Workshop J Clinical Immunology

Abt. Rheumatologie u. Klinische Immunologie, Med. Universitatsklinik, 7S00 Freiburg, Germany

J.1 Immunophenotypical alterations in a subset of patients with common variable immunodeficiency (CVI)

ELISABETH BAUMERT, G. WOLFF-VORBECK, J. A. RUMP, ANGELIKAJAHREIS, M. SCHLESIER, and H. H. PETER

Common variable immunodeficiency is a heterogeneous primary immunodeficiency disease within which various pathogenetic mechanisms may be responsible for an insufficient immun­globulin production. In this study we investigated the expression of surface molecules on lymphocytes from 20 patients with CVI and 40 controls. Lymphocytes were analyzed by dual color flow cytometry. We identified a subset of patients (8 out of 20) characterized by low CD4/CDS-ratio « 1,1), expansion of T cells coexpressing CD57. Expression of the adhesion molecules LFA-3 (CD5S) and ICAM-l (CD 54) also was significantly increased in this subgroup. In addition, within the CD4+ T cells the percentage of CD29+ (<<memory») cells was increased, while the CD45 and LAM-l (LeuS) antigens were depressed. These results indicate that in a subgroup of CVI patients T cells are activated in vivo and the CD5r CDS+ lymphocyte subpopulation, supposed to comprise functional suppressor T cells, is expanded. We suggest a chronic viral infection in these patients, but it is not clear whether this assumed infection is a consequence of the defective immune system or whether a viral infection is - at least in some patients with CVI - responsible for the development of immunodeficiency. There was no correlation of the immunophenotypical alterations neither to clinical features nor to a classification of CVI patients on the basis of in vitro immunglobulin synthesis.

Institute of Immunology, Dept. of Internal Medicine, University, 8000 Munich, Germany

J.2 Measurement of circulation anti-endothelial-cell-antibodies (AECA) in vasculitis patients by flow-cytometry

C. BROCKMEYER, R. GRUBER, E. JESCHKE, G. SCHMID, E. Kopp, G. RIETHMULLER, and H. E. FEUCHT

The presence of AECA in the serum of patients with various vasculitic diseases, systemic sclerosis, multiple sclerosis and other demyelinating disorders has been reported. In these studies, antibodies were detected by complement dependent lysis, radioimmunoassay, and recently, by ELISA. Frequently, human umbilical endothelial cells (HUVEC) grown in gelatine coated tissue culture plates served as antigen and bovine serumalbumine (BSA), fetal

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calf serum or gelatine were used as blocking reagents. However, since high levels of serum antibodies against BSA and gelatine are a common finding in several immunological disorders, such systems seem to be inappropriate for the measurement of AECA. Therefore a protocol using flow cytometry without the need of blocking has been developed and studies were conducted to evaluate the specificity of this test system. Thus, high levels of AECA were detected in subgroups of patients with SLE or primary glomerulonephritis (GN), but not in forms of vasculitis unrelated to SLE or GN. In addition, it has been investigated, whether heat­inactivation of sera, or activation of HUVEC by cytokines would influence the test results. It appeared, that prior treatment of EC with TNF had no effect on the detection of AECA, whereas heat-inactivation of sera at 56°C for 30 min resulted in positive AECA in several vasculitis patients when no AECA were found in the native serum. This increment in detectable AECA seems to be related to the heat-induced formation of immunoglobulin­aggregates.

Diabetes Research Institute, University, 4000 Dusseldorf 1, Germany

J.3 Endothelial cell derived oxygen radicals may contribute to islet cell destruction in diabetes development

