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Workshop J Regional Immunity
WORKSHOP J
Regional Immunity
!Department of Clinical Immunology and 2Department of Dermatology, Medical School of Hannover, Hannover, Germany
J.1 Detection of MxA protein in leukocytes and skin of SLE patients A. N. AL-MASRI!, T. WERFEL 2, P. PANAGIOTOU!, and P. v. Wussow! The 78 kd intracellular MxA-protein is an excellent marker for detecting type-IInterferon activity in humans. SLE patients are known to chronically have a type-I Interferonemia. The cause of this Interferonemia is unknown. Lysed blood samples, blood smears and formaline-fixed and parafine-embedded tissue sections of skin biopsies from 12 patients with systemic lupus erythematosus were collected to investigate their MxA contant. Blood smears and tissue sections were stained with a specific monoclonal antibody employing the APAAP technique. In lysed blood MxA-protein was measured in a specific ELISA. From the twelve patients studied their leukocytes including granulocytes, monocytes and lymphocytes were MxA-positive at an antibody concentration of 0.5 ~g/ml. In contrast, blood leucocytes (except granulocytes) from healthy persons were MxA negative at a concentration of 0.5 f-lg/ml. Also epidermis and subcutaneous lymphocytic infiltrations from the 12 patients showed a positive staining for MxA (0.5 ~g/ml). Scin controls from cancer patients did reveal an almost negative staining. These data show that the MxA protein is expressed both in leukocytes and in skin of SLE patients and suggests an activation of the IFN system not only in blood, but also in the skin of lupus patients.
Federal Institute of Health Protection of Consumers and Veterinary Medicine,
Jena Branch, Jen a, Germany
J.2 Flow cytometric characterization of non-lymphoid cell populations in the bronchoalveolar lavage of pigs after immunization and infection with Pasteurella multocida (P.m.) A. BERNDT and G. MULLER By means of the flow cytometric light scattering and green own fluorescent characteristic, the following three non lymphoid cell populations of porcine bronchoalveolar lavage (BAL) were determined and investigated after aerogeneous immunization and intra-
Joint annual meeting 1995 OGAI/GFI . 193 tracheal infection with P. m.: 1. large high-fluorescent cells (LHC), 2. small highfluorescent cells (SHC), 3. small low-fluorescent cells (SLC). In comparison to the control animals the percentage of the LHC (65.0 ± 12.9 % vs. 38.2 ± 7.3 %) and SHC (28.2 ± 5.6 % vs. 13.9 ± 7.8 %) within the hole BAL macrophage population was decreased, whereas the SLC (8.3 ± 1.4 % vs. 21.7 ± 5.8 %) was significantly enhanced after infection. In order to investigate the phenotype of these cell populations monoclonal antibodies against porcine antigens (SWC1, SWC3a, MHC class II, 3A2, 2G6) in combination with FACS analysis were used. All three cell populations reacted with the SWC3a antibody (LHC: 96.5 ± 4.2 %; SHC: 96.0 ± 2.1 %; SLC: 80.4 ± 7.7 %) and showed no significant changes after immunization or infection. The SWC1 positive cells of LHC (18.2 ± 8.4 % vs. 53.3 ± 4.4 % vs. 75.1 ± 6.2 %) and SHC (7.7 ± 5.3 o/" vs. 55.8 ± 10.6 % vs. 77.7 ± 3.6 %) were significantly increased after immunization and infection. The binding of the MHC class II antibody was unchanged after immunization but decreased after infection (LHC: 96.5 ± 4.2 % vs. 95.4 ± 1.6 % vs. 48.7 ± 8.1 %; SHC: 94.3 ± 2.5 % vs. 90.2 ± 3.6 % vs. 58.7 ± 6.9 %; SLC: 38.7 ± 15.6 % vs. 43.8 ± 10.5 % vs. 12.0 ± 2.5 %). The number of 2G6 and 3A2-positive cells within the LHC (2G6: 97.3 ± 0.8 % vs. 92.7 ± 2.8 % vs. 33.2 ± 9.5 %; 3A2: 59.5 ± 2.3 % vs. 79.8 ± 11.8 % vs. 21.1 ±9.7%) and SHC (2G6: 96.5±2.6% vs. 89.5±10.4% vs. 51.2±1.5%; 3A2: 80.3 ±10.9% vs. 87.7 ±6.2% vs. 33.4±1.7%) were only reduced after infection while within the SLC (2G6: 4.5 ± 4.5 % vs. 35.5 ± 16.1 % vs. 6.1 ± 0.6 %; 3A2: 5.7 ± 3.4 % vs. 21.8 ± 10.4 % vs. 2.3 ± 0.5 %) the number of the 2G6 and 3A2 positive cells were increased after immunization but unchanged after infection.
j Institute of Virology and 2Institute of Pathology, Friedrich Schiller University J ena, Jena; 3Institute of Immunology, Philipps University Marburg, Marburg, Germany
J.3 Induction of cytokine gene expression by Coxsackie virus B3 (CVB3) in the acute phase of myocarditis in NMRI-mice B. GLDCK\ M. SCHMIDTKE 1,1. SCHUMANN \ U. GABLER 2, A. STELZNER \ and D. GEMSA 3 Coxsackievirus B3-induced myocarditis in mice was used as a model to investigate interrelationships between virus replication and development of acute and chronic enteroviral heart disease. The presence of heterogenous inflammatory cell populations in the hearts of infected mice implies the existence of a complex cytokine microenvironment, which may contribute to pathogenesis. In this study the pattern of cytokine gene expression was investigated in heart, spleen and thymus during the acute phase of myocarditis in NMRI-mice. Polymerase chain reaction (PCR) has been used to examine selective cytokine mRNA's in conjunction with CVB3-RNA kinetic. The detection of CVB3 active viral replication in heart and pancreas was observed between days 2 and 14 post infection (p.i.). CVB3 viral genomes (VP1 region) could be detected in heart, spleen and thymus tissue using PCR and seminested PCR corresponding to the TCID so titers. Restriction fragment pattern were as predicted and led to the correct identification of the PCR products. Histologically CVB3 myocarditis was manifested by inflammation of the myocardium characterized by infiltration of round cells and tissue necrosis during the first two weeks p.i. Changes in the connective tissue marked by interstitial fibrosis peaked on day 14. We found IL2 mRNA levels from day 4 p.i., but no IL4 mRNA was detectable, implying that the responding cells belong to the Th j subset of CD4+ T helper cells. ILl-
194 . Joint annual meeting 1995 OGAIIGFI
a, IFN-~ as common mediators of the immune systems were transcribed in heart and spleen tissue. These results demonstrate that cytokine regulation is important in determining the histopathological and immunological responses of mice to cardiotropic virus infection.
