Workshop L Cell Adhesion — Cell Traffic

'Department of Dermatology, University of Freiburg, and 2Institut fUr Genetik, Forschungszentrum Karlsruhe, Germany

L. 1 C044 is the principal hyaluronate receptor on activated human mono· cytes

TH. AHRENS I,]. C. SIMONI, A. C. RENK I,]. MOLL 2, B. H. MAIl, E. SCHOPF I, and]. M. WEISS' During cutaneous inflammation, activated monocytes (Mo) migrate into skin where they interact with ECM components like hyaluronate (HA), produced in high amounts at inflammatory sites. We wished to determine whether activation affects the capacity of Mo to interact with HA and to express putative HA-receptors i.e. the constant form of CD44 (CD44s) and CD44 isoforms generated by alternative splicing (CD44v), ICAM-l or RHAMM. Mo freshly purified from peripheral blood bound little HA and expressed CD44s but not epitopes encoded by CD44 variant exons, ICAM-l or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-l, CD44s, epitopes encoded by CD44 variant exons v3, v4, vS, v6, v7, v9 but not RHAMM, all of which were further augmented by IFN-y or LPS, while RHAMM remained uninducible. LPS or IFN-y-induced HA binding was inhibited by more than 90% with mAbs directed against N-terminal HA binding domains of CD44s but not by a panel of mAbs against other CD44 epitopes including those encoded by CD44 variants or ICAM-l. Tunicamycin, an inhibitor of N-linked glycosylation, reduced IFN-y-induced HAavidity and binding of panCD44 mAbs recognizing the HA-binding domains. This tunicamycin effect was due to a down-modulated expression of mature glycosylated forms of CD44 rather than a reduction in CD44 gene transcription as shown in Western and Northern blot, respectively. In lesional skin of contact dermatitis, but not in normal skin, activated infiltrating Mo displayed high HA-avidity and expressed panCD44, CD44v, ICAM-l, but no RHAMM, thus resembling exactly in vitro activated Mo. In conclusion, we show that upon in vitro or in vivo activation Mo enhance their capacity to bind HA. This is critically dependent upon the expression of CD44-isoforms containing distinct glycosylated N-terminal HA-binding domains. Such regulated CD44-HA interactions may be important for the ability of Mo to migrate into and within sites of cutaneous inflammation and induction of their effector functions.

German Cancer Research Center, Heidelberg, Germany, and IUniversity of Virginia, Charlottesville, USA

L. 2 C024 on human tumor cells supports adhesion to and rolling on p. selectin in the absence of PSGL-l S. AIGNER, M. LAWRENCE', K. LEY I and P. ALTEVOGT Leukocyte recruitment in inflammation is initiated by the capture of leukocytes from the blood stream followed by their rolling along the endothelium. This first and essential step of the recruitment paradigm is mediated by the binding of selectins to their ligands. The best character-

302 . 28th Annual Meeting 1997 ized selection ligand is P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 appears to account for all or most of the P-selectin dependent rolling of myeloid cells in vivo and in vitro. Here we investigated the P-selectin dependent binding and rolling of a breast carcinoma cell line (KS) that is negative for PSGL-l. We show that CD24, a mucin type GPI-linked glycoprotein on this cell line can promote shear stress dependent rolling on coated P-sel-IgG under flow. The rolling was dependent on divalent cations and was abolished after treatment with an anti-P-selectin antibody. Incubation of KS cells with Phosphatidylinositol-Phospholipase C (PI-PLC), which specifically cleaves GPI-linked molecules, including CD24, blocked adhesion to P-selectin by appro 66% and caused a clear change in the rolling behavior of single cells. In the presence of soluble CD24 (sCD24) KS.celis showed a 50% decreased attachment rate. P-selectin transfectants (300.19P) adhered to coated sCD24 in a P-selectin dependent manner, suggesting further that CD24 can serve as a ligand for P-selectin. Furthermore, we could demonstrate that the attachment rate of KS cells to P-sel-IgG correlates with the CD24 expression level. Cells sorted for high and low expression of CD24 showed normal or decreased binding, respectively. However, CD24 seems to play no role on HL-60 promyelocytes. Soluble CD24 and PI-PLC treatment failed to inhibit rolling of HL-60 cells, whereas an antibody against PSGL-1 completely abolished the rolling. These results establish a physiological role of CD24 as a novel functional counter-receptor for P-selectin in the absence of PSGL-l. Since CD24 is re-expressed by many human carcinomas, this binding pathway could be important in tumor dissemination during hematogenous metastasis.