v. BURKART, H.-H. BRENNER, and H. KOLB

The pathogenesis of human type I (insulin-dependent) diabetes is characterized by a continuous loss of insulin-producing pancreatic beta-cells. In animal models of the disease it was shown, that cellular effector mechanisms essentially contribute to the beta-cells destruc­tion. On the other hand, islet cells have a reduced capacity to scavenge toxic oxygen radicals. Therefore we tested the susceptibility of islet cells against the toxic effects of reactive oxygen intermediates (ROIs) generated by xanthineoxidase (XO), an enzyme which is known to be associated with endothelial cells. The XO (0.025U/ml)/hypoxanthine (HX, 0.5 mM) system releases ROIs in vitro as detected by luminol (2 x 10-4M) and lucigenin (5 x 10-sM) in a chemiluminescence analyzing system. Islet cells labeled with -3H-Ieucin were lysed to 38 + 8 % during 15 h coincubation with the XO/HX system. Only background lysis was observed when islet cells were incubated with HX (0±3%) or XO (7±10%) alone. The addition of the radical scavenger nicotinamide (20 mM) inhibited the ROI-formation from XO/HX by 54 % and suppressed the specific lysis of islet cells by 61 %. Our results show that enzymatically produced ROIs are toxic for islet cells. We conclude that endothelial cells are involved in the destruction of islet cells via formation of ROIs.

Rheumaklinik, 2357 Bad Bramstedt, and Abt. fur Klinische Rheumatologie, Med. Universitat, 2400 Lubeck, Germany

J.4 Autoantibodies to lysosomal proteins in sera of patients with vasculitis and glomerulonephritis

BRIGITTE K. FLESCH, ELENA CSERNOK, MICHAEL LAMPE, and WOLFGANG L. GROSS

Recent data demonstrate the occurrence of a new class of autoantibodies (antineutrophil cytoplasmic autoantibodies - ANCA) against myeloid lysosomal proteins that are associated with vasculitis and glomerulonephritis. The aim of our study was to investigate the antigen

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specificities of pANCA (perinuclear ANCA) and atypical fluorescence and to evaluate the clinical significance of these autoantibodies. We tested 15324 sera from 9170 patients on alcohol fixed granulocytes by indirect immunofluorescence. 475 sera exhibited an atypical fluorescence pattern and were tested for the presence of autoantibodies directed against proteinase 3 (PR3), myeloperoxidase (MPO), elastase (HLE), lactoferrin (La), cathepsin G (CG) and lysozyme (Ly) using ELISA and immunoblotting.

Among these sera we found antibodies directed against MPO (16 %), PR3 (2 %), HLE (2%), CG (6%), La (4%), and Ly (4%). Antibodies against MPO were associated with rapidly progressive glomerulonephritis (RPGN) (47%) and microscopic polyarteritis (MPA) (40 %). Anti PR3 antibodies were restricted to vasculitis. Antibodies against HLE, CG, La, Ly were associated with vasculitic (WG, MPA, panarteritis nodosa), renal and collagen vascular diseases.

In contrast to PR3 and MPO autoantibodies the clinical significance of autoantibodies directed to other lysosomal proteins still needs to be investigated.

Institute of Diabetes «G. Katsch», 2201 Karlsburg, Germany

J.5 Rejection of allogeneic skins grafted into BB/OK rats: Effect of glycaemia

H. J. HAHN, J. KLOTING, and B. KUTTLER

The glucose metabolism of splenocytes is markedly altered in diabetes-prone BB rats. The BB rats are characterized by an immunedysregulation accompanied by a delayed allograft rejection. To examine if an altered glycaemic state modifies the immune response of BB/OK rats we investigated the survival of full-thickness BB.lA skin allografts in normoglycaemic as well as hyperglycaemic BB/OK rats. When normoglycaemic BB/OK rats were grafted the rejection occurs at 14.3±0.7 d (n = 10). A hyperglycaemia for 14d prolonged the graft survival to 20.2 ± 2.5 d in streptozotocin-diabetic and to 27.7 ± 3.7 d in spontaneously-diabetic recipients. When the skin grafts were performed in 12 weeks spontaneously diabetic BB/OK rats the graft survival was prolonged to > 80 d. The results demonstrated that the mean survival time of grafted skins depends on the glycaemic state. Hyperglycaemia suppressed the immune response already in 2 weeks diabetic animals and this effect is more pronounced in long-term diabetic recipients supporting the assumption, that the hyperglycaemic state exerts immunmodulating effect in BB/OK rats.

