting now for more than 12 months in 1 pat. 4 pat. responded to the treatment clinically, but relapsed after 6 weeks. Anti CD4+ therapy may be of value as a second line treatment of inflammatory bowel disease when conventional therapy has failed.

96 . 23rd Meeting of the Society of Immunology Institute for Immunology, University, 2300 Kiel, Germany

G.18 Severely deficient T cell response in uremic patients

J.

STEINMANN, C. HERWARTZ, and W. MOLLER-RuCHHOLTZ

The immunodeficiency patients with chronic renal failure is manifested through a relatively high incidence of infections and a low response to vaccines. On the other hand, uremic patients seem to benefit from their immunodeficiency in allotransplantation. It has been demonstrated by others that the monocyte function is impaired under uremia, and that uremic immunodeficiency may be enhanced by soluble factors present in uremic serum. Here we show by means of functional in vitro T cell assays, that uremic immunodeficiency is demonstrable on the T cellleve!. Mixed lymphocytes cultures (MLC) and limiting dilution assays (LDA) for cytotoxic T lymphocyte precursors (CTL-p) were performed in RPMI with 15 % prescreend pooled (non-uremic) human AB-serum according to the consensus protocol of the European CTL-p workshop. The mean stimulation index of 8 uremic patients in 16 mixed lymphocyte culture combinations was only 4,9 versus 45 in healthy controls. The mean cytotoxic T cell precursor frequency in the same combinations was only 18 per million versus 500 per million in the controls. After kidney transplantation the immunodeficiency remained in all patients due to the immunosuppression. We conclude that uremia causes T cell dysfunction. Our investigation of uremic T cell dysfunction is aimed at supplemental cytokine therapy.

Euroimmun, 2400 Lubeck;

I

Dept. of Medicine, University, 8000 Munchen, Germany

G.19 Autoantibodies to granulocytes in chronic inflammatory bowel disease are not correlated with antibodies to intestinal goblet cells in ulcerative colitis and to pancreatic juice in Crohn's disease W. STOCKER, S. OLBRICH, W. SCHLUMBERGER, A. BROHMANN, E. MOLLER-KuNERT, and P. C. SCRIBAl Autoantibodies to the cytoplasma of granulocytes were observed in sera of patients with ulcerative colitis (UC) and, with a lower prevalence, of patients with Crohn's disease (CD); the immunofluorescence pattern was perinuclear (pANCA; 1). On the other hand, disease specific autoantibodies are directed against intestinal goblet cells in CU (GAb; 2, 3) and against pancreatic juice in CD (PAb; 3). We analyzed if pANCA were correlated with GAb or PAb. Sera were tested by indirect immunofluorescence, using frozen sections of human fetal intestine and adult pancreas as well as granulocyte preparations as antigenic substrates. Fluorescein labelled antihuman IgA + IgG + IgM was the second antibody. Only patients with agreeing clinical, endoscopical and histological diagnoses of UC or CD were regarded. UC patients exhibited pANCA in 66.7% of 21 GAb+ve and in 52.7% of 55 GAb-ve cases. CD patients exhibited pANCA in 6.7% of 15 PAb+ve and in 7.4% of 54 PAb-ve cases. Thus, a close correlation of GAb or PAb with pANCA can be excluded. GAb in UC were preponderant in males (prevalence ratio malelfemale 3.3). For PAb in CD the ratio was 1.1, for pANCA in UC 0.9. All five pANCA +ve of 69 CD patients were women. The additional testing of pANCA enhanced the predictive value of the serological diagnosis in UC from 28% (GAb; 3) to 66% (GAb plus pANCA) and in CD from 39% (PAb; 3) to 46% (PAb plus pANCA). However, pANCA are not restricted to one of the two diseases, whereas GAb show a close association with UC and PAb with CD. 1. SAXON, A. et a!.: J. Allergy. Clin. Immuno!. 86,202 (1990), 2. BROBERGER, O. and B. PERLMANN: J. expo Med. 115, 13 (1962), 3. STOCKER, W. et a!.: Scand. J. Gastroent. 22, Supp!. 139,41 (1987)