I Abteilung Gastroenterologie and 2 Abteilung Rheumatologie, Zentrum fiir Innere Medizin, Medizinische Hochschule Hannover, Hannover, Germany
J.4 Detection of a soluble form of P-selectin in sera of patients with inflammatory bowel disease and patients with rheumatoid arthritis
J. C. HOFFMANN2 , M. N. GOKE\ H. ZEIDLER2 ,J. EVERS\ andM. P. MANNS! The adhesion receptor P-selectin is expressed on activated endothelium and platelets. It mediates binding of neutrophiles to activated endothelium and is strongly expressed on platelets in chronic inflammation. In addition to the membrane anchored form there exists a secreted soluble form. The purpose of the present study was to measure sPselectin in sera of patients with inflammatory bowel disease (IBD) and in sera and synovial fluids of patients with rheumatoid arthritis (RA). sP-selectin was measured by a sandwich type ELISA in 64 patients with inflammatory bowel disease (Crohn's disease 41, ulcerative colitis 23), 29 patients with RA, and 22 healthy controls (HC). sP-selectin levels were correlated to standard clinical and biochemical markers of disease activity. Synovial fluid (SF) levels were determined in 10 of the 29 RA patients. Significantly increased sP-selectin levels were found for IBD patients (mean ± SEM: 381 ± 24.4 ng/ml) and for RA patients (449 ng/ml ± 62.9) as compared to He (251.5 ± 34.4 ng/ml) (p = 0.001 and p = 0.02). In contrast, sP-selectin levels in SF were significantly reduced compared to sera of HC or RA patients (each p < 0.001). No correlation could be found between sP-selectin and laboratory or clinical markers of disease activity for patients with IBD. A positive correlation was found between sPselectin and rheumatoid factor for patients with RA (r = 0.73, P = 0.001). The correlation between SF and serum sP-selectin levels was not significant (r = 0.55, P = 0.1). In conclusion, sP-selectin is increased in patients with IBD and patients with RA. Since significantly reduced levels were found in SF of RA patients elevated sP-selectin levels do not appear to originate from inflammed joints. Increased systemic sP-selectin might, however, compete with increased membranous P-selectin in order to limit inflammatory reactions.
Joint annual meeting 1995 OGAIIGFI
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Institute of Diabetes «G. Katscb Karlsburg, University of Greifswald, Greifswald, Germany
J.5 In vitro and in vivo Poly I: C-induced changes of antigen expression on pancreatic ~-cells B. KUTTLER and H. J. HAHN Aberrant expression of MHC class II antigen (MHC II) on ~ cells caused by cytokines is one of the hypotheses to explain ~ cell destruction. Since Poly I: C is known to accelerate overt diabetes possibly by stimulating cytokine release, we investigated in vitro and in vivo its effects on MHC antigen expression on pancreatic ~ cells. Isolated islets obtained from 8 to 12 d old BB/OK rats were cultivated in the presence of Poly I: C (10,100,1000 !!g/ml) for 4,7, 14, and 21 d. Furthermore, normoglycaemic diabetes-prone BB/OK rats (nBB/OK) were treated with Poly I: C (10 !!g/g b. w., 3 x weekly for 7, 14, and 21 d). Surface antigen expression was measured on single islet cells obtained from cultivated islets, untreated or Poly I: C-treated nBB/OK rats by FACS analysis using OX18 (MHC I), OX6 (MHC II), ICAM-1 (adhesion molecule) and K14D10 (f:l cell reactive). In vitro, Poly I: C up regulated both ICAM-1 on f:l cells and MHC I antigen density already after 4 d of culture in the presence of the lowest Poly I: C concentration. Up to 7 d a further increase was observed. After that (14, 21 d), antigen expression decreased, but was still significantly (p < 0.01) higher than on control cells. We never observed an increase of MHC II+ ~ cells, neither with 1000 !!g/ml PolyI: C nor after 21 d of culture. In vivo, treatment with Poly I: C enhanced MHC I antigen density and ICAM-1+ ~ cells significantly (p < 0.01), already after 7 d. A further increase was observed after 21 d. MHC II+ ~ cells increased significantly (p < 0.05) after 21 d of treatment. Conclusion: As shown in vitro, Poly I: C itself induces changes of MHC I and ICAM -1 expression on ~ cells, but has no effect on the MH C II expression. The observed increase of MHC II+ ~ cells after in vivo application is, therefore, not a direct effect, but a Poly I: C-mediated phenomenon.
1 Department of Dermatology, 2Institute for Immunology, 3Laboratory for Immunogenetics, and 4Max von Pettenkofer Institute, Ludwig Maximilians University, Munich, Germany
J.6 Clonal T cell expansion in skin lesions of psoriasis vulgaris indicates that an antigen-driven immune reaction provides the pathogenetic basis of psoriatic inflammation A. MENSSEN\ P. TROMMLER 1, S. VOLLMER 1, D. SCHENDEL 2, E. ALBERT3 , L. GORTLER\ G. RIETHMOLLER 2, G. PLEWICl, andJ. C. PRINZ 1
Psoriasis vulgaris is a chronic inflammatory, hyperproliferative skin disease. The pathogenetic basis is poorly understood but cellular immune responses are thought to be involved. To establish whether T lymphocytes that infiltrate lesional dermis and epidermis reflect an ongoing local immune response we analyzed the TCR-beta-chain variable region (V~) gene usage in chronic plaque-type psoriasis. By semiquantitative PCR we could show an elevated expression of V~6 and/or V~2 gene families in the plaques in
196 . Joint annual meeting 1995 OGAI/GFI comparison to blood lymphocytes of these patients. Nucleotide sequence analysis of the complementarity-determining region 3 (CDR3) revealed that the overexpression corresponded to a striking oligoclonality. Furthermore we found common motifs among different patients in the amino acid sequences of the CDR 3 regions. Biopsies taken up to two years later at different anatomical sites from the same patients still contained the predominant clonotypes in both dermal and epidermal compartments. These results suggest that the pathogenesis of psoriasis vulgaris crucially involves a restricted T cell response that is directed against a common antigen in the skin of psoriasis patients.
Max Planck Institute for Psychiatry, Neuroimmunology, Martinsried, Germany
J.7 MHC class II induction on microglial cells is controlled by sodium-dependent neuronal activity T. MISGELD, H. NEUMANN, and H. WEKERLE Intact brain tissue does not show MHC class II antigen expression. In contrast, under a variety of pathological conditions, MHC class II antigen is found within the central nervous system. MHC class II is most readily inducible in microglia cells, but is also noted in astrocytes and brain endothelial cells. We studied the expression of MHC class II antigens on microglial cells in organotypic hippocampal slice cultures of newborn Lewis rats. We used semiquantitative image analysis and confocal laser microscopy of whole immuno-stained slice cultures. After one week of culture the hippocampal slices exhibited good neuronal differentiation. In the absence of stimulation, only very few scattered MHC class II positive microglial cells were found. Stimulation with IFN-y resulted in strong induction of MHC class II antigens on microglial cells in the injured superficial areas of the slice cultures. In the central parts of the slices containing most intact neurons, only very low levels of MHC class II immunoreactivity were detected. After blocking of sodium-dependent neuronal activity by tetrodotoxin (TTX) IFN-y-inducible MHC class II antigen levels on microglial cells were raised, however, without reaching the levels found in the areas depleted of active neuronal elements. Our results indicate, (i) that electrically active neurons suppress inducibility of MHC class II genes in microglial cells; (ii) that this suppression can be neutralized by TTXblockage of neuronal activity, and (iii) that, however, additional factors - probably associated with neuronal degeneration - are required to reach maximal expression levels in microglial cells.
