Workshop L: Effector Mechanism in Allergic Diseases

Division of Immunology and Allergology, Medical Institute of Environmental Hygiene, Heinrich Heine University, Dusseldorf, Germany

L. 1 Prevention and treahnent of nickel allergy in mice by tolerance induction S. ARTIK, K. HMRHUIS, X. Wu, E. ELIEYIOGLU, C. VON VULTEE, P. GRIEM, and E. GLEICHMANN We have established a reproducible model for induction of contact hypersensitivity to nickel (Ni), using higher Ni oxidation states, i.e., Ni(III) and Ni(IV), instead of Ni(II), for priming mice. Contact hypersensitivity to Ni(II) was assessed by the ear-swelling test. We propose that Ni(II) provides an adequate signal 1 to T cells, but apparently an insufficient signal 2. In contrast, Ni(III) and Ni(IV) seem capable of providing signal 2 in addition to signal 1. As it is known that signal 1 provided in the absence of signal 2 will induce tolerance, we asked whether administration of Ni(II) via the oral or intraperitoneal (i.p.) route would induce tolerance. For oral tolerization, mice were given 10 mM or 2 mM Ni(II)Cl z in drinking water for 5 and 10 weeks, resulting in nickel doses of 350 and 70 mg/kg bw/week, respectively, of which only 10% are resorbed. For i.p. tolerization, mice were injected 3 times/week with 50 111 Ni(II)Cl z for 4 weeks, resulting in nickel doses of 4.43 and 0.44 mg/kg bw/week, respectively. After the tolerization period, mice were primed with Ni(III) and 10 days later the ear-swelling response to Ni(II)Cl z was measured. In mice pretreated with Ni(II)Cl z, the ear-swelling response was significantly reduced compared to non-tolerized mice. After having successfully tolerized non-sensitized mice, we tried to establish tolerance in sensitized mice: mice were sensitized to Ni(III), left untreated for 5 weeks, tolerized with 10 mM Ni(II) for 2 or 5 weeks, and then challenged with Ni(II). Interestingly, the ear-swelling response was significantly reduced in sensitized mice tolerized with Ni(II) compared to non-tolerized mice; thus tolerance could be induced in mice already sensitized to Ni. Using a mouse model ofNi allergy that involves priming of mice with Ni(III) or Ni(IV), we demonstrated that treatment with Ni(II), orally or i.p., renders both non-sensitized and sensitized mice tolerant to subsequent induction of hypersensitivity to Ni. By adoptively transferring cells from allergic and tolerant mice we now investigate the cell type, cell amount and cytokines involved in Ni-induced contact dermatitis and tolerance.

30th Annual Meeting 1999 . 507 lInstitute of Virology and Immunobiology, University ofWiirzburg, Wiirzburg, Germany and 2Department of Molecular and Cell Biology, Universiry of California, Berkeley, USA

L. 2 Tissue distribution of the mouse NK cell receptor MAFA (mast cell function-associated antigen) N.B. BEYERSDORF l , L. CORRAL 2, D.H. RAULET 2, and T. HANKEl The recently identified mouse homologue of the lectin-like inhibitory receptor MAFA (mast cell function-associated antigen) is expressed on a subpopulation (approx. 40%) of splenic NK cells. Here, we have analyzed the tissue distribution of MAFA by flow cytometry and immunohistology. MAFA expression was readily detectable in secondary lymphoid organs (spleen, lymph nodes), but not in non-hematopoietic tissues (brain, liver, lung, testis, kidney, eye). In spleen and lymph node, MAFA expressing cells were clustered next to, but not in the T- and B cell areas, consistent with the notion that MAFA is expressed predominantly by NK cells. MAFA was not expressed substantially on fetal liver cells or fetal thymocytes. During ontogeny, relative and absolute numbers of splenic MAFA positive NK cells increased until adulthood. Interestingly, the subset size of MAFA expressing NK cells varied significantly in different lymphoid organs. Activation ofNK cells by Poly-I:C in vivo or IL-2 in vitro did not induce MAFA expression, ruling out MAFA as a bona fide activation marker. In addition to NK cells, MAFA was also expressed on a subpopulation ofT cells «3% of COB T cells, <2% of CD4 T cells) most if not all of which displayed memory phenotype. Unexpectedly, MAFA was also on the surface of a subpopulation of splenic macro phages and on in vitro generated bone marrow derived macrophages, but not on mast cells. These data show that in the mouse, MAFA is mainly, if not exclusively expressed by lymphocytes and macrophages.

Research Toxicology, Bayer AG, Wuppertal, Germany

L. 3 TaqMan-PCR analysis of cytokine expression pattern during irritant and allergic skin reactions in interleukin-4 transgenic mice

J.

BLOMEL, H.]. AHR, and H.-W VOHR

The interleukin (IL)-4 transgenic (tg) mice used in our study express IL-4 under the control of major histocompatibiliry (MHC) class I regulatory sequences continuously at a low level in all tissues. This IL-4 over-expression results in immunological dysregulation, e.g. B cell hyperreactiviry, increased MHC class II expression and elevated IgE serum levels. In previous investigations we found differences in the Oxazolone-induced primary local contact hypersensitivity (CHS) between IL-4 tg and wild-rype mice: The IL-4 tg mice developed in comparison to wildtype mice an epidermal hyperreactiviry associated with a decreased activation of the skin-draining lymph nodes, a phenorype which is very similar to the phenotype found during Croton oilinduced local irritant contact dermatitis or the phenotype of atopic diseases in humans. In the present study we investigated the !xpression pattern of different cytokines during Oxazoloneinduced local primary CHS and Croton oil-induced local irritant contact dermatitis in both