23rd Meeting of the Society of Immunology . 97 2nd Med. University Clinic, 2300 Kiel; Dept. Internal Medicine, 2370 Rendsburg, Germany

G.20 Treatment-free remission in severe SLE - update of a pilot trial U. M. SCHWAB, P. HARTEN, E. TESKE,J. O. SCHROEDER,J. D. HERRLINGER, and H. H. EULER Standard treatment for severe SLE consists of prednisone (Prd)and cyclophosphamide (Ctx). Treatment-free remissions have thus not been reported. Plasmapheresis (PI) as an adjunct to long-term immunosuppression does not improve the results in controlled trials. We applied PI with subsequent pulse Ctx, aiming at the largest possible deletion of pathogenic clones during compensatory lymphocyte activation «
1 Med. Klinik Innenstadt, Universitat, 8000 Miinchen;

2 4

Med. Poliklinik, 3 Dermatologische Klinik und Poliklinik, Universitats-Hautklinik, 5000 Kiiln, Germany

G.21 Autoimmunity and bacterial infection: antibodies against pore forming peptides of E. coli hemolysin in Systemic Lupus Erythematosus R. L. OROPEZA WEK),RLEl, K. KROGER 2 , M. SCHATTENK1RCHNER2 , M. MEURER 3 , T. KRIEG\ and H. FRICKE 1 We previously identified two pore forming peptide sequences of the hemolysin of E.coJi (1). We now demonstrate by ELISA that patients with Systemic Lupus Erythematosus (SLE, n = 60) have enhanced immunoglobulin titers against native E.coJi hemolysin, and that much of this reactivity is focussed on the pore forming peptide sequence 310-317 (peptide 002). Less frequently, antibodies against another pore forming peptide sequence (pos. 300-309, peptide 001) were found. The anti-hemolysin and -peptide response is not seen in sera from healthy donors, nor from patients with rheumatoid arthritis (n = 12), Wegener's granulomatosis (n=6), nor scleroderma (n=6). Finally, SLE sera showed only low binding with a control synthetic peptide. We discuss a possible role of the anti-pore forming peptide antibodies in the pathogenesis of human SLE. Supported by Wilhelm Sander Foundation (Fricke 91.028.1). 1. OROPEZA WEKERLE et al.: Mol. Microbiol. 6, 115-121 (1992)

98 . 23rd Meeting of the Society of Immunology ! Institute of Med. Microbiology, University, 6500 Mainz; 2 Institute of Immunology, University, 6900 Heidelberg; 3 Dept. of Rheumatology, University, 6000 Frankfurt/Main, Germany

G.22 A complete selective Clq deficiency: Characterization of the nonfunctional protein and analysis of the genes F. PETRY!, D. T. LEI, M. KIRSCHF!NK2,

J.

P. KALTwASSER3, and M. Loos!

A case of selective functional C1q deficiency has been recently reported (Behring Inst. Mitt. 84: 55, 1989). C1 activity could be restored by addition of purified normal C1q, illustrating the presence of functional C1r and C1s in the patient's serum. The individual protein chains of C1q could be detected by Western blotting with polyclonal anti-C1q. Partial antigenic identity with normal C1q was demonstrated by double immunodiffusion. While the reduced protein chains of C1q migrated with a molecular weight similar to normal C1q in SDS-PAGE, C1q eluted with the IgG peak in gel filtration of the patient's plasma on Superdex 200. Ultracentrifugation in sucrose density gradients revealed that C1q in the patient's plasma is present in a low molecular weight from with a sedimentation constant lower than IgG (7 S) but higher than albumin (4, 6 S). No 11 S C1q was detectable. For further characterization of this deficiency the patient's DNA was analyzed by Southern blotting. Restriction of chromosomal DNA with 10 endonucleases and hybridization with DNA probes coding for the three individual chains of C1q, revealed no differences in comparison with normal individuals. These data indicate that no substantial insertions, deletions or invertions of DNA fragments, which should be detectable as restriction fragment length polymorphisms, are present in the patient'S C1q genes. Oligonucleotides have been designed that flank the coding region of the largest exon (exon 2) of the genes coding for the A-, B-, and C-chain of C1q. We are currently amplifying the patient's DNA by the polymerase chain reaction and sequencing the fragments after subcloning into a plasmid vector. Differences in the sequence information to the published data will be confirmed be a second analysis of an independent amplification and sequencing reaction. A number of potential mutated sites have been observed and are presently under consideration as possible causes of this deficiency.