Joint annual meeting 1995 OGAI/GFI . 197 Research Center for Infectious Diseases, University of Wiirzburg, W iirzburg; 1Institute of Clinical Microbiology and Immunology, University of Erlangen-Niirnberg, Erlangen, Germany
J.8 Inducible nitric oxide synthase is not expressed in murine epidermal Langerhans cells H. MOLL, C. BOGDAN, 1 and C. BLANK In Leishmania-infected macrophages, the formation of nitrogen oxides is critical for intracellular killing of the parasites. Their production is mediated by the inducible nitric oxide synthase (iNOS). We have recently shown that, in addition to macrophages, epidermal Langerhans cells can phagocytose L. major. It was therefore of interest to analyze whether Langerhans cells display the same leishmanicidal effector mechanism. For this purpose, pure Langerhans cells were collected, using single-cell picking, and were infected with L. major and/or activated with different cytokines in the presence or absence of LPS in vitro. Subsequently, the cells were analyzed for expression of iNOS mRNA using RT-PCR. In contrast to macrophages, Langerhans cells did not express iNOS mRNA. On the other hand, significant levels of iNOS mRNA could be detected in unselected epidermal cells, the majority of which consists of keratinocytes. These results indicate that in the L. major-infected skin, activated macrophages and keratinocytes, but not Langerhans cells have the ability to express iNOS activity. The implications of this finding for the interaction of Langerhans cells with intracellular pathogens will be discussed.
Departments of 1 Clinical Immunology and 2Dermatology, Medical School of Hannover, Hannover; 3Department of Rheumatology and Clinical Immunology, Freiburg, Germany
J.9 Elevated MxA level in SLE, but not in systemic skleroderma, M. Wegener or cryoglobulinemia P. PANAGIOTOUl, A. N. AL-MASRIl, T. WERrEL2, A. KAPp2, H. H. PETER 3, R. E. SCHMIDTi, and P. v. Wussow i MxA is an IFN type I-induced protein. Its increased level in leukocytes indicates the presence of biologically active IFN in the circulation. Blood MxA level of 51 patients with SLE, 13 patients with skleroderma, 11 patients with Wegener's Granulomatosis and 10 patients with cryoglobulinemia were determined in a MxA ELISA employing specific monoclonal antibodies. 45 of 51 SLE patients had MxA level above 4 m U / 1000 L. The remaining six patients with normal MxA level «3 mU / 1000L) had a completely inactive disease. In SLE disease activity and blood MxA level significantly correlated at a p-value of 0.01. In contrast, 9 of 11 patients with skleroderma had normal MxA level «3 mU / 1000L). The remaining two patients showed elevated MxA level, but also signs of a clinically relevant vasculitis. Furthermore, in the absence of infectious complications all patients with Wegener's disease did not possess elevated MxA level. Six of these patients had active disease with lung infiltrates (n=4) and nephritis (n=3); one patient showed
198 . Joint annual meeting 1995 OGAI/GFI significantly elevated MxA level only during a febrile episode indicating that these patients can produce MxA. Also patients with cryoglobulinemia (n = 10) did not show elevated MxA level. From some of the above mentioned patients skin biopsies were taken for MxA immunostaining. Only in SLE-, but not in skleroderma or cryoglobulinemia the skin tissue showed an intensive MxA staining. Not only the epidermis, but also lymphocytic infiltrations in the subcutis showed an intensive MxA staining in these specimen. We conclude that the IFN system is activated in SLE, but not in the other autoimmune diseases investigated. Furthermore, not only leukocytes in the blood stream, but also keratinocytes and other cells in the skin express the MxA protein.
Institute of Immunology and Transfusion Medicine, Medical School, University of Lubeck, Lubeck; 1Department of Internal Medicine, University of Rostock, Rostock, Germany
J.10 Impaired local immunity in inflammatory bowel diseases M. SEYI'ARTH, M. KNOCHE, K. KISSING, andJ. EMMRICH There are not enough informations about the local immunity in inflammatory bowel diseases. We are interested in both humoral and cell-mediated immunity. We investigated 9 pat. with Colitis ulcerosa (CU), 8 pat. with Crohn's disease (CD) and 23 controls for their local immune reactivity only in not affected areas of colon mucosa. The S-IgA-concentration (humoral immunity) was lower in patients with UC and CD than in controls (p < 0.05). In controls we found the highest values of S-IgA in the colon transversum. There was neither age nor sex differences. Lamina propria lymphocytes of the patients differed in their cytokine secretion pattern. We found increased levels of interleukin (IL) 6 in the unstimulated supernatants of UC and CD patients and increased levels of IL 1~ in UC, whereas CD pat. and controls produce only small amounts of IL 1~. No significant differences could be shown in the TNFa production. It was possible also to inhibit the IL 6 and TNFa production by specific antibodies. The results shown suggest that T cells, in particular, may be involved in the disorders in both local humoral and local cell-mediated immunity.
Department of Urology and ENT, University of Vienna, Vienna, Austria
J.11 Increased expression of interferon-y in benign prostatic hyperplasia G. E. STEINER, B. KNERER, H. MARTINEK, G. KRAMER, and M. MARBERGER Previously we demonstrated that benign prostatic hyperplasia (BPH) is frequently associated with lymphocytic infiltration. This observation suggested to us that the close contact of prostatic stromal cells (PSC) with activated lymphocytes might modify their cytokine responsiveness. Therefore normal (n=2), BPH (n=5) and BPH-PSC clones
Joint annual meeting 1995 OGAI/GFI . 199 (n = 11) were established and stimulated with various cytokines. In summary, normal PSC revealed no proliferative response to IL-2, -7, a slight response to IFN-y, and dramatic inhibition induced by IL-4 and TNF-u. By contrast, BPH-PSC lines consistently showed hyperresponsiveness when stimulated with IL-2, -7 and IFN-y (135-147%). Analyses regarding the heterogeneity of PSC in BPH using PSC clones revealed that 7 of those 11 clones exhibiting rapid proliferation had an even greater responsiveness to IFN-y and IL-2 compared to the polyclonal mother line. Therefore normal, BPH and cancer tissues were screened for anti-IL2 and -IFN-y reactivity by immunohistology revealing no staining for IL-2 and intense reactivity with the anti-IFNy mAb. The specificity was confirmed by nonsense isotype control mAb as well as dosedependent inhibition with rIFN-y. To analyze if IFN-y is produced intra-prostatically, total RNA was prepared from all tissues, reverse transcribed into c-DNA and PCR amplification was performed using IL-2 and IFN-y-specific primers. The specificity of PCR reaction was confirmed by Southern blotting and hybridization with internal either IFN-y and IL-2-specific oligos. Similar to the staining results both normal and the 5 malign prostatic tissues, although clearly positive, exhibit much less IFN-y message when compared to the amount expressed in BPH tissue whereas probing for IL-2 revealed completely negative results.