508 . 30th Annual Meeting 1999 mouse mains using the highly sensitive Real-time RT-PCR method (TaqMan-Assay). We focused on epidermal-derived IL-l~, tumor necrosis factor (TNF)-a, and macrophage inflammatory protein (MIP)-2 - cytokines which are known to be involved in the induction of maturation and migration of epidermal antigen-presenting cells (APC). Furthermore, we analyzed the expression of APC-derived IL-6 in the skin and skin-draining lymph nodes, a cytokine which is exclusively produced by APC during the onset of immune reaction. Comparing wild-type and IL-4 tg mice, no differences in the cytokine expression pattern were found during Croton oilinduced local irritant contact dermatitis. In contrast, a strongly increased expression of epidermal-derived TNF-a and MIP-2 was detected in IL-4 tg mice during Oxazolone-induced local primary CHS. Focusing on the expression of APC-derived IL-6, we found an increased expression in the skin yet decreased expression in the skin-draining lymph nodes of IL-4 tg mice, indicating a decreased migration of epidermal APC towards the skin-draining lymph nodes. We concluded that the altered cytokine expression in the skin of IL-4 tg is responsible for this decreased migration of epidermal derived APC towards the skin-draining lymph nodes leading to the observed irritant-like phenotype during primary local CHS. Overall, epidermal hyperreactivity and immunological alterations of IL-4 tg mice might serve as a helpful rodent model for the investigation of the pathophysiology of human atopic diseases.

1Physiological Chemistry II, Biocenter of the University, 3Molecular Pathology, Institute of Pathology, University ofWlirzburg, Wlirzburg, and 2Tumor Immunology, German Cancer Research Center, Heidelberg, Germany

L. 4 The environmental pollutant pyrene has proallergic effects H. BOEMMEL 1, M. LI-WEBER 2 , E.S. SERFLING 3, and A. DUSCHL 1 The prevalence of type I allergies in developed countries has increased in recent decades. It is conceivable that a modern life style provides factors which modify immune responses. Since IL-4 is crucial for the development ofT1l2 cells and for class switching of B cells to IgE type antibodies, factors which lead to higher IL-4 levels will increase the risk for allergic diseases. Epidemiological studies and experiments with mouse models suggest that exposure to the particulate fraction of air pollution increases IgE levels and other allergic parameters. Extracts of polyaromatic hydrocarbons (PAH) associated with such particles have also been shown to promote allergy in experimental systems. We have investigated whether PAH affect the transcriptional activity of the IL-4 promoter. Reporter gene constructs containing the human or mouse IL-4 promoter were transfected into Jurkat or EI4 cells, and the transfected cells were treated with PAH. We have found that pyrene, but not several other related PAH, enhanced basal transcription of the mouse and human IL-4 promoter. Pyrene did not affect transcription from the IL-4 regulated IgE class switching promoter, or from an oligomeric Stat6 binding site. When peripheral blood lymphocytes were stimulated with ConA, costimulation with pyrene led to increased IL-4 secretion. IL-4 was detected in this case by ELISA in the supernatant, which demonstrates that pyrene can enhance production of the protein. Our data suggest, that IL-4 production is increased by pyrene, while IL-4 dependent Stat6 activation is not affected. An increase in basal IL-4 expression following exposure to pyrene should enhance Th2 differentiation and IgE class switching. Pyrene is present in many sources,

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including emissions from domestic heating and industry, various foods, and cigarette smoke. Our continuous exposure to this substance may be one of the factors which associate the modern life style with increased incidence of type 1 allergies.

lInsitute of Immunology and Allergology, Inselspital, University of Bern, Bern, Switzerland and 2Department of Pharmacology and Therapeutics, University of Liverpool, UK

L. 5 Reactivity of T cell clones to drugs and their metabolites C. BURKHART l , B. SCHNYDER 1, S. VON GREYERZ 1, D.]. NAISBITT 2, K. SCHNYDER-FRUTIG 1, M. PIRMOHAMED 2 , B.K. PARK 2 , and W]. PICHLER 1

Drugs represent a group oflow molecular weight non-peptide antigens which can be recognized by a~ TCR-positive T cells. Over the past years, specific T cell clones have been generated from patients allergic to the antibiotic sulfamethoxazole (SMX) suggesting an important role for T cells in the manifestation of drug hypersensitivity. Allergic reactions to SMX are usually thought to be a consequence ofbioactivation to the hydroxylamine metabolite (SMX-NHOH) and further oxidation to the ultimately reactive metabolite, nitroso-sulfomethoxazole (SMX-NO). SMX-NO is then able to covalently modifY self proteins which in turn might be recognized as neo-antigens by the immune system. Recent investigations, however, revealed additionally an MHC-restricted but processing- and metabolism-independent pathway of drug presentation. Antigen-presenting cells (APCs) preincubated with the drug overnight and subsequently washed were not able to activate SMX-specific a~ TCW T cells. The drug could, however, be efficiently presented even by glutaraldehydefixed APC when it was continuously present in the culture. Moreover, T cell clones responded to non-covalenrly presented SMX by a rapid downregulation of their T cell receptors. These observations were best explained by a labile, low-affinity binding of SMX to MHC-peptide complexes on APCs and a processing-independent antigen presentation. In order to study the role of covalent versus non-covalent drug presentation in SMX-allergy, a) we analyzed whether T cells from allergic and non-allergic individuals react with SMX and the metabolites SMX-NO or SMX-NHOH; b) we generated T cell clones to SMX and the chemically reactive metabolites and investigated the crossreactivity in detail; and c) we assessed the kinetics ofTCR downregulation in T cell clones crossreactive with SMXJSMX-NO. Our data indicate, that crossreactive T cells can be detected in and isolated from the peripheral T cell pool of drug-allergic individuals but that their appearance differ between individuals. The overall pattern of antigen-reactivity suggests that in SMX-hypersensitivity already the primary stimulation can be directed to the non-covalenrly bound SMX.

510 . 30th Annual Meeting 1999 1Department of Dermatology an[Allergology, Medical School, Hannover, Germany and 2LeukoSite Inc., Cambridge, USA

L. 6 Dermal fibroblasts are a natural source of CC chemokine receptor 3 ligands activating eosinophils

Y. DULKYS l , H. PETERING l , C. KLUTHE l , D. KIMMIG l , P. PONATH 2, A. ELSNER

1

l

KApp , and J.