Institute of Clinical Immunology, 6900 Jena, Germany

G.23 CD45-isotypes in patients with common variable immunodeficiency (CVID) H. VOGELSANG, C. DIENER, B. HEYDEN-RYNSCH, B. JUNKER, and L. JAGER Beside other factors, T-cell abnormalities may be responsible for the missing or decreased production of immunoglobulines in patients with CVID. Starting from a decrease in CD4/ CD45R ratio shown earlier in several patients, we further differentiated the CD45R isotypes with monoclonal antibodies to CD45RA, CD45RO (Becton-Dickinson), and CD45RB (DAKO), both in CD4+ and CD8+ cells. In 10 controls we found the following distribution amongst CD4+ cells: CD45RA (naive cells) 38 ± 11 %; CD45RO (memory cells) 54 ± 14 %; CD45RB 87 ± 9 % and CD45RB++ 48 ± 18 %. In five of ten CVID patients there was a considerable decrease of CD45RA (1-6 %) and an increase of CD45RO (76-92 %). These same patients showed a diminished CD4/CD45RB++ population (8-28 %) which some authors correlate with the TH1 population. Within the CD8+ population the relation between CD45 isotypes and functional groups is not yet clear. In this subgroup we also found some deviations. However, in this cases the changes in CD45RA and CD45RO showed no clear reciprocal course. It will be necessary to define the functional activities of the different subpopulations in CVID.

23rd Meeting of the Society of Immunology . 99 Central Laboratory, Medical Clinic and Policlinic, University, 6900 Heidelberg

G.24 Rheumatoid factors: Hemagglutination, nephelometry and isotype differentiation by ELISA E. WERLE, M. BLAZEK, and W. FIEHN Rheumatoid factors (RF) are autoantibodies directed against antigenic determinants in the Fc region of immunoglobulin G (IgG) which is altered in its tertiary structure. RF are used to strengthen a clinical diagnosis of rheumatoid arthritis and they may be important for the pathogenesis and prognosis of this autoimmune disease. During a period of one year 5598 sera from 4665 patients were tested for RF in our laboratory. In 4779 sera both the Waaler-Rose hemagglutination test (Cellognost RF micro, Behring, Marburg, FRG) and a nephelometric assay (Behring) were performed. In the hemagglutination test the antigen source are rabbit antibodies whereas the nephelometric assay is based on latex particles coated with human IgG. 88.6 % and 4.7 % of the patients had negative and positive results in both tests, respectively. 192 patients (6.2 %) out of 4095 had a positive result only in the nephelometric RF assay, whereas in 23 patients (0.56 %) only the Waaler-Rose test was positive (up to 50 IU/ml). Random samples of sera could further be tested for RF of the IgA and IgM class with an ELISA using microtiter plates coated with Fc fragments of human IgG. Thereby, one could investigate whether differing results might be method-dependent artifacts or result from the species specificity of the patients' antibodies. In conclusion we propose to determine RF with nephelometry and to use the Waaler-Rose hemagglutination test only in case of a positive nephelometric assay result except when the clinical diagnosis of RA is evident.