Department of Internal Medicine I, University Medical Center Regensburg, Regensburg, Germany
J.12 Endogenous norepinephrine and opioids control splenic IL-6 secretion in mice R. H. STRAUB, M. HERRMANN, G. BERKMILLER, W. FALK,]. SCHOLMERICH, andB. LANG In a previous study we demonstrated a newly developed technique which allows the investigation of nerve-immune cell interaction in spleen (1). To study further aspects of this neuronal-immune interplay, effects of propranolol and opioid antagonists were investigated. The ~-adrenergic antagonist propranolol decreased the electrically induced inhibition of IL-6 secretion in bacteria-containing medium but increased the electric effect in bacteria-free medium (for the difference p = 0.0023). The opioid antagonist naloxone decreased the electrically induced inhibition of IL-6 secretion in a medium irrespective of the presence of bacteria (bell-shaped curve with Emax at 10-6 M, p=0.0046). Further investigation using the ~-specific opioid receptor antagonist cyprodime hydro bromide revealed involvement of ~-opioid receptors (bell-shaped curve with Emax at 10-8 M, p=0.0014). The 6-opioid receptor antagonist ICI 174,864 was not able to decrease the electrically induced inhibition of IL-6 but showed a trend to increase the electric effect (p = 0.1422). In conclusion, these results demonstrate a synergistic effect of norepinephrine and enkephalins to inhibit the splenic IL-6 secretion in bacteriacontaining medium. However, in bacteria-free medium the colocalized transmitters had opposing effects on splenic IL-6 secretion. Thus, during stress due to septicemia both colocalized transmitters of the sympathetic nervous system feedback IL-6 secretion to dampen the inflammatory response whereas during stress aseptic conditions no such increased attenuation of IL-6 secretion exists. 1. STRAUB, R. H. et al. 1995. ]. Neuroimmunology, in press.
200 . Joint annual meeting 1995 OGAIIGFI Institute of Immunology and 1Lung Diseases and Tuberculosis Hospital, Zagreb, Croatia
J.13 C038 antigen on bronchoalveolar (BAL) T lymphocytes correlate with activation status in interstitial pulmonary diseases (ILO) I. SVOBODA BEUSAN 1, R. AGBABA PRIMORAC, D. GOMERCld, and Z. PRAHIN 1 As shown in our previous OGAI reports, CD38 antigen plays a relevant role in sarcoidosis. In this study we were interested in evaluating the role of CD38 in the pathogenesis of other ILD. BAL was obtained from patients with sarcoidosis'f (n=31), fibrosis':":' (n = 8) and controls':":":' (n = 14). The CD38+ T cell levels was assesed by double immunofluorescence staining and analyzed by FACS. Total CD38 was highly elevated in AS (40 ± 20 %), in comparison to IS, F and C samples (= 20 %). In AS CD38 is predominantly expressed on CD4 (:S 70 %), whereas in IS and F CD38 is equally expressed on CD4 and CD8 lymphocytes (= 30 %). T subset analysis revealed that CD4+CD38+ cells were highly elevated in AS (29 ± 17 %) as compared to IS (5 ± 3) and F (6 ± 4), whereas CD8+CD38+ were generally low. Eight patients underwent 2-6 repeated lavages and this follow-up study indicated that CD4+CD38+ are elevated on the onset of the disease, decreased after therapy and increased again in relapse of the disease. Steroid therapy slowly increase CD8+CD38+ up to 10 %. As local CD4+CD38+ expansion correlates with the onset of alveoli tis. Erythrema nodosum and disease progression, our results indicate that this subpopulation may playa relevant role in ILD and could serve as an indicator of disease activity. ':. active and inactive sarcoidosis (AS and IS, respectively); ,:.,:. fibrosis (F); ,:.,:.,:. control (C)
Department of Clinical Immunology, Medical School of Hannover, Hannover, Germany
J.14 Elevated blood MxA level correlate with disease activity in patients with SLE and Sharp syndrome P. v. Wussow, A. N. AL-MASRI, P. PANAGIOTOU, U. NOLTE, and R. E. SCHMIDT MxA is an IFN type I-induced protein. Its increased level in leukocytes indicates the presence of biologically active IFN in the circulation. Blood MxA level and disease activity were monitored in 51 patients with systemic lupus erythematosus and 12 patients with Sharp syndrome over two years. The patients investigated were categorized into three groups: patients with a completely inactive, with a moderately active or an exacerbated disease. In addition, the SLE patients were classified according to the SLEDAI index. 6 patients had a completely inactive disease and normal MxA level « 3 mU / 1000 L). In contrast, all patients with minimally or moderately active disease (n=31) showed elevated MxA level between 4.2 and 23 mU /1000 L. Finally 14 patients experienced an exacerbation of their disease in the observation period. Their MxA level ranged between 12 and 48 mU /1000 L. In all patients studied disease activity as assessed by the SLEDAI correlated with the blood MxA level at a value of p = 0.Q1.
Joint annual meeting 1995 OGAI/GFI . 201 Prednisolone treatment in the 14 patients with exacerbation led to drastic MxA level reductions within 1-2 months (mean 18.3 to 4.1 mU /1000 L). Upon lowering the steroid dosis six patients who experienced an early relapse of their symptoms had a significant increase of their MxA level to almost pretherapy values (mean 4.6 to 12.5 mU / 1000 L). In contrast, patients with a sustained response to prednisolone showed only moderate elevations of their blood MxA level (mean 3.3 to 6.7 mU 11000 L). In summary, the elevated MxA level in both SLE and Sharp syndrome demonstrate the activation of the IFN-system in these patients. Apparently, MxA level correlate with disease activity in such patients. Specifically, blood MxA level are probably capable of identifying patients with a completely inactive disease.
Basel Institute for Immunology, Basel, Germany
J.1S The peripheral blood is not representative of the recirculating immune system A. J. YOUNG, W. L. MARSTON, L. DUDLER, and W. R. HEIN Lymphocytes continually traffic between the blood and the lymph in vivo. This process is absolutely required for adequate immune surveillance, and the dissemination for immunological memory. This migration does not appear to occur randomly, but it has been shown that lymphocytes will traffic specifically through different tissues. Although this specificity has been known for some time, the relative contribution of the peripheral blood to the recirculating pool has not been addressed. The experiments reported here have directly examined the migratory properties of peripheral blood lymphocytes (PBLs). PBLs were labeled in vitro and reinjected intravenously in sheep. Lymph was collected from a variety of tissues, as well as peripheral blood, and analyzed for the concentration of labeled cells. Despite the large numbers of lymphocytes known to migrate through intestinal tissues, labeled PBLs were detected in greater concentrations in subcutaneous efferent lymph. Peak recovery of labeled cells in lymph occurred 24 hours after reinjection. Surprisingly, the concentration of labeled lymphocytes found in peripheral blood was significantly higher than that found in lymph. This is the reverse of what is observed when lymphocytes collected from lymph are labeled. These data suggest that the peripheral blood is not representative of the mature recirculating lymphocyte pool, and that a large proportion of peripheral blood lymphocytes do not recirculate under normal conditions. Because of the importance of lymphocyte recirculation in the physiology of immunological surveillance and memory, these data suggest that the peripheral blood is not an effective site to measure overall immune competence.