Increased numbers of eosinophils are found in different autoimmune diseases, particularly in sclerotic disorders of the skin. The reason for this phenomenon is still unclear. It is well known, however, that fibroblasts are a major source of CC chemokines responsible for the attraction of eosinophils at the site of inflammation. On the other hand, eosinophils are capable to release cytokines activating dermal fibroblasts leading in turn to an amplification of this immune response. In this study we investigated the interaction of dermal fibroblasts and eosinophils with respect to the biological activity of CC chemokines. RT- PCR revealed that cytokine-stimulated dermal fibroblasts are able to synthesize mRNA for RANTES, eotaxin, and MCP-4, whereas mRNA for eotaxin-2 could not be detected. To investigate the biological potency of these CC chemokines, actin polymerization, chemotaxis and release of reactive oxygen species were assessed. In all experiments eotaxin-2 was as potent as eotaxin. Moreover, eotaxin-2-induced release of reactive oxygen species and intracellular calcium transients could be blocked by a monoclonal antibody against CCR3 in the same range as eotaxin-stimulated eosinophils. This study demonstrates that there exists a discrepancy between the natural source of CC chemokines and their biological role in the activation of human eosinophils: Eotaxin-2 is one of the most potent CC chemokines for eosinophils, however, it could not be detected in vivo in this test system. Therefore, the most relevant CC chemokines for the interaction between dermal fibroblasts and eosinophils, so far, seem to be eotaxin and RANTES.

1 Forschungszentrum Borstel, Borstel, 2Hautklinik, Medizinische Universitat zu Lubeck, Lubeck, and 3Institut fur Physiologische Chemie, Tierarztliche Fakultat, Ludwig-MaximiliansUniversitat, Munchen, Germany

L. 7 Lectins: Trigger molecules for a Th2 response and IgE-mediated allergy H. HAAS l , EH. FALCONE l , G. SCHRAMM l , K. HAISCH l , B.E GIBBS 2,]. KLAUCKE l , M. POPPELMANN 1, W-M. BECKER l , H.-J. GABIUS 3, and M. SCHLAAK 1 Lectins are common components of plant, animal, fungal, and microbial cells. Since concanavalin A (ConA) has been found to trigger human basophils to release histamine, presumably via crosslinking receptor-bound IgE, we analyzed the capacity of 16 lectins, to induce human basophils to secrete interleukin- (IL-)4 and IL-13. Indeed, ConA and several other lectins triggered basophils to release IL-4 at concentrations of up to 1 ng/10 6 basophils. Lectins with high IL-4-inducing capacity also induced the release of IL-I3 and histamine (l). Some of these lectins stimulated a rat basophil/ma~· cell line (RBL-2H3) only in the presence and some also in

30th Annual Meeting 1999 . 511 the absence of rat IgE to release hexosaminidase and IL-4, suggesting different ways of basophil activation by lectins. Taken together, lectins can trigger human basophils to release IL-4 and IL-13, presumably by more than one mechanism. Since lectins are widely distributed as constituents of foods and pathogenic organisms, some of them might trigger basophils and mast cells to produce the so-called early IL-4 which biases the immune response towards a Th2 response and type I allergy. (1) Haas, H. eta!. (1999), Eur.]. Imrr·.r,ol. 29:918-927

lImmunobiology and 2Pulmonary Function and Pharmacology, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover, Germany

L. 8 Comparison of the respiratory allergenicity in a rat model of allergic asthma at two time points M. HECHT', O. MACKEl, A. EMMENDORFFER', and H.G. HOYMANN 2 To investigate the allergic potential of different substances e.g. pharmaceuticals we developed a rat model of allergic asthma using ovalbumin as allergen. In this study we were interested in changes of the reaction measured at two different time points after antigen challenge. We investigated the reaction to ovalbumin in comparison to a chemical powder, substance X. Male 8/9week old Brown Norway rats were given s. c. substance X, ovalbumin (OA) plus alum, or vehicle on day 0, 2, and 7. Antigen challenge by inhalation was performed under anesthesia on day 14 or 21 respectively. For challenge with substance X we used two concentrations (X low, X high) and the ovalbumin sensitized animals were challenged with 40 Jlg OA (POS). A negative control group (NEG) was sensitized with the vehicle and challenged with the high concentration of substance X. Airway hyperreactivity was measured 22-24 h after the challenge using acetylcholine. Bronchoalveolar lavage (BAL) was performed 44-48 h after the challenge and serum was collected to determine the IgE antibody concentration. In the BAL lavage we examined the total cell counts and the percentage of eosinophils. At both time points we observed an increased hyperreactivity and an elevation in-eell number. The percentage of eosinophils in the BAL as well as the IgE level in the serum were increased in the POS group compared to the NEG group or the substance X treated groups. This increase was more pronounced at day 21 especially for the IgE level. The treatment with substance X did not result in any changes compared to the control group. Our results show that the chemical powder substance X did not induce an allergic reaction and has no allergic potential. This study also demonstrated that the assessment of allergenicity should be performed on day 21 after sensitization.

512 . 30th Annual Meeting 1999 Clinical Research Unit, Department of Dermatology, Johannes Gutenberg-University, Mainz, Germany

L. 9 Evaluation of the hu-PBL-SCID mouse as a model for studies of therapies of type I allergy E.R. JARMAN, K. PERSCHKE, E. MONTERMANN, R. Ross, J. KNop, and A.B. RESKE-KuNZ Human IgE responses were established in C.B-17 severe combined immunodeficiency (SCID) mice following intraperitoneal transfer of PBMC from atopic patients with the aim of using these mice to test therapeutic approaches to downregulate IgE production. Total serum IgE levels as well as allergen (Dermatophagoides pteronyssinus or Phleum pratense)-specific IgE levels were dependent on the presence of allergen. Immunohistochemical studies performed using tissue sections obtained from hu-PBL-SCID revealed infiltration of murine organs notably spleen, thymus, lungs by human CD3+ T cells. To enumerate human cells in mouse tissues with high sensitivity, a quantitative RT-PCR was established. Using EllSPOT tests we found that stimulation of cell suspensions, prepared-from peritoneum and spleen on day 7 to 21 after cell transfer, with the relevant allergen did not induce human IL-4, IL-5 or IFN-y production. However, maximal numbers of cytokine-producing cells were obtained, when antigen presenting cells, allergen as well as IL-2 were added to the culture. These data suggest that the T cells present in hu-PBL-SCID peritoneum and spleen are in a state of anergy as early as day 7 of adoptive transfer. Thus our data caution against the use of the hu-PBL-SCID mouse model in studies of therapeutic approaches for type I allergy.