Institute of Diabetes, 2201 Karlsburg, and IBeriin-Chemie AG, Germany

J.6 Monoclonal antibody-based quantitative detection of porcine proinsulin in insulin preparations using the monoclonal antibody (MAB) SPI14D4

J. HAMANN!, M. STURM!, B. ZIEGLER, F. LUHDER, and M. ZIEGLER

Commercial porcine insulin preparations are contaminated with proinsulin. After using administration of insulin solutions these impurities are responsible for increased production of insulin antibodies in patients, which influence the bioreactivity of insulin. The aim of this study was to develop an immunoassay, which makes it possible to quantify specifically the

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proinsulin in insulin preparations. MAB were generated by hybridoma technique after immunization of BALB/c mice with porcine proinsulin. One hybridoma, SPI14D4, is producing an antibody which specifically binds to the proinsulin molecule. This antibody does not cross-react either with insulin or C-peptide. With this antibody we developed a competi­tive solid phase immuno-enzymetric assay. The detection limit of porcine proinsulin in an insulin solution of 20mg/ml amounts to 0.03 parts per million (ppm). The interassay variation coefficient amounts to 8.8 %. Surprisingly, the SPI14D4 shows a crossreactivity of 96 % with human proinsulin. This makes it possible to quantify human proinsulin with the same immunoassay in insulin preparations. Furthermore, this antibody is suitable to demonstrate the beta cell loss in type 1 diabetes.

! Abt. Klinische Immunologie, 2 Abt. Medizinische Psychologie, Medizinische Hochschule, 3000 Hannover 61, Germany

J.7 Stress induced alterations of cellular immunity

R. JACOBS!, G. STRATMANN!, M. SCHEDLOWSK!2, and R. E. SCHMIDT!

To investigate stress induced alterations of the immune system cellular parameters of 40 parachutists were studied. Blood samples were drawn from each jumper directly before parachute (= base line [= A J), immediately after [= BJ, and 1 Y, to 2 hours after landing [= Cl. White blood cell (WBC) differential counts, phenotypes of peripheral blood lymphocytes, natural killing (NK) activity, antibody dependent cellular cytotoxicity (ADCC), and PHA induced interleukin 2 (IL2) production were determined. There were significant differences in regard to all parameters of each parachutist's WBC sample. We observed an increase of WBC count (mean values for the three samples: A: 8,lX103/yl, B: 1l,2xl03/f,1l, C: 9,5X103/f,1l) often with an additional increase of lymphocytes after landing and a decrease in the last sample (mean of lymphocytes: A: 2,65x103/f,1l, B: 3,7X103/f,1l, C: 2,1 X 103/f,1l). In addition we detected considerable changes in the lymphocyte subsets of peripheral blood at the different time points. Corresponding to the NK cell increase characteristic differences in NK activity and ADCC were observed with an increase of activity in the second sample (B) and a decrease down below base line values in the last sample (C). A parallel development with maximal production of IL2 in the second specimens (B) was observed in PHA stimulated cells. Further studies will address the question whether these data merely reflect cell distribution phenomena or direct activation by hormones and neuropeptides.

Supported by Volkswagen Stiftung.

Med. Klinik, Klinikum Steglitz, 1000 Berlin 45, ! Chirurgische und 2Med. Klinik, Klinikum Rudolf Virchow/Charlottenburg, Freie Universitat, 1000 Berlin, Germany

J.8 Human mononuclear liver cells: Isolation and preliminary characterization

H. u. JAHN, R. ULLRICH, H. L. SCHIEFERDECKER, D. C. SCHMIDT, P. NEUHAUs2, U. HOPFI,

E. O. RIECKEN, and M. ZEITZ

Human mononuclear liver cells are still poorly characterized. Therefore, we developed a new method to isolate mononuclear cells from liver tissue obtained from explanted livers of