Regional Immunity
!Department of Clinical Immunology and 2Department of Dermatology, Medical School of Hannover, Hannover, Germany
J.1 Detection of MxA protein in leukocytes and skin of SLE patients A. N. AL-MASRI!, T. WERFEL 2, P. PANAGIOTOU!, and P. v. Wussow! The 78 kd intracellular MxA-protein is an excellent marker for detecting type-IInterferon activity in humans. SLE patients are known to chronically have a type-I Interferonemia. The cause of this Interferonemia is unknown. Lysed blood samples, blood smears and formaline-fixed and parafine-embedded tissue sections of skin biopsies from 12 patients with systemic lupus erythematosus were collected to investigate their MxA contant. Blood smears and tissue sections were stained with a specific monoclonal antibody employing the APAAP technique. In lysed blood MxA-protein was measured in a specific ELISA. From the twelve patients studied their leukocytes including granulocytes, monocytes and lymphocytes were MxA-positive at an antibody concentration of 0.5 ~g/ml. In contrast, blood leucocytes (except granulocytes) from healthy persons were MxA negative at a concentration of 0.5 f-lg/ml. Also epidermis and subcutaneous lymphocytic infiltrations from the 12 patients showed a positive staining for MxA (0.5 ~g/ml). Scin controls from cancer patients did reveal an almost negative staining. These data show that the MxA protein is expressed both in leukocytes and in skin of SLE patients and suggests an activation of the IFN system not only in blood, but also in the skin of lupus patients.
Federal Institute of Health Protection of Consumers and Veterinary Medicine,
Jena Branch, Jen a, Germany
J.2 Flow cytometric characterization of non-lymphoid cell populations in the bronchoalveolar lavage of pigs after immunization and infection with Pasteurella multocida (P.m.) A. BERNDT and G. MULLER By means of the flow cytometric light scattering and green own fluorescent characteristic, the following three non lymphoid cell populations of porcine bronchoalveolar lavage (BAL) were determined and investigated after aerogeneous immunization and intra-
Joint annual meeting 1995 OGAI/GFI . 193 tracheal infection with P. m.: 1. large high-fluorescent cells (LHC), 2. small highfluorescent cells (SHC), 3. small low-fluorescent cells (SLC). In comparison to the control animals the percentage of the LHC (65.0 ± 12.9 % vs. 38.2 ± 7.3 %) and SHC (28.2 ± 5.6 % vs. 13.9 ± 7.8 %) within the hole BAL macrophage population was decreased, whereas the SLC (8.3 ± 1.4 % vs. 21.7 ± 5.8 %) was significantly enhanced after infection. In order to investigate the phenotype of these cell populations monoclonal antibodies against porcine antigens (SWC1, SWC3a, MHC class II, 3A2, 2G6) in combination with FACS analysis were used. All three cell populations reacted with the SWC3a antibody (LHC: 96.5 ± 4.2 %; SHC: 96.0 ± 2.1 %; SLC: 80.4 ± 7.7 %) and showed no significant changes after immunization or infection. The SWC1 positive cells of LHC (18.2 ± 8.4 % vs. 53.3 ± 4.4 % vs. 75.1 ± 6.2 %) and SHC (7.7 ± 5.3 o/" vs. 55.8 ± 10.6 % vs. 77.7 ± 3.6 %) were significantly increased after immunization and infection. The binding of the MHC class II antibody was unchanged after immunization but decreased after infection (LHC: 96.5 ± 4.2 % vs. 95.4 ± 1.6 % vs. 48.7 ± 8.1 %; SHC: 94.3 ± 2.5 % vs. 90.2 ± 3.6 % vs. 58.7 ± 6.9 %; SLC: 38.7 ± 15.6 % vs. 43.8 ± 10.5 % vs. 12.0 ± 2.5 %). The number of 2G6 and 3A2-positive cells within the LHC (2G6: 97.3 ± 0.8 % vs. 92.7 ± 2.8 % vs. 33.2 ± 9.5 %; 3A2: 59.5 ± 2.3 % vs. 79.8 ± 11.8 % vs. 21.1 ±9.7%) and SHC (2G6: 96.5±2.6% vs. 89.5±10.4% vs. 51.2±1.5%; 3A2: 80.3 ±10.9% vs. 87.7 ±6.2% vs. 33.4±1.7%) were only reduced after infection while within the SLC (2G6: 4.5 ± 4.5 % vs. 35.5 ± 16.1 % vs. 6.1 ± 0.6 %; 3A2: 5.7 ± 3.4 % vs. 21.8 ± 10.4 % vs. 2.3 ± 0.5 %) the number of the 2G6 and 3A2 positive cells were increased after immunization but unchanged after infection.
j Institute of Virology and 2Institute of Pathology, Friedrich Schiller University J ena, Jena; 3Institute of Immunology, Philipps University Marburg, Marburg, Germany
J.3 Induction of cytokine gene expression by Coxsackie virus B3 (CVB3) in the acute phase of myocarditis in NMRI-mice B. GLDCK\ M. SCHMIDTKE 1,1. SCHUMANN \ U. GABLER 2, A. STELZNER \ and D. GEMSA 3 Coxsackievirus B3-induced myocarditis in mice was used as a model to investigate interrelationships between virus replication and development of acute and chronic enteroviral heart disease. The presence of heterogenous inflammatory cell populations in the hearts of infected mice implies the existence of a complex cytokine microenvironment, which may contribute to pathogenesis. In this study the pattern of cytokine gene expression was investigated in heart, spleen and thymus during the acute phase of myocarditis in NMRI-mice. Polymerase chain reaction (PCR) has been used to examine selective cytokine mRNA's in conjunction with CVB3-RNA kinetic. The detection of CVB3 active viral replication in heart and pancreas was observed between days 2 and 14 post infection (p.i.). CVB3 viral genomes (VP1 region) could be detected in heart, spleen and thymus tissue using PCR and seminested PCR corresponding to the TCID so titers. Restriction fragment pattern were as predicted and led to the correct identification of the PCR products. Histologically CVB3 myocarditis was manifested by inflammation of the myocardium characterized by infiltration of round cells and tissue necrosis during the first two weeks p.i. Changes in the connective tissue marked by interstitial fibrosis peaked on day 14. We found IL2 mRNA levels from day 4 p.i., but no IL4 mRNA was detectable, implying that the responding cells belong to the Th j subset of CD4+ T helper cells. ILl-
194 . Joint annual meeting 1995 OGAIIGFI
a, IFN-~ as common mediators of the immune systems were transcribed in heart and spleen tissue. These results demonstrate that cytokine regulation is important in determining the histopathological and immunological responses of mice to cardiotropic virus infection.