Immunobiology Department, Clinic for Dermatology, Bonn University, Bonn, Germany

L. 10 Evidence for coregulation of the high affinity receptor for IgE and MAFA on human antigen-presenting cells S. KOCH, S. KRAFT, E. GEIGER, J.-P. ALLAM, and T. BIEBER Human antigen-presenting cells (APC) variably express the high affinity receptor for IgE, FceRI. Other than in mast cells or basophils, where the FceRI complex consists of one alpha, one beta and rwo gamma chains, FceRI on APC is lacking the beta chain. Again in contrast to its function on basophils and mast cells, FceRI on APC functions efficiently as allergen focusing molecule. A novel molecule, MAFA, mast cell function associated antigen, is expressed on rat mast cells and basophils, and negatively regulates the function of FceRI on these cells. It was not known whether MAFA was also expressed on human APC. The functional interference berween these rwo structures led us to study their regulation during in vitro differentiation of human dendritic cells (DC). Monocyces are known to express FceRI at variable levels closely correlating to the atopic status of the donor. We observed that during differentiation of monocytes to DC under IL-4 and GM-CSF the alpha chain of the FceRI was slightly down regulated at the cell surface, but still present at moderate levels. Interestingly, it accumulated intracellularly to form a pool, a situation we have recently reported in Langerhans cells. Expression of the FceRI gamma

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chain remained unchanged at high levels. We found MAFA on human monoeytes expressed to varying degrees by cells from diffe~nt donors. MAFA expression is regulated similarly to FCERI expression during in vitro differentiation of monoeytes to DC: We observed a slight down regulation of surface levels during differentiation, whereas intracellular levels dramatically increased. Culture of CD34+ stem cells from cord blood with GM-CSF, TNF-a and SCF led to the spontaneous expression of FceRI at the cell surface. Adding high doses ofTGF-~ to these cells resulted in a down regulation of the FceRI alpha chain at the cell surface with a concomitant increase of an intracellular pool. These common regulatory principles of FceRI and MAFA may suggest a possible role of MAFA in the function of FceRI on APc.

1 Department of Dermatology, MEB, FWU Bonn, Bonn, 3Department of Dermatology, Ludwig-Maximilians-University, Munich, Germany, and 2Department of Dermatology, Kyoto Prefectural University of Medicine, Kyoto, Japan

L. 11 Aggregation of FceRI on human dendritic cells and monocytes induces NF-xB activation

Antigen-presenting cells (APC) like dendritic cells (DC), especially epidermal Langerhans cells (LC), and monocytes express high affinity IgE receptors (FceRI) lacking the FceRI beta chain. In contrast to mast cells/basophils exhibiting the complete structure of FceRI, signal transduction in APC is poorly characterized. However, it is interesting because IgE-mediated delayedtype hypersensitivity reactions are suggested to participate in the pathophysiology of atopic diseases. The transcription factor NF-xB activates many genes involved in inflammatory processes (e.g. proinflammatory cytokines). Its subunits associate to active hetero-/homodimers after various stimuli. Using gel shift assays, we observed a strong activation of NF-xB after FceRI ligation on LC. Supershift analyses revealed a complex composed of the p50, ReiB and c-Rel subunits, whereas p52 and p65 were not detectable. This phenomenon was only observed in LC showing high FceRI expression. FceRI-mediated NF-xB DNA-binding could also be observed in monocytes and monocyte-derived DC, with supershift analyses showing a different complex containing p50 and p65. However, by immunoblotting we could find all five NF-xB subunits to be expressed in these cells. FceRI ligation induced serine phosphorylation and degradation of the inhibitor I-xBa. Inhibition of NF-xB by NAC abrogated the FceRI-mediated synthesis of IL-l~, IL-6, IL-8 and TNF-a, indicating the functionality of this pathway. Thus, while NF-AT is involved in signal transduction induced by FCERI ligation on basophils and mast cells, crosslinking of this receptor rather implicates NF-xB which in turn may activate a series of genes involved in the regulation of inflammatory reactions.

514 . 30th Annual Meeting 1999 Department of Respiratory Medicine, Medical School, Hannover, Germany

L. 12 Increased Th2-cytokine profile of ytJ-T cells in asthma N. KRUG, V ERPENBECK, K. BALKE, J. PETSCHALLIES, J. HOHLFELD, and H. FABEL Cytokine producing T cells play an important role in allergic asthma, however, little is known about cytokine production of yo-T cells. In this study we have obtained T cells from bronchoalveolar lavage before and 24 h after segmental saline and allergen provocation. In a group of 11 atopic mild asthmatics and 9 cont-rol subjects we have assessed the ability of yO-T cells to produce IFN-y, IL-2, IL-4, IL-5, and IL-13. After stimulation with PMA and ionomycin a flow cytometric method of intracellular cytokine detection was employed which permits the analysis of cytokine production in single yO-T cells. The percentage of yO-T cells was 3.0±2.6% in asthmatics and 2.3± 1.1 % in controls without significant changes after saline or allergen challenge. The percentage of IL-5 and IL-13 producing yo-T cells before allergen challenge was significantly increased in asthmatics (p<0.05). After allergen, but not after saline challenge, there was a significant decrease in IFN-y and IL-2 producing yO-T cells in asthmatics (p
Pulmonary Pharmacology, Corporate Research ASTA Medica AG, Arzneimittelwerk Dresden GmbH, Radebeul, Germany

L. 13 The role of phosphodiesterases in cytokine release from human nasal polyp cells and human blood

S. KOSTERS, S. HEER,

J.