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patients with different liver diseases undergoing liver transplantation. Methods: Liver tissue was taken from one patient each with hepatitis B, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis and from normal liver, which could not be transplanted. About 30 g of tissue was taken from the freshly explanted liver, cut into small pieces, and washed with medium. Using a scalpel, liver tissue was cleared of connective tissue and minced. After washing, the liver tissue fragments were passed through a mesh, suspended in culture medium containing collagenase, trypsin inhibitor, and DNAse and were gently shaken in an incubator for three hours. From the resulting cell suspension mononuclear liver cells were obtained by a two step density gradient centrifugation. After washing, mononuclear cells were counted and tested for viability by trypan blue exclusion test, followed by a microscopical morphological examination. Mononuclear liver cells were stained with conjugated monoclonal antibodies and analysed with a fluorescence activated flow cytometer (FACScan). Following mitogen stimula­tion, proliferation was measured by 3H-thymidin incorporation. Results: Using this method, 3-12 x 106 mononuclear liver cells could be isolated from about 30 g of liver tissue. The viability was 2: 95 %. FACScan analysis revealed a CD4/CD8 ratio of < 0.5. In diseased liver, a high percentage of activated (HLA DR-positive) CD8-positive lymphocytes was found. 20 to 40 % of the cells expressed Leu-7 (natural killer cells). After mitogen stimulation for three days mononuclear liver cells had a proliferation index of about 10. Conclusions: Our newly developed method allows the isolation of viable mononuclear cells from human liver. Whether the phenotypical differences to peripheral blood lymphocytes are disease specific, or indicate specialized liver-associated function remains to be clarified.

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

J.9 TGFj3 overexpression in systemic vasculitis

J. KEKOW, C. SZYMKOWIAK, and W. L. GROSS

Wegener's Granulomatosis (WG) is characterized by systemic vasculitis, granuloma forma­tion, and vascular/tissue necrosis. Several cytokines have been found to be elevated, especially in acute, generalized WG, but also in polyarteritis nodosa (PAN). Inducers of vasculitis and granuloma may be, at least in part, Interleukin-l (IL-l) and TNF-u. In addition, a poor T- and B-cell response of mononuclear cells (MNC) can be observed despite signs of hyperreactivity of the immune system (e.g. hypergammaglobulinemia, high levels of soluble Interleukin 2 receptors). Recently we could detect high levels of transforming growth factor ~ (TGF~) in HIV infected individuals contributing to immune dysfunction (PNAS 87, 8321, 1990). On the basis of these findings we studied TGF~ expression in WG and PAN. Bioactive TGF~ was determined after acidification by the CLL64 assay in 24 h supernatants of MNC using a recombinant human TGF~ standard. Specificity was demonstrated by preincubation of samples with TGF neutralizing antibodies. The TGF~ 1 isoform was found to be elevated in patients with active, generalized WG (x ± sem in 5 cases with WG: 4070 ± 1730 vs control 390 ± 150 pg/ml, p < 0.05). 4 patients with PAN showed inconsistent TGF~ levels (minimax: 60-3800 pg/ml). Performing PCR for TGF~ 1 and 2 expression in MNC showed evidence for elevated mRNA levels. These data demonstrate the overexpression of the immunosuppressive cytokine TGF~ in WG, and to a lesser extent, in PAN. These findings may indicate, that TGF~ is involved especially in granuloma formation.

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Padiatrische Hamatologie u. Onkologie, Med. Hochschule, 3000 Hannover 61, Germany

J.l0 G-CSF production and G-CSF receptor expression in patients with severe congenital neutropenia

U. KYAS, T. PIETSCH, K. MEMPEL, P. BEENKEN, and K. WELTE

Severe congenital neutropenia (SCN) is characterized by a maturation arrest of neutrophil precursors on the level of promyelocytes. Our hypothesis on the pathophysiology of SCN includes a defective G-CSF production or a defective response to G-CSF. Our clinical trial includes 40 SCN patients. We investigated G-CSF production by monocytes and G-CSF receptor expression on neutrophils from SCN patients. G-CSF production was determined in NFS-60 bioassay and Western-blot analysis. G-CSF receptor expression was measured in binding assays using 125I-rhG-CSF and subsequent scatchard analysis. Monocytes from SCN patients were able to produce normal amounts of G-CSF. We found increased serum levels of G-CSF prior to (up to 0.9 ng/ml) and normal levels during G-CSF therapy. Produced G-CSF was biological active as could be demonstrated in the NFS-60 bioassay. Neutrophils from SCN patients during G-CSF therapy expressed elevated G-CSF receptor numbers (2000-3000/cell) as compared to neutrophils from healthy donors (800-1200/cell). The affinity of G-CSF to its receptor was similar in patients and controls. From this data we conclude that there is no defective G-CSF receptor expression and G-CSF production in SCN patients. However, these data do not exclude that there might be a defective signal transduction of the G-CSF receptor in SCN.