I Abteilung Gastroenterologie and 2 Abteilung Rheumatologie, Zentrum fiir Innere Medizin, Medizinische Hochschule Hannover, Hannover, Germany
J.4 Detection of a soluble form of P-selectin in sera of patients with inflammatory bowel disease and patients with rheumatoid arthritis
J. C. HOFFMANN2 , M. N. GOKE\ H. ZEIDLER2 ,J. EVERS\ andM. P. MANNS! The adhesion receptor P-selectin is expressed on activated endothelium and platelets. It mediates binding of neutrophiles to activated endothelium and is strongly expressed on platelets in chronic inflammation. In addition to the membrane anchored form there exists a secreted soluble form. The purpose of the present study was to measure sPselectin in sera of patients with inflammatory bowel disease (IBD) and in sera and synovial fluids of patients with rheumatoid arthritis (RA). sP-selectin was measured by a sandwich type ELISA in 64 patients with inflammatory bowel disease (Crohn's disease 41, ulcerative colitis 23), 29 patients with RA, and 22 healthy controls (HC). sP-selectin levels were correlated to standard clinical and biochemical markers of disease activity. Synovial fluid (SF) levels were determined in 10 of the 29 RA patients. Significantly increased sP-selectin levels were found for IBD patients (mean ± SEM: 381 ± 24.4 ng/ml) and for RA patients (449 ng/ml ± 62.9) as compared to He (251.5 ± 34.4 ng/ml) (p = 0.001 and p = 0.02). In contrast, sP-selectin levels in SF were significantly reduced compared to sera of HC or RA patients (each p < 0.001). No correlation could be found between sP-selectin and laboratory or clinical markers of disease activity for patients with IBD. A positive correlation was found between sPselectin and rheumatoid factor for patients with RA (r = 0.73, P = 0.001). The correlation between SF and serum sP-selectin levels was not significant (r = 0.55, P = 0.1). In conclusion, sP-selectin is increased in patients with IBD and patients with RA. Since significantly reduced levels were found in SF of RA patients elevated sP-selectin levels do not appear to originate from inflammed joints. Increased systemic sP-selectin might, however, compete with increased membranous P-selectin in order to limit inflammatory reactions.
Joint annual meeting 1995 OGAIIGFI
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Institute of Diabetes «G. Katscb Karlsburg, University of Greifswald, Greifswald, Germany
J.5 In vitro and in vivo Poly I: C-induced changes of antigen expression on pancreatic ~-cells B. KUTTLER and H. J. HAHN Aberrant expression of MHC class II antigen (MHC II) on ~ cells caused by cytokines is one of the hypotheses to explain ~ cell destruction. Since Poly I: C is known to accelerate overt diabetes possibly by stimulating cytokine release, we investigated in vitro and in vivo its effects on MHC antigen expression on pancreatic ~ cells. Isolated islets obtained from 8 to 12 d old BB/OK rats were cultivated in the presence of Poly I: C (10,100,1000 !!g/ml) for 4,7, 14, and 21 d. Furthermore, normoglycaemic diabetes-prone BB/OK rats (nBB/OK) were treated with Poly I: C (10 !!g/g b. w., 3 x weekly for 7, 14, and 21 d). Surface antigen expression was measured on single islet cells obtained from cultivated islets, untreated or Poly I: C-treated nBB/OK rats by FACS analysis using OX18 (MHC I), OX6 (MHC II), ICAM-1 (adhesion molecule) and K14D10 (f:l cell reactive). In vitro, Poly I: C up regulated both ICAM-1 on f:l cells and MHC I antigen density already after 4 d of culture in the presence of the lowest Poly I: C concentration. Up to 7 d a further increase was observed. After that (14, 21 d), antigen expression decreased, but was still significantly (p < 0.01) higher than on control cells. We never observed an increase of MHC II+ ~ cells, neither with 1000 !!g/ml PolyI: C nor after 21 d of culture. In vivo, treatment with Poly I: C enhanced MHC I antigen density and ICAM-1+ ~ cells significantly (p < 0.01), already after 7 d. A further increase was observed after 21 d. MHC II+ ~ cells increased significantly (p < 0.05) after 21 d of treatment. Conclusion: As shown in vitro, Poly I: C itself induces changes of MHC I and ICAM -1 expression on ~ cells, but has no effect on the MH C II expression. The observed increase of MHC II+ ~ cells after in vivo application is, therefore, not a direct effect, but a Poly I: C-mediated phenomenon.
1 Department of Dermatology, 2Institute for Immunology, 3Laboratory for Immunogenetics, and 4Max von Pettenkofer Institute, Ludwig Maximilians University, Munich, Germany
J.6 Clonal T cell expansion in skin lesions of psoriasis vulgaris indicates that an antigen-driven immune reaction provides the pathogenetic basis of psoriatic inflammation A. MENSSEN\ P. TROMMLER 1, S. VOLLMER 1, D. SCHENDEL 2, E. ALBERT3 , L. GORTLER\ G. RIETHMOLLER 2, G. PLEWICl, andJ. C. PRINZ 1
Psoriasis vulgaris is a chronic inflammatory, hyperproliferative skin disease. The pathogenetic basis is poorly understood but cellular immune responses are thought to be involved. To establish whether T lymphocytes that infiltrate lesional dermis and epidermis reflect an ongoing local immune response we analyzed the TCR-beta-chain variable region (V~) gene usage in chronic plaque-type psoriasis. By semiquantitative PCR we could show an elevated expression of V~6 and/or V~2 gene families in the plaques in
196 . Joint annual meeting 1995 OGAI/GFI comparison to blood lymphocytes of these patients. Nucleotide sequence analysis of the complementarity-determining region 3 (CDR3) revealed that the overexpression corresponded to a striking oligoclonality. Furthermore we found common motifs among different patients in the amino acid sequences of the CDR 3 regions. Biopsies taken up to two years later at different anatomical sites from the same patients still contained the predominant clonotypes in both dermal and epidermal compartments. These results suggest that the pathogenesis of psoriasis vulgaris crucially involves a restricted T cell response that is directed against a common antigen in the skin of psoriasis patients.
Max Planck Institute for Psychiatry, Neuroimmunology, Martinsried, Germany
J.7 MHC class II induction on microglial cells is controlled by sodium-dependent neuronal activity T. MISGELD, H. NEUMANN, and H. WEKERLE Intact brain tissue does not show MHC class II antigen expression. In contrast, under a variety of pathological conditions, MHC class II antigen is found within the central nervous system. MHC class II is most readily inducible in microglia cells, but is also noted in astrocytes and brain endothelial cells. We studied the expression of MHC class II antigens on microglial cells in organotypic hippocampal slice cultures of newborn Lewis rats. We used semiquantitative image analysis and confocal laser microscopy of whole immuno-stained slice cultures. After one week of culture the hippocampal slices exhibited good neuronal differentiation. In the absence of stimulation, only very few scattered MHC class II positive microglial cells were found. Stimulation with IFN-y resulted in strong induction of MHC class II antigens on microglial cells in the injured superficial areas of the slice cultures. In the central parts of the slices containing most intact neurons, only very low levels of MHC class II immunoreactivity were detected. After blocking of sodium-dependent neuronal activity by tetrodotoxin (TTX) IFN-y-inducible MHC class II antigen levels on microglial cells were raised, however, without reaching the levels found in the areas depleted of active neuronal elements. Our results indicate, (i) that electrically active neurons suppress inducibility of MHC class II genes in microglial cells; (ii) that this suppression can be neutralized by TTXblockage of neuronal activity, and (iii) that, however, additional factors - probably associated with neuronal degeneration - are required to reach maximal expression levels in microglial cells.