RUDERT,..A. WACHS, and D. MARX

To characterize new chemical entities it is imperative to investigate their effects on human cells. The primary culture of freshly isolated human nasal polyp cells consists of epithelial cells, monocytes, macrophages, mast cells and infiltrating cells such as lymphocytes, neutrophils and a large number of eosinophilic granulocytes. Accordingly, human blood consists of various cell types that are involved in inflammatory events. Both cells culture systems are suitable to investigate the inflammatory interplay of the different cell populations. The aim of this study was to investigate the role of the PDE isoenzymes 1, 2, 3, 4 and 5 in inflammatory events. Thus the effect of selective inhibitors on the release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating-factor (GM-CSF), a cytokine responsible in eosinophil proliferation and activation, from sensitized human nasal polyp cells was studied. In addition, the PDE-inhibitors were tested on the lipopolysaccharide-induced release ofTNF from 1:5 diluted human whole blood.

30th Annual Meeting 1999 . 515 Inhibition of PDEI or PDE2 by vinpocetine and EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), respectively, had no or only very weak effects on.the release ofTNF from polyp cells and whole blood, or on the release of GM-CSF from polyp cells. Similarly, zaprinast, a selective PDE5 inhibitor, as well as theophylline, a non-selective PDE inhibitor, decreased the cytokine release only weakly without an obvious dose-dependency. Inhibition of the PDE3 isoenzyme by milrinone slightly decreased the release of TNF from both cell cultures in a dose-dependent manner (polyp cells: IC so 25 /-Lmolll; blood: IC so 21 lJ.rnolll), but had hardly any effect on GMCSF release (21 % inhibition by 100 lJ.rnolll). In contrast, selective PDE4 inhibitors drastically reduced the release of TNF from whole blood (IC so : rolipram 1.21J.rnolll, RPR 73401 0.08IJ.rnol/l, RS-25344 0.1 /-Lmolll, SB-207499 7 /-Lmolll, AWD 12-281 0.8 lJ.rnolll) as well as the release ofTNF (IC so : rolipram 0.1 lJ.rnolll, RPR 73401 0.014 lJ.rnolll, RS-25344 0.015 lJ.rnolll, SB-207499 0.1 /-Lmolll, AWD 12-281 0.09 /-Lmolll) and GM-CSF (IC so : rolipram 0.7 lJ.rnol/l, RPR 73401 0.022 lJ.rnolll, RS-25344 0.037 /-Lmolll, SB-207499 0.5 lJ.rnolll, AWD 12-281 ca. 0.8 /-Lmolll) from the polyp cells. In conclusion, our present results confirm the hypothesis that selective inhibition of PDE4 is a promising therapeutic strategy in the treatment of inflammatory and allergic disorders.

1Institute of Clinical Immunology-and Transfusion Medicine, 2Department of Pediatrics, University of Leipzig, Leipzig, and 3Center for Environmental Research (UFZ), Leipzig, Germany

L. 14 Suppression of the Th 1 cell response by indoor mould exposure I. LEHMANN 1, A. SEIFFART 1, A. MOLLER3, U. DIEZ3, H. WETZIG 2, F. EMMRICH 1, M. BORTE 2, and O. HERBARTH 3

There is an increasing evidence that indoor exposure to mould is associated with an impairment of health. Components of mould spores are known to induce respiratory symptoms. In children indoor exposure to moulds results in an increased incidence of infections and skin symptoms. Although th<: toxic effects of mould, especially mycotoxins, are well investigated, little is known about their influence on the immune system. Within a longitudinal cohort study (<
516 . 30th Annual Meeting 1999 ed immune responses. This is of importance since dominant Th2-reactivity promotes the development of allergic diseases in general and suppresses the host defense against virus infections.

Institute of Immunology, Johannes Gutenberg University, Mainz, Germany

L. 15 Inhibition of Th2-mediated allergen induced airway eosinophilia by Th 1 cells K. LINGNAU, S. GERECHT, A. MENZEL, and E. SCHMITT Airway eosinophilia is one of the characteristic features of asthma. Activation of this type of inflammatory cells is thought to cause at least the considerable tissue damage that accompanies asthmatic reactions. Eosinophils were shown to be recruited from the circulation into the lung tissue via several chemokines that were expressed by lung epithelial cells. Furthermore, the expression of these chemokines was shown to be regulated by Th2 cytokines, namely IL-4 and IL-9. Thus, Th2 cells playa key role with respect to the induction of an airway eosinophilia. Herein, we have investigated the role and cross-regulatory properties of transgenic OVA-peptidespecific Th 1 and Th2 cells with respect to the induction and maintenance of an allergen-induced airway eosinophilia. Thl and Th2 cells were established in vitro and transferred i.v. into BALB/c mice that were subsequently treated intranasally with OVA for 7 days (50 fJg/day). The number of eosinophils was determined in the BAL on day 8 after Th cell transfer. Transfer ofTh2 cells resulted in a profound lung eosinophilia concomitant with a considerable mucus production. OVA-peptide-specific ex vivo activation of lung cells and lung lymph node cells resulted in the production of IL-4 and IL-5 indicating that the transferred Th2 cells migrated to the lung compartment. By contrast, transfer ofIhl cells could not induce eosinophilia or mucus production. OVA-peptide-specific activation of lung cells and lung lymph node cells led to the production of huge amounts of IFN-y, thus indicating that the transferred Thl cells also migrated to the lung compartment. The transfer of a mixture ofThl and Th2 cells led to a strongly reduced eosinophilia when equal numbers ofThl and Th2 cells were used. Detailed analyses revealed that Th 1 cells can inhibit Th2-mediated airway eosinophilia in a concentration-dependent manner. Assessment of the cytokine production ex vivo using lung lymph node cells revealed that there was no difference in the production of IL-4 and IL-5 between Th cells from mice that received only Th2 cells or Th2 cells in combination with Th 1 cells. Therefore, these results indicate that Th 1 cells did not suppress airway eosinophilia via the inhibition of the eosinophiliapromoting cytokines IL-4 and IL-5.