Stadtkrankenhaus, 5450 Neuwied, and Deutsches Krebsforschungszentrum, 6900 Heidelberg, Germany

J .11 Impaired interactions of immunocompetent cells in Whipple's Disease (W. D.)

T. MARTH, G. E. FEURLE, M. Raux, and S. C. MEUER

WD is a rare disorder in which almost all body compartments can be invaded by rod-like bacteria, partly free in tissue, partly in phagocytes. An impaired cell-mediated immune response has been previously postulated on the basis of skin testing, E-rosette counts, and T­cell proliferation test.

We now have examined cell mediated immunity in a larger group of patients (pt) with newer techniques. Proliferation, rosette forming, and expression of surface antigens using 17 mono­clonal antibodies were tested in 27 pt with WD and matched controls. 7 pt had florid WD (stage I), 6 pt were in clinical remission but had positive histology (stage II), and 14 pt were in remission (stage III).

The rosette-forming capacity and the response of the T-cells of the pt to PHA and SRBC were reduced when tested in pt-serum (p < 0.05). This suppression was partially reversed when control serum was used. There was an increased reaction when the alternative pathway of T-cell activation was stimulated (CD2 {T11IA + T11 2 } + CD2R-mAk; p < 0,05); the reac­tion of the TCR pathway (anti CD3-Sepharose) was not significantly altered. The expression of the cell adhesion molecule CDllb and the marker of naive T-cells CD45RA were reduced (p < 0.01, < 0.05 resp). These findings were dependent on the different clinical stages, i.e. the changes were most evident in stage 1.

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These data, therefore, revealed a soluble factor, present in patient-serum, inhibiting the interaction of antigen presenting cells with T-cells, particularly the CD2-LFA-3-mechanisms. This inhibition was reversed by antibodies with high affinity to the CD2 (T11 IA -mAk). The decreased number of CDllb positive cells indicates a reduced cellular adhaesion capacity to T -cells and obsonized bacteria.

Our findings indicate an impaired interaction between phagocytes and T-cells which may explain the reduced capacity of patients with WD to eliminate bacteria.

Forschungsinstitut Borste!, 2061 Borste!, and I Med. Kern- u. Poliklinik, Universitats­krankenhaus Eppendorf, 2000 Hamburg, Germany

J.12 CD26 expression in HIV patients

T. MATTERN, H. ALBRECHT!, H. J. STELLBRINK1, and A. J. ULMER

The CD26 antigen dipeptidyl peptidase IV (DPPIV) is known to be an activation marker of T lymphocytes. Beside its distribution on the membrane of T cells, CD26 is expressed in several mammalian tissues. Soluble forms of CD26 could be detected in body fluids like serum or urine. We have now investigated the levels of soluble CD26 in the plasma or the expression of membrane-bound CD26 on T cells and different T cell subtypes in patients with HIV infection. Soluble CD26 was measured by the specific hydrolysis of the DPPIV substrate Glycyl-Prolyl-p-Nitroanilide. In the plasma of 13 HIV patients in different states of infection and 18 healthy control persons we found equal amounts of DPPIV activity (patients: 162 ± 24 ng/ml, control group: 163 ± 14 ng/ml). Membrane-bound CD26 was determined in a Cyto­fluorograph by staining peripheral blood mononuclear cells (PMNC) with the monoclonal CD26 antibodies Tal (Coulter Clone) and TIl 19-4-7. We found that the total number of CD26 positive lymphocytes in HIV patients did not differ from the control group (patients: 16 ± 7 %, control group: 19 ± 8 % positive cells). Although the total number of CD26-positive cells is equal in both groups, there were differences in the distribution of CD26-positive cells in the T cell subpopulations. HIV patients had less CD4+CD26+ cells than the healthy control group, but more CD8+CD26+ and CDI6+CD26+ T cells. While the ratio of CD4+CD26 to CD8+CD26+ T cells reflects the reduced CD4/CD8 ratio in HIV patients, the enhanced number of CDI6+CD26+ NK cells seems to be the result of a preactivation of these cells by the human immunodeficiency virus itself or by opportunic infections.