Joint annual meeting 1995 OGAI/GFI . 197 Research Center for Infectious Diseases, University of Wiirzburg, W iirzburg; 1Institute of Clinical Microbiology and Immunology, University of Erlangen-Niirnberg, Erlangen, Germany
J.8 Inducible nitric oxide synthase is not expressed in murine epidermal Langerhans cells H. MOLL, C. BOGDAN, 1 and C. BLANK In Leishmania-infected macrophages, the formation of nitrogen oxides is critical for intracellular killing of the parasites. Their production is mediated by the inducible nitric oxide synthase (iNOS). We have recently shown that, in addition to macrophages, epidermal Langerhans cells can phagocytose L. major. It was therefore of interest to analyze whether Langerhans cells display the same leishmanicidal effector mechanism. For this purpose, pure Langerhans cells were collected, using single-cell picking, and were infected with L. major and/or activated with different cytokines in the presence or absence of LPS in vitro. Subsequently, the cells were analyzed for expression of iNOS mRNA using RT-PCR. In contrast to macrophages, Langerhans cells did not express iNOS mRNA. On the other hand, significant levels of iNOS mRNA could be detected in unselected epidermal cells, the majority of which consists of keratinocytes. These results indicate that in the L. major-infected skin, activated macrophages and keratinocytes, but not Langerhans cells have the ability to express iNOS activity. The implications of this finding for the interaction of Langerhans cells with intracellular pathogens will be discussed.
Departments of 1 Clinical Immunology and 2Dermatology, Medical School of Hannover, Hannover; 3Department of Rheumatology and Clinical Immunology, Freiburg, Germany
J.9 Elevated MxA level in SLE, but not in systemic skleroderma, M. Wegener or cryoglobulinemia P. PANAGIOTOUl, A. N. AL-MASRIl, T. WERrEL2, A. KAPp2, H. H. PETER 3, R. E. SCHMIDTi, and P. v. Wussow i MxA is an IFN type I-induced protein. Its increased level in leukocytes indicates the presence of biologically active IFN in the circulation. Blood MxA level of 51 patients with SLE, 13 patients with skleroderma, 11 patients with Wegener's Granulomatosis and 10 patients with cryoglobulinemia were determined in a MxA ELISA employing specific monoclonal antibodies. 45 of 51 SLE patients had MxA level above 4 m U / 1000 L. The remaining six patients with normal MxA level «3 mU / 1000L) had a completely inactive disease. In SLE disease activity and blood MxA level significantly correlated at a p-value of 0.01. In contrast, 9 of 11 patients with skleroderma had normal MxA level «3 mU / 1000L). The remaining two patients showed elevated MxA level, but also signs of a clinically relevant vasculitis. Furthermore, in the absence of infectious complications all patients with Wegener's disease did not possess elevated MxA level. Six of these patients had active disease with lung infiltrates (n=4) and nephritis (n=3); one patient showed
198 . Joint annual meeting 1995 OGAI/GFI significantly elevated MxA level only during a febrile episode indicating that these patients can produce MxA. Also patients with cryoglobulinemia (n = 10) did not show elevated MxA level. From some of the above mentioned patients skin biopsies were taken for MxA immunostaining. Only in SLE-, but not in skleroderma or cryoglobulinemia the skin tissue showed an intensive MxA staining. Not only the epidermis, but also lymphocytic infiltrations in the subcutis showed an intensive MxA staining in these specimen. We conclude that the IFN system is activated in SLE, but not in the other autoimmune diseases investigated. Furthermore, not only leukocytes in the blood stream, but also keratinocytes and other cells in the skin express the MxA protein.
Institute of Immunology and Transfusion Medicine, Medical School, University of Lubeck, Lubeck; 1Department of Internal Medicine, University of Rostock, Rostock, Germany
J.10 Impaired local immunity in inflammatory bowel diseases M. SEYI'ARTH, M. KNOCHE, K. KISSING, andJ. EMMRICH There are not enough informations about the local immunity in inflammatory bowel diseases. We are interested in both humoral and cell-mediated immunity. We investigated 9 pat. with Colitis ulcerosa (CU), 8 pat. with Crohn's disease (CD) and 23 controls for their local immune reactivity only in not affected areas of colon mucosa. The S-IgA-concentration (humoral immunity) was lower in patients with UC and CD than in controls (p < 0.05). In controls we found the highest values of S-IgA in the colon transversum. There was neither age nor sex differences. Lamina propria lymphocytes of the patients differed in their cytokine secretion pattern. We found increased levels of interleukin (IL) 6 in the unstimulated supernatants of UC and CD patients and increased levels of IL 1~ in UC, whereas CD pat. and controls produce only small amounts of IL 1~. No significant differences could be shown in the TNFa production. It was possible also to inhibit the IL 6 and TNFa production by specific antibodies. The results shown suggest that T cells, in particular, may be involved in the disorders in both local humoral and local cell-mediated immunity.
Department of Urology and ENT, University of Vienna, Vienna, Austria
J.11 Increased expression of interferon-y in benign prostatic hyperplasia G. E. STEINER, B. KNERER, H. MARTINEK, G. KRAMER, and M. MARBERGER Previously we demonstrated that benign prostatic hyperplasia (BPH) is frequently associated with lymphocytic infiltration. This observation suggested to us that the close contact of prostatic stromal cells (PSC) with activated lymphocytes might modify their cytokine responsiveness. Therefore normal (n=2), BPH (n=5) and BPH-PSC clones
Joint annual meeting 1995 OGAI/GFI . 199 (n = 11) were established and stimulated with various cytokines. In summary, normal PSC revealed no proliferative response to IL-2, -7, a slight response to IFN-y, and dramatic inhibition induced by IL-4 and TNF-u. By contrast, BPH-PSC lines consistently showed hyperresponsiveness when stimulated with IL-2, -7 and IFN-y (135-147%). Analyses regarding the heterogeneity of PSC in BPH using PSC clones revealed that 7 of those 11 clones exhibiting rapid proliferation had an even greater responsiveness to IFN-y and IL-2 compared to the polyclonal mother line. Therefore normal, BPH and cancer tissues were screened for anti-IL2 and -IFN-y reactivity by immunohistology revealing no staining for IL-2 and intense reactivity with the anti-IFNy mAb. The specificity was confirmed by nonsense isotype control mAb as well as dosedependent inhibition with rIFN-y. To analyze if IFN-y is produced intra-prostatically, total RNA was prepared from all tissues, reverse transcribed into c-DNA and PCR amplification was performed using IL-2 and IFN-y-specific primers. The specificity of PCR reaction was confirmed by Southern blotting and hybridization with internal either IFN-y and IL-2-specific oligos. Similar to the staining results both normal and the 5 malign prostatic tissues, although clearly positive, exhibit much less IFN-y message when compared to the amount expressed in BPH tissue whereas probing for IL-2 revealed completely negative results.