Institute for Biomedical Aging Research, Austrian Academy of Sciences; 2 Institute for General and Exp. Pathology, 3 Institute for Neurology, University, 6020 Innsbruck, Austriy

1

G.25 Relationship of serum antibodies to heat shock protein 65 and atherosclerosis Q. Xul, R. KLEINDIENS,-l, G. LUEF3 , M. MAROSI 3 , and G. WICK1.2 We have shown previously that arteriosclerotic lesions can be induced in normocholesterolemic rabbits by immunization with heat shock (hsp) 65, a stress protein found to be expressed in high levels also in human atherosclerotic lesions. If an immune reaction to hsp65 plays a role also in human atherogenesis it would be conceivable to find anti-hsp 65 antibodies in patients with atherosclerosis. In order to study this issue and a possible relationship of immune reactions to hsp65 and atherosclerosis, 867 individuals aged 40 to 79 years from the Bruneck region, South Tyrol, were randomly selected from 13,534 inhabitants for the determination of serum antibodies against hsp65, nuclear antigens (ANA), thyroglobulins (TG-AAb), thyroid microsomal antigens (MS-AAb), and immunoglobulins (RF), and simultaneous sonographic assessment of carotid atherosclerotic lesions. Our data showed that serum hsp65 antibodies were significantly (p = 0.0016) increased in subjects aged 60 to 79 years with carotid atherosclerosis compared to those without lesions. This increased antibody level was independent of age and sex. On the other hand, the incidence and titers of various autoantibodies (ANA, TG-AAb, MS-AAb and RF) did not correlate with the occurrence of carotid atherosclerotic lesions. Our data provide the first evidence of a strong correlation of hsp65 antibodies with carotid atherosclerosis, suggesting that hsp65 might be involved in pathogenesis of atherosclerosis and that a determination of antibodies to hsp65 is possibly a new and potentially useful parameter for diagnosis and prognostic evalution of atherosclerosis.

100 . 23rd Meeting of the Society of Immunology 1 Universitats-Kinderklinik, 6000 Frankfurt/Main; 2 Abt. Angewandte Immunologie, Deutsches Krebsforschungszentrum, 6900 Heidelberg, Germany

G.26 Restoration of immunglobulin production by IL-2 is limited in children with common variable immunodeficiency (CVI) S. ZIELEN\ T.

J.

DENGLER2 , P. BAUSCHER\ and S. C. MEUER2

CVI is an acquired disease of unknown cause characterized by a defect in antibody production. In adult patients the severity of B-cell defect has been assessed by secretion pattern of immunglobulin isotypes in response to IL-2 and polyclonal B-cell activators. Patients are divided in to groups: neither IgM nor IgG (patient group CVI-A); IgM but no IgG (CVI-B), IgM and IgG (CVI-C). However, it is not clear whether the B cell defect in children with CVI follows the same pattern. Therefore, the extent of B cell defect was investigated in 8 children with CVI aged 5-6 years (4 males, 4 females). Lymphocytes (PBL) and purified B-cells were stimulated with SAC 0,001 % and the CD 40 system with increasing doses of IL-2 (10-1000 U), respectively. In addition, IL-2 and IL-6 secretion were measured by ELISA in supernatants of PBL and monocytes after incubation with various stimuli (PHA, CD3, PWM). Preliminary results confirm lower IL-2 production in CVI (n = 8): median 128 (range < 10-564) vs. controls (n= 10): median 610 (range 53-1464 pg/ml). However, IL-6 levels in unstimulated cultures were significantly enhanced in CVI: median 2661 (range 161-12404) compared to controls: median 252 (range < 100-6121 pg/ml). Though IL-2 levels were diminished in most children with CVI, in vitro immunglobulin secretion stimulated by SAC and CD40 could not be restored to normal even by increased doses of IL-2. Only 2 patients showed significant secretion of IgM and are classified as group B. This functional unresponsiveness to IL-2 might reflect a more severe disturbance of B-cell function in these children.