This work was supported by BMFT, grant no. III-006-89.

University Hospital, Center of Internal Medicine, 6000 Frankfurt/Main 70, Germany

J.13 Profiles of CD26 positive peripheral blood cells and of DAP-IV in normal persons and patients with acquired immunodeficiency

A. NOCKHER, L. BERGMANN, and J. E. SCHERBERICH

In order to assess distribution profiles of peripheral blood cells expressing the CD26 epitope FACS analyses were performed in 11 normal controls and 23 patients with +HIV-serology. In addition, dipeptidylpeptidase-IV (DAP-IV; EC 3.4.14-) was analyzed in serum by an enzyme

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kinetic method. The following major data emerged: In normal controls 59 % of all CD4+ lymphocytes also expressed CD26 compared to 40 % in HIY+ patients (pts). In HIY+ pts. CD26/CD4+ cells decreased dramatically (ii. 71 % vs. 25,5 %); this decrease was in proportion with the drop of total CD4+ lymphocytes. CD26 was also coexpressed by CD4+ monocytesl macrophages, which were slightly reduced in HIY+ pts (57 % vs. 80 %, P < 0.05); however, cell counts and distribution profils of total CD4+ macrophages/monocytes in HIY+ pts. did not differ compared to those of healthy controls. Whereas granulocytes positive for CD26 were slightly reduced in HIY+ pts (ii. 80 % vs. 91 %) CD13+ granulocytes carrying the CD26 epitope remained unchanged (ii. 92 % vs. 92 %). DAP-IY activity of serum specimens showed no significant changes in HIY+ pts compared to controls, and DAP-IY enzyme activities were not correlated with the amount of CD26+ peripheral blood cells (PBC). The data indicate that 1.) not only lymphocytes but also macrophages/monocytes as well as granulocytes may express CD26 epitopes, which were initially detected on activated T-Iymphocytes 2.) CD26+ behave in a different manner in HIY+ patients 3.) DAP-IY, as a «soluble serumprotein», carrying the CD26 epitope is not related to the amount of CD26+-PBC.

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

J.14 Determination of soluble interleukin-2 receptor (sIL-2R) in Wegener's Granulomatosis (WG): Indicator ofT-cell activation

W. H. SCHMITT, C. HEESEN, A. RAUTMANN, and W. L. GROSS

The significance of cell-mediated immune reactivity for the pathogenesis of WG is unclear. Since granuloma in WG consist in part of T cells (mainly CD4+) we studied sIL-2RS in WG with the question, whether sIL-2R could be a useful marker of disease activity and indicate T cell activation. Using a sandwich-ELISA (T-cell sciencesR, USA), we determined sIL-2Rs in 102 patients (pat.) with biopsy proven WG. 37 pat. had an initiallocoregional form; 70 patients suffered from generalised WG. In each group, the sIL-2R levels of the active patients were significantly elevated compared with the inactive form (sIL-2R ini.-act.: x (median) = 640, ini.-inact.: x = 504, P < 0.05; gen.-act. x = 3240, gen.-inact.: x = 820, P < 0.001; control values: x = 420, all U/ml). Being in complete clinical remission, 7 pat. with generalised WG relapsed after 3 (1 to 6) months. Before the relapse their mean c-reactive protein (CRP) and cANCA levels were not increased in comparison with pat. in full remission (CRP <10 mg/l, cANCA titer < 1 :8), whereas sIL-2Rs were found to be elevated (x = 1200 U/ml). Later during relapse we observed a further rise in sIL-2R (x = 2550), now accompanied by increasing CRP levels (x = 30 mg/l) and cANCA titers (x = 1:32). Thus, sIL-2R determination may be an early indicator of a reactivation of the disease. In WG-patients, it can be difficult to distinguish intercurrent infection from relapse: In contrast to CRP, sIL-2Rs were only slightly influenced by infections. Furthermore, we studied 19 pat. with clinically active WG and low cANCA or CRP levels. SIL-2Rs (x = 1400) were significantly elevated thus confirming high disease activiry. In conclusion, elevated sIL-2Rs during complete remission suggest that immunologic alterations are present which are not detected by conventional measures. sIL-2R may be useful as a new marker of disease activity, especially when cANCA and CRP levels are still low. Continuous sIL-2R determination may indicate imminent relapse and may help to manage difficult WG-patients.