Department of Internal Medicine I, University Medical Center Regensburg, Regensburg, Germany
J.12 Endogenous norepinephrine and opioids control splenic IL-6 secretion in mice R. H. STRAUB, M. HERRMANN, G. BERKMILLER, W. FALK,]. SCHOLMERICH, andB. LANG In a previous study we demonstrated a newly developed technique which allows the investigation of nerve-immune cell interaction in spleen (1). To study further aspects of this neuronal-immune interplay, effects of propranolol and opioid antagonists were investigated. The ~-adrenergic antagonist propranolol decreased the electrically induced inhibition of IL-6 secretion in bacteria-containing medium but increased the electric effect in bacteria-free medium (for the difference p = 0.0023). The opioid antagonist naloxone decreased the electrically induced inhibition of IL-6 secretion in a medium irrespective of the presence of bacteria (bell-shaped curve with Emax at 10-6 M, p=0.0046). Further investigation using the ~-specific opioid receptor antagonist cyprodime hydro bromide revealed involvement of ~-opioid receptors (bell-shaped curve with Emax at 10-8 M, p=0.0014). The 6-opioid receptor antagonist ICI 174,864 was not able to decrease the electrically induced inhibition of IL-6 but showed a trend to increase the electric effect (p = 0.1422). In conclusion, these results demonstrate a synergistic effect of norepinephrine and enkephalins to inhibit the splenic IL-6 secretion in bacteriacontaining medium. However, in bacteria-free medium the colocalized transmitters had opposing effects on splenic IL-6 secretion. Thus, during stress due to septicemia both colocalized transmitters of the sympathetic nervous system feedback IL-6 secretion to dampen the inflammatory response whereas during stress aseptic conditions no such increased attenuation of IL-6 secretion exists. 1. STRAUB, R. H. et al. 1995. ]. Neuroimmunology, in press.
200 . Joint annual meeting 1995 OGAIIGFI Institute of Immunology and 1Lung Diseases and Tuberculosis Hospital, Zagreb, Croatia
J.13 C038 antigen on bronchoalveolar (BAL) T lymphocytes correlate with activation status in interstitial pulmonary diseases (ILO) I. SVOBODA BEUSAN 1, R. AGBABA PRIMORAC, D. GOMERCld, and Z. PRAHIN 1 As shown in our previous OGAI reports, CD38 antigen plays a relevant role in sarcoidosis. In this study we were interested in evaluating the role of CD38 in the pathogenesis of other ILD. BAL was obtained from patients with sarcoidosis'f (n=31), fibrosis':":' (n = 8) and controls':":":' (n = 14). The CD38+ T cell levels was assesed by double immunofluorescence staining and analyzed by FACS. Total CD38 was highly elevated in AS (40 ± 20 %), in comparison to IS, F and C samples (= 20 %). In AS CD38 is predominantly expressed on CD4 (:S 70 %), whereas in IS and F CD38 is equally expressed on CD4 and CD8 lymphocytes (= 30 %). T subset analysis revealed that CD4+CD38+ cells were highly elevated in AS (29 ± 17 %) as compared to IS (5 ± 3) and F (6 ± 4), whereas CD8+CD38+ were generally low. Eight patients underwent 2-6 repeated lavages and this follow-up study indicated that CD4+CD38+ are elevated on the onset of the disease, decreased after therapy and increased again in relapse of the disease. Steroid therapy slowly increase CD8+CD38+ up to 10 %. As local CD4+CD38+ expansion correlates with the onset of alveoli tis. Erythrema nodosum and disease progression, our results indicate that this subpopulation may playa relevant role in ILD and could serve as an indicator of disease activity. ':. active and inactive sarcoidosis (AS and IS, respectively); ,:.,:. fibrosis (F); ,:.,:.,:. control (C)
Department of Clinical Immunology, Medical School of Hannover, Hannover, Germany
J.14 Elevated blood MxA level correlate with disease activity in patients with SLE and Sharp syndrome P. v. Wussow, A. N. AL-MASRI, P. PANAGIOTOU, U. NOLTE, and R. E. SCHMIDT MxA is an IFN type I-induced protein. Its increased level in leukocytes indicates the presence of biologically active IFN in the circulation. Blood MxA level and disease activity were monitored in 51 patients with systemic lupus erythematosus and 12 patients with Sharp syndrome over two years. The patients investigated were categorized into three groups: patients with a completely inactive, with a moderately active or an exacerbated disease. In addition, the SLE patients were classified according to the SLEDAI index. 6 patients had a completely inactive disease and normal MxA level « 3 mU / 1000 L). In contrast, all patients with minimally or moderately active disease (n=31) showed elevated MxA level between 4.2 and 23 mU /1000 L. Finally 14 patients experienced an exacerbation of their disease in the observation period. Their MxA level ranged between 12 and 48 mU /1000 L. In all patients studied disease activity as assessed by the SLEDAI correlated with the blood MxA level at a value of p = 0.Q1.
Joint annual meeting 1995 OGAI/GFI . 201 Prednisolone treatment in the 14 patients with exacerbation led to drastic MxA level reductions within 1-2 months (mean 18.3 to 4.1 mU /1000 L). Upon lowering the steroid dosis six patients who experienced an early relapse of their symptoms had a significant increase of their MxA level to almost pretherapy values (mean 4.6 to 12.5 mU / 1000 L). In contrast, patients with a sustained response to prednisolone showed only moderate elevations of their blood MxA level (mean 3.3 to 6.7 mU 11000 L). In summary, the elevated MxA level in both SLE and Sharp syndrome demonstrate the activation of the IFN-system in these patients. Apparently, MxA level correlate with disease activity in such patients. Specifically, blood MxA level are probably capable of identifying patients with a completely inactive disease.
Basel Institute for Immunology, Basel, Germany
J.1S The peripheral blood is not representative of the recirculating immune system A. J. YOUNG, W. L. MARSTON, L. DUDLER, and W. R. HEIN Lymphocytes continually traffic between the blood and the lymph in vivo. This process is absolutely required for adequate immune surveillance, and the dissemination for immunological memory. This migration does not appear to occur randomly, but it has been shown that lymphocytes will traffic specifically through different tissues. Although this specificity has been known for some time, the relative contribution of the peripheral blood to the recirculating pool has not been addressed. The experiments reported here have directly examined the migratory properties of peripheral blood lymphocytes (PBLs). PBLs were labeled in vitro and reinjected intravenously in sheep. Lymph was collected from a variety of tissues, as well as peripheral blood, and analyzed for the concentration of labeled cells. Despite the large numbers of lymphocytes known to migrate through intestinal tissues, labeled PBLs were detected in greater concentrations in subcutaneous efferent lymph. Peak recovery of labeled cells in lymph occurred 24 hours after reinjection. Surprisingly, the concentration of labeled lymphocytes found in peripheral blood was significantly higher than that found in lymph. This is the reverse of what is observed when lymphocytes collected from lymph are labeled. These data suggest that the peripheral blood is not representative of the mature recirculating lymphocyte pool, and that a large proportion of peripheral blood lymphocytes do not recirculate under normal conditions. Because of the importance of lymphocyte recirculation in the physiology of immunological surveillance and memory, these data suggest that the peripheral blood is not an effective site to measure overall immune competence.