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Universitats-Hautklinik, Freiburg, Germany

L. 16 Contact hypersensitivity induced by dendritic cell immunization is mediated by Tc 1 cells S. MARTIN and J.e. SIMON Our study aimed at the identification and characterization of the effector T cells induced after sensitization of mice for contact hypersensitivity (CHS) with hapten-modified bone-marrow derived dendritic cells (DC). We have used bone marrow-der~ed DC chemically modified with TNP to sensitize C57BL/6 mice for CHS. Five days later the same hapten was painted on both ears and ear swelling measured after 24 h. After TNP challenge on the ears, we detected an ear swelling response only in mice immunized with TNP-modified but not with unmodified De. When cells from draining axillary, auricular and maxillary lymph nodes (LN) ofTNP-DC immunized mice were anylyzed 72 h after ear challenge, we found a significant induction of IFN-y producing CDB+ effector cells as shown by ELISPOT and ELISA assays. Little TNP-specific lL-4 production was detected. These effector cells were clearly cytolytic towards TNP-modified EL-4 cells as shown after a three day restimulation in vitro with TNP-modified syngeneic spleen cells. They also lysed target cells pulsed with various synthetic TNP-modified peptides. To our surprise, we also found TNP-specific cytolytic CDB+ effector T cells after restimulation of lymph node cells from mice immunized with unmodified DC and then challenged by TNP painting on both ears. The TCR V~ repertoire did not show any significant bias. In conclusion, these results show that the hapten TNP is an unusually strong antigen which elicits a polyclonal T cell response mediated by cytotoxic, IFN-y producing T c1 cells. We speculate that the strong T cell response is due to a very high precursor frequency ofTNP-specific CDB+ T cells as seen in preliminary ELISPOT assays. This work was supported by the K1inische Forschergruppe «Pathomechanismen der allergischen Entziindung" BMBF FKZ: OIGC9701/7.

Department of Dermatology and Allergology, Medical School, Hannover, Germany

L. 17 Effects of the immunosuppressive cytokine IL·1 0 on human eosinophils H. PETERING, Y. DULKYS, D. KIMMIG, A. KApP, and J. ELSNER The production of reactive oxygen species by activated human eosinophils leads to an extensive destruction of the surrounding tissue at the site of inflammation and amplifies the severity of chronic inflammatory diseases such as atopic dermatitis and allergic asthma. These disorders exhibit a Th2-like cytokine pattern including IL-I 0 and are characterized by a peripheral blood and tissue eosinophilia. IL-I0 is a cytokine with immunosuppressive and anti-inflammatory effects produced by B cells, Th2-like T cells, monocytes/macrophages, and eosinophils that exhibits diverse activities on different cell types. On stimulated macrophages, IL-IO inhibits the synthesis of other cytokines, down-regulates the expression of MHC class II antigens and sup-

518 . 30th Annual Meeting 1999 presses the release of reactive oxygen species (ROS). Whereas effects of IL-l 0 on other hematopoetic cells are well-known, there exist only few data regarding the role of IL-l 0 for the activation of human eosinophils. The present study was performed to investigate whether IL-1 0 is able to modulate the respiratory burst in eosinophils. Therefore, human eosinophils were preincubated with IL-I0 and stimulated with eotaxin, MCP-4 and C5a. The respiratory burst of eosinophils stimulated by CC chemokines or by C5a was not influenced by IL-10. As a related event, actin polymerization induced by the same stimuli was measured in presence or absence ofIL-lO. Using NBD-phallacidin staining, no effects ofIL-l 0 on eosinophil activation could be observed. To investigate whether the expression of CD69, an activation marker for human eosinophils, can be influenced by IL-I0, flow cytometry was performed demonstrating that IL-I0 has no effect on CD69 expression on eosinophils primed with GM-CSF and IFN-y. In addition, the expression of CCR3 and the C5a receptor was analyzed showing that IL-10 does not affect the expression of both receptors. Finally, IL-10 receptor and mRNA expression was investigated in human eosinophils by measurement of [Ca2+]j and RT-PCR analysis. Both assays provided no evidence for the expression of IL-1 OR on eosinophils. Therefore, this study demonstrates that IL-l 0 does not affect the respiratory burst of human eosinophils and the expression of important cell surface receptors. Supported by DFG-Grant (EL 16016-1) and by the Hochschulinterne Leistungsforderung.

1Department of Immunology and Cell Biology, 2Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Borstel, and 3Division of Enviromental Dermatology and Allergology GSF/TUM, Munich, Germany

L. 18 Interaction of human peripheral blood eosinophils with bacterial lipopolysacharide S. PLOTZ l , H. BEHRENDT3, A LENTSCHAT l , L. HAMANN l , E.TH. RIETSCHEL 2, H.-D. hAD l , and AJ. ULMER l Eosinophils are important inflammatory cells in allergic diseases. The interaction of eosinophilic granulocytes with the bacterial lipopolysaccharide (LPS, endotoxin) has not been studied in detail so far. The aim of our investigation was to examine the activation of and binding to human peripheral blood eosinophils by LPS. Human blood eosinophils from healthy, non-atopic volunteers were isolated by density centrifugation on Ficoll-Paque and negative immunomagnetic selection using an anti-CD 16 mAb. This purification procedure resulted in an essentially pure eosinophil population (>98%). Eosinophils were cultivated in the presence of human serum for 16 h and stimulated by LPS and its endotoxic component, lipid A In the supernatants TNF-o. expression was determined by ELISA and the release of the eosinophil-specific granula protein ECP was estimated by RIA. The results show expression ofTNF-o. and ECP-release after stimulation with LPS and lipid A in a dose dependent manner. In blocking experiments with an anti-CD14 mAb (clone MEM-18) and the synthetic lipid A partial structure 406, the release ofTNF-o. and ECP by LPS- and Lipid A- stimulated eosinophils could be blocked in a dose-dependent manner. Binding studies with radioactively labeled LPS showed dose dependent binding of 3H-LPS to eosinophils. The 3H_ LPS binding was considered to be specific because preincubation with unlabeled LPS, com-

30th Annual Meeting 1999 . 519 pound 406 and also anti-CD 14-antibodies inhibited binding of 3 H-LPS to eosinophil granulocytes in a dose dependent manner. By flow cytometry using various directly and indirectly labeled antibodies and with the PCR-technique, CD 14 expression was found. Furthermore, mRNA expression of Toll-like receptors (TLR) 2 and TLR 4 was detected. The results indicate that eosinoe,!lils can be stimulated by LPS and are capable of binding LPS in a CD 14 dependent manner. Hence, in addition to allergens, eosinophils can interact with endotoxin, thereby possibly exacerbating ongoing inflammatory processes. Supported in part by DFG (SFB 367, project C5)