Supported by BMFT, grant 01 YM 8622.

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1 Institute of Immunology and Transfusion Medicine and Z Clinic of Geriatrics and Angiology, University Medical School, 2400 Lubeck, Germany

J.15 Investigations of the lymphokine system in elderly individuals

JURGEN SINDERMANNl, HANS-JOACHIM FRERCKS2, RUDOLF M. SCHUTZz, and HOLGER KIRCHNERl

Elderly people are at risk for an increased incidence of infections. We have studied the correlation between the production of interferon gamma (IFN-gamma), interferon alphaz (IFN-alphaz), interleukin-2 (IL-2), soluble interleukin-2 receptors (sIL-2R) and interleukin-6 (IL-6) in young (25-34 years old) and old (over 65 years) persons. In our institute we had the opportunity to obtain blood from 48 young and 45 former blood donors. Furthermore we took blood from 16 geriatric patients (> 65 years old). All persons were selected according to the basic concept of the «SENIEUR protocol». Heparinized venous blood was taken and cultured in a whole blood assay. For induction of IFN-gamma, IL-2, sIL-2R and IL-6, the cells were stimulated with pyhtohaemagglutinin (PHA Boroughs Wellcome), IFN-alphaz was induced by Newcastle Disease Virus (NDV). Supernatants were taken after 48 h for IL-2, 72 h for IFN-alphaz and 96h for IFN-gamma, sIL-2R and IL-6. All parameters were also investigated in unstimulated control cultures. The determination of these parameters was done by ELISA techniques. We found significantly decreased levels of sIL-2R and IFN-alphaz after stimulation whereas the values of IFN-gamma, IL-2 nd IL-6 showed no significant difference between elderly and young persons. Additionally there were no differences in the supernatants of the non-stimulated cultures. We also studied the lymphocyte subpopulations T4, T8 and NK-cells by flow cytometry after labelling with monoclonal antibodies. Elderly individuals showed a significantly increased T4/T8 ratio, caused by a decreased T8 level. These results show, that the elderly have decreased values of some innunological parameters that might be of relevance for the infections.

Institute of Immunology, Universitat, 6900 Heidelberg, and 1 Frauenklinik, 6800 Mannheim, Germany

J.16 yo T cell subsets in HIV infection

DANIELA WESCH, KLAUS FRIESE l, ANGELIKA HERM, and DIETER KABELITZ

The distribution of T cell subsets was determined in 61 HIV-infected individuals as well as in 30 HIV-negative control blood donors. Apart from the expected decline of CD4+ T cells, significant alteration of the yo T cell subset were observed in HIV+ individuals. Both the percentage and the absolute number of yo T cells were preserved, even in advanced clinical stages of HIV infection (CDC IV). More importantly, significant changes in the relative proportion of Vy9 and VI\1 subsets were noted in HIV+ donors. While Vy9+ (7 A5+) cells dominated among peripheral blood yO T cells in healthy adults, VOl+ (oTCSl+) yO T cells constituted the major yO population in HIV-infected individuals, thus giving rise to dramatic alterations of the Vy9IVOI ratio. These results suggest that VOl-expressing yO T cells might playa role during HIV infection. Current experiments are aimed at analyzing this question in some detail. To this end, V01+ cell lines are being established from HIV+ individuals. A potential effector function of these yo T cell lines will be tested against autologous CD4+ cells

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and EBV -transformed B cells, both in the absence or presence of recombinant HIV proteins. In addition, we attempt to selectively activate and expand yo T cells from HIV+ donors by stimulation with single anti-CD2 mAbs as recently described (WESSELBORG et aI., J. Exp. Med. 173,297, 1991).

I Klinik fur Chirurgie, Med. Universitat, 2400 Lubeck, and 2Institut fUr Anaesthesiologie, Universitat, 8700 Wurzburg, Germany

J.17 Predictive value of skin test response soon after multiple trauma as compared with routine clinical parameters

A. WOLTMANN1 and H. G. KRESS2

Impaired immune function has been assumed to be associated with the development of infection and sepsis in trauma victims. In the present study the predictive value of the cutaneous delayed type hypersensitivity (DTH) response was compared with routine clinical parameters. Using the Multitest® device, the DTH was sequentially tested at defined time intervals in 35 multiple trauma patients. The intensive care unit mortality rate in these patients was 23 %. Mortality significantly increased with sepsis. Interestingly the DTH response and the severity of trauma (assessed by the Injury Severity Score) did not show interdependence. Immediately after admission the DTH testing failed to correlate with either infection or the mortality rate as compared with routine clinical parameters: Sensitivity, specificity, positive and negative predictive value for fraction of inspiratory oxygen (Fi02), lactate, lymphocyte and promyelocyte count were all together higher than those for DTH response. Beginning on the 4th day after trauma, however, DTH scores below 5 mm defined a population with a high incidence of developing a clinical important septic episode. Consequently, FiOb lactate and white blood cell counts are early indicators of an impending poor outcome, whereas the skin test response is not. In the later course, the sequentially determined DTH testing may substantially contribute to the identification of multiple trauma patients at increased infectious and mortality risk.

Med. Klinik Borstel, 2061 Borstel, and I Nephrol. Abt., 1. Med. Universitatsklinik, Schittenhelmstr. 12,2300 Kiel 1, Germany

J.IS Deficiency of monokine secretion and changes in monocyte subsets in patients with renal failure

P. ZABEL, G. LEIMENSTOLL\ W. NIEDERMAYER\ and M. SCHLAAK

Immunodeficiency in uremic patients seems to be caused by defective monocyte functions. We, therefore, investigated the capacity of spontaneous and LPS-induced monokine secretion as well as monocyte subsets with a panel of monoclonal antibodies in patients with different stages of renal failure. We found both the spontaneous and LPS-induced interleukin-l (IL-l) and interleukin-6 (IL-6) secretion of isolated monocytes significantly reduced in dialysis patients as compared with normal controls, whereas secretion of tumor-necrosis-factor-alpha (TNF) was unchanged in both groups. Deficiency of IL-l and IL-6 secretion was associated

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with changes in monocyte subsets. Especially Ki-M8-, KiM5-, and LEU-M3-positive mono­cytes were significantly decreased in patients on dialysis. Interestingly, changes in monocyte subsets as well as defective monokine secretion were restored after renal transplantation. Deficiency of IL-l and IL-6 secretion was found to be stage-dependent in patients with renal failure and could only be demonstrated in patients with a serum creatinine> 6 mg/dl. Because of the stage-dependency and reversibility of these effects, we conclude that uremic toxins might be responsible for the defective monokine secretion. The correlation of defective monokine secretion with changes in monocyte subsets argues in favor of different functions of different monocyte subsets.

I. and III. Med. Div., Dept. of Pneumology, University, 6500 Mainz, Germany

J.19 Analyses ofT cell clones from different compartments of patients with sarcoidosis

G. ZISSEL, B. FLEISCHER, and J. MOLLER-QUERNHEIM

Sarcoidosis is a systemic granulomatous disorder characterized by an alveolar macrophage/ lymphocyte alveolitis. There is evidence that an unknown antigen elicits an inflammatory response in the alveolar space. We investigated in five patients with sarcoidosis and one patient with pulmonary tuberculosis (pTb) T-cells obtained from peripheral blood (PB), lung biopsy (LB) and bronchoalveolar lavage fluid (BAL) at a clonal level. The generated clones exhibited in three of five patients with sarcoidosis a variably increased proportion of T -cell-receptor (TCR) V~8+ and V~5+. These changes were not concordant in the three compartments examined. In pulmonary tuberculosis the percentage of V ~5+ clones derived from PB as well as from LB were substantially increased. The CD4/CD8 ratio of clones from LB varied from 0.5 to 8 and was significantly different from those of the BAL (2-29) but not from clones derived from the PB (0.2-6.3). We conclude that the putative antigen of sarcoidosis positively selects T-cell bearing V~5 and V~8 TCR. In spite of the systemic nature of the disease this process differs in the compartments evaluated.