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

L. 19 Structure-function analysis of the cytoplasmic domain of the mast cell function-associated antigen (MAFA) R. SIEGEL, O. ROCKS, K. SCHLESSINGER, R. Xu, and I. PECHT Immunological stimulation of mast cells is initiated by clustering the type I FCE-Receptor (FcERI). A biochemical cascade is coupling it to the secretory response via the immunoreceptor tyrosine based activation motif (ITAM) located within the intracellular domain of the FCERI ~­ and y-chains. This cascade has recently been shown to be regulated by receptors containing a sequence called immunoreceptor tyrosine based inhibitory motif (lTIM). One of these lTIMbearing receptors, a type II transmembrane protein, was discovered in our lab in the rat mucosal-type mast cell line RBL-2H3 and named mast cell function-associated antigen (MAFA). It inhibits the FcERI induced signaling cascade and hence the cell's secretion. In order to deepen the understanding of the functional role of MAFA's specific structural elements we designed recombinant proteins containing different features of the MAFA sequence related to its function. The cDNA-sequences were cloned into an eucaryotic expression vector containing a selectable marker. First we stably transfected RBL-2H3 cells with a chimeric molecule consisting of the transmembrane and intracellular domains of MAFA and the extracellular domain of human CD23 (FcERII). Expression of this chimera was demonstrated by flow-cytometry using a FlTC-labeled hCD23 specific antibody. Secretion assays, performed on the clones of the highest expression levels, clearly showed an inhibitory effect upon clustering with antihCD23. In order to identifY MAFA's sites of interaction with other intracellular components we produced chimeric molecules with specific changes at MAFA's cytoplasmic domain. A chimera constituted of the hCD23 extracellular domain and MAFA transmembrane domain, but lacking its cytoplasmic tail was transfected into RBL-2H3 cells. Clones expressing this molecule at satisfactory levels were again tested for function by secretion assay. No inhibition of the secretory response could be resolved upon clustering this chimera. Taken together with the inhibition observed by the chimera containing the complete intracellular domain, these results provide clear evidence for localizing the inhibitory function to the cytoplasmic domain. Further constructs with single amino acid changes within the lTIM sequence (YSTL) were now cloned and their expression and functional screening are currently investigated.

520 . 30th Annual Meeting 1999 Support is acknowledged from the Israel National Science Foundation and the U.S.-Israel Binational Foundation (I.P.) and from the Ministerium filr Wissenschaft und Forschung des Landes Notdthein-Westfalen (R.S.)

lImmunobiology, Fraunhofer Institute of Toxicology and Aerosol Research and 2Department of Pediatric Pneumology, KinderkIinik, Medical School, Hannover, Germany

L. 20 Influence of immunization with Mycobacterium bovis Bacille-CalmetteGuerin (BeG) on the sensibilization to inhaled allergens after infection with respiratory syncytial virus (RSV) R.P. VONBERG 2, M. MOLLER 2, A. EMMENDORFFER 1, and]. FREIHORST 2 RSV represents an important infection of the lung in early infancy and may play an important role in the allergic diathesis by creating a Th2-rype immune response. BCG is discussed to induce a typical Thl type immune response and has been shown to reduce airway eosinophilia in a mouse model. To determine whether vaccination with mycobacterium bovis Bacille-Calmette-Gulrin is able to suppress the allergic potential of respiratory syncytial virus we vaccinated BALBI c-mice with BCG bacteria, infected with RSV and challenged with ovalbumin. Mice were sacrificed on day 6, 13 and 27 after allergen exposition. Alveolar macrophages and lymphocytes from spleen and lungassociated lymphnodes were investigated for cytokine-production and proliferation. Serum was tested for allergen-specific IgE. Chemokine- and cyrokine-RNA oflung tissue has been analyzed by RNAse-protection-assay. We could show an increased secretion of OVA-specific serum-IgE in all groups. IL-4-secretion by spleen lymphocytes after ConA-stimulation was five-times increased after BCG-vaccination on day 6 after allergen exposition. The production decreased gradually in the vaccinated group, increased in the non-vaccinated group and reached equal levels at the end of the observation period as tested by ELISA. IFN-y-secretion by spleen lymphocytes of the vaccinated group continued to raise over time, while no effects could be shown in the non-vaccinated group. Proinflammatory cyrokines (IL-6 and TNF-a) were significantly increased and prolonged in the vaccinated group on day 13 and 27 as tested by bioassay. Proliferation of thoracic lymphnode cells after in vitro stimulation started earlier and to a greater extent in the vaccinated group as tested by 3H-incorporation. No difference in chemokine- or cytokine-RNA of lung tissue between RSV-infected animals regardless of previous BCG-vaccination was found. However, RNA,:leveis ofTNF-a, IL-l, IL-lra, and IFN-y were significantly elevated in a control group that had received BCG-immunization but no RSV-infection. MCSF- and Len-levels were also higher in this group although levels failed to reach statistical significance. Our data demonstrate increased activation levels of all tested cell compartments concerning Th 1 and Th2 type cyrokines after BCG-vaccination. Further studies are needed to discover whether IL-4-secretion switches after day 27. Although lung eosinophilia was diminished by

BCG-immunization, no influence on IgE-leveis could be shown indicating that local inhibition of allergic symptoms, bur not systemic protection has been established.

30th Annual Meeting 1999 . 521 11nstitute of Clinical Immunology and Transfusion Medicine, University of Leipzig and 2Center for Environmental Research (UFZ), Leipzig, Germany

L. 21 Modulation of in vitro cytokine production by aromatic hydrocarbons (AHs): Indications for participation of AHs in the development of allergies

Objectives: Environmental pollutants, especially AHs, are assumed to be factors in the recently increasing incidence of IgE-mediated allergies. The mechanisms are poorly understood but may involve increased synthesis of cytokines. Thus our aim was to measure immunomodulating effects of AHs on human cells in vitro. Methods: We developed a suitable cell culture system for measurement of immunomodulating effects of AHs on human peripheral blood mononuclear cells (PBMC). The Ficoll Hypaqueseparated cells of healthy blood donors were prestimulated (LPS from E.coli [055:B5], monoclonal antibodies against CD3£ [UCHT-1] and CD40 [B-B20], each at 100 ng/ml) before adding the dimethylsulfoxide-solved AHs. The following AHs were tested: anthracene, benzo[a]pyrene, benzo[e]pyrene, dibenzo[a, h]anthracene, 7,12-dimethylbenzo[a]anthracene, p-benzoquinone. As indicators of immunomodulation we chose proliferation eH-thymidine incorporation) and the secretion of cytokin~ (IFN-y, IL-1~, -2, -4, -5, -6, TNF-a) and immunoglobulins (IgA, IgE, IgG, IgM), each measured with highly sensitive sandwich ELISAs. Results: All AHs increased the cytokine production only of stimulated PBMC in a dosedependent manner and caused individual different cytokine-profiles (CPs), either Th 1- or Th2dominated. These opposite CPs are correlated with a corresponding immunoglobulin production, i.e. under influence of the above mentioned AHs in concentrations like those present in our environment, the PBMC from Th2-dominated donors showed an increased IgE-production. Conclusions: The donor-dependent modulation of cytokine production by AHs supports the idea of individually different regulatory effects of AHs. Furthermore, it provides an indication for a participation of AHs in the induction of IgE synthesis via enhanced cytokine production. Antigen-nonspecific regulatory effects of AHs are leading in vitro to significant increased IgEsynthesis of PBMC only from Th2-dominated donors (almost half of the donors). Therefore AHs might at least partially be responsible for the development of allergies by susceptible human beings. Supported by the UFZ Leipzig/Halle, Leipzig, Germany, Grant-No. UFZ-12/95.

522 . 30th Annual Meeting 1999 1 Division of Immunopathology, Institute of General and Experimental Pathology, University of Vienna, Vienna, Austria and 21nstitut fur Laboratoriumsmedizin und Pathobiochemie, Charite, Campus Virchow-Klinikum, Berlin, Deutschland

L. 22 Intranasal tolerance induction with Bet v 1 and non anaphylactic frag-

ments of Bet v 1, the major birchpollen allergen, in a murine model of allergic asthma

u. WIEDERMANN 1, S. VRTALA 1, U. HERZ2, U. NEUHAUS-STEINMETZ2, H. RENZ2, R.

VALENTA 1, C. EBNER 1, and D. KRAFT 1

The prevalence of type I allergy, frequently elicited by airborne allergens, has constantly increased within the recent years. Birchpollen (BP), and its major allergen Bet vi, represent an important source of eype I allergens. Recently, Bet vi, the major birch pollen allergen, was expressed in E.coli in two halfs, together covering the sequence of the whole Bet v 1 molecule. In contrast to the entire Bet v 1, the two fragments lost their IgE binding capaciey, which was demonstrated in the human system and also recently in BP-sensitized BALB/c mice. We have established a murine model of aerosol sensitization to BP leading to type I allergic immune responses and airway hyperresponsiveness in BALB/c mice. We demonstrated that mucosal administration of recombinant Bet v 1 prior to sensitization led to allergen-specific suppression ofT and B cell responses in vivo and in vitro, reduction of eosinophilic airway infiltration and inhibited airway hyperresponsiveness. Subsequently we used the two non-anaphylactic fragments of Bet v 1 for intranasal tolerance induction. Intranasal administration prior to sensitization of the fragments, in particular of the one containing the major T cell epitope of Bet v 1 in BALB/c mice, led ro significantly reduced lymphoproliferative responses, diminished IFN-y and IL-5 production in vitro. Moreover, eype I skin tests were weaker or negative in the majority of the pretreated compared to the sensitized mice. In conclusion, mucosal/intranasal treatment with such non-anaphylactic allergens could provide a promising and risk-free alternative for conventional immunotherapy in type I allergy.

Department of Dermatology and Allergology, Medical School, Hannover, Germany

L. 23 CD40L is a main regulator for IL-12 production by human monocytes M. WITTMANN, P. SCHMIDT, G. BEGEMANN, A. KApP, and T. WERFEL IL-12 is a key eytokine in skewing immune responses towards Thl like reactions. It is well known that an optimal IL-12 production is only achieved ifIL-12 producing cells are stimulated with two signals. The first one is the «priming signal» (IFN-y or GM-CSF), the second one the «challenging" or «second signal». Human antigen presenting cells produce high amounts of bioactive IL-12 if the priming signal precedes the second signal (e.g. LPS). We have previously shown that preincubation with LPS before the priming and second signal can efficiently and selectively suppress the production of IL-12 by human monoeytes. We now show that this finding apparently represents a general regulatory mechanisms also relevant for physiologic stimuli

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in vivo. An almost complete suppression of IL-12 production can be observed if the cells are preincubated with TNF-a, sCD40L or CD40L+ T cells. This suppression of IL-12 is observable on the mRNA and protein level and is not due to endogenous production ofIL-10, IL-4, PGE 2 or an increased number of cells undergoing apoptosis. As CD40L+ T cells are present in early eczematous skin lesions shown by immunohistochemical staining we here provide evidence for a mechanism responsible for a Th2-like eytokine milieu in early eczematous skin lesions. This mechanism may also help to explain phenomena observed in infectious diseases and may have an impact on therapeutical approaches for autoimmune and allergic disorders.

lInstitut filr Immunologie, Ludwig-Maximilians-Universitat, Milnchen, Germany and 2Department of Molecular Immunology, Reseatch Institute of Microbial Diseases, Osaka University, Osaka, Japan

L. 24 IgE production in vivo without T·8 cell collaboration and Th2 cytokine production P. yu l and H.

KIKUTANI

2

It has been discussed that at an early stage IgE production in allergic patients might be induced by viral infections. Here we present a mouse model for IgE induction in vivo by a retrovital infection. To study this topic we have infected CD40-deficient mice with BM5-def retrovirus, which is the causative agent for a acquired immunodeficiency syndrome in mice (MAIDS). We show that MAIDS infection promotes class switch to IgG 1 and IgE even in CD40-deficient mice. Furthermore we present data that the class switch duting direct infection of B cells by the virus is not accompanied by upregulation ofTh-2 eytokines in vivo.