Department of Dermatology, University of Wiirzburg, Medical School, Wiirzburg, Germany

L. 3 Chemokines are differentially expressed during normal healing of human wounds E. ENGELHARDT, A. TOKSOY, M. GOEBELER, E. B. BROCKER, R. GILLITZER

Leukocytes (i.e. macrophages, neutrophils and lymphocytes) are increasingly considered as important producers of growth factors during wound healing. To better understand the role of leukocytes during wound healing spacial distribution and time kinetics of leukocyte immigration were correlated with the expression profile of chemotactic cytokines (chemokines) in a human model of artificial wounds (day 0, 1,2,4,7, 10, 14 and 21) employing in situ hybridisation. Maximum levels of interleukin-8 (lL-8) message were detected on the wound surface after 1-2 days and declined thereafter. Growth related oncogene (GRO) mRNA expression was weaker and additionally also detectable at the wound edges whereas ENA-78 mRNA expression was negligible. Expression of IL-8 and GRO was timely and spatially correlated with neutrophil recruitment indicating that both are important regulators of neutrophil chemotaxis during wound healing. Strong infiltration of macrophages (day 1-10) coincided with monocyte chemoattractant protein-1 (MCP-1) expression whereas expression of other macrophage attractant chemokines (MIP-1a/~, RANTES, MCP-3) was quiescent. Initially, MCP-1 was highly expressed in basal keratinocytes at the wound edge (proliferative compartment) as well as within the wounded areas with high macrophage densities. Infiltration of lymphocytes was correlated with high levels of both MCP-1 and monokine induced by IFN-y (MIG) expression, the latter being maximally expressed after 4-7 days. Expression patterns of IP-10 resembled those of MIG but showed weaker intensity. In summary, our data demonstrate that the differential recruitment of leukocytes is associated with distinct expression patterns of IL-8, GRO, MCP-1 and MIG suggesting an important role for chemokines in wound healing.

28th Annual Meeting 1997 . 303 Department of Dermatology, University of Wiirzburg and 'Institute Immunology, University of Witten/Herdecke, Germany

L. 4 Divergent migratory properties and phenotypes emerging from in vitro generated human dendritic cells upon culture in three Climensional collagen lattices

P. FRIEDL, P. KEIKAVOUSSI, W. FRIES, M. GUNZER 1, K. ZANKER!, E. B. BROCKER and E. KAMPGEN Human dendritic cells (DC) have become a promising adjuvant tool for various tumor vaccination protocols. The immunostimulatory potency of DC encompasses their capacity to migrate into lymphatic tissue and present MHC/peptide complexes in conjunction with several costimulatory signals. To analyse the phenotypic stability of in vitro generated human DC within an in vitro tissue environment, we assessed DC morphology, migration and expression of surface markers upon culture within 3-D collagen lattices using time-lapse videomicroscopy and computer assisted cell tracking. Spontaneously motil DC, obtained from monocytic precursor cells with GM-CSF and IL-4, ceased migration within the collagen lattice and developed into sessile «epitheloid»-like cells of remarkable length (40->100 Jlm) and bi- to tri-polar morphology. These cells, when carefully released from the matrix, showed reduced expression of MHC class II and costimulatory molecules (e.g. B7) assessed by FACS analysis, whereas monocytic markers (e.g. CDIIS) were upregulated. In contrast, DC cultured with a monocyteconditioned-medium (MCM) remained motile over a prolongued time-period and displayed a more stable DC morphology and receptor expression. These results suggest that, depending on the culture conditions, in vitro generated DC may develop into different morphological and functional phenotypes the outcome of which may crucially affect the specificity and efficacy of DC based vaccination approaches.

Institute of Immunology, University of Witten/Herdecke, 'Department of Dermatology, University of Wiirzburg, Germany

L. 5 Leukoc~s (T lymphocytes, dendritic cells, monocytes) utilize ~ 1 integrin independent migration strategies for polarization, interaction with collagen fibers, and migration

P. FRIEDL', E. KAMPGEN', F. ENTSCHLADEN, B. NIGGEMANN, E.-B. BROCKER', and K. S. ZANKER Migration of different cell types within the tissues is thought to involve the coordinated integrin-mediated adhesion to and deadhesion from ligands of the extracellular matrix. Using a highly sensitive cell migration approach combining a 3-D collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking, and confocal laser-scanning and reflection microscopy, we describe T cell migration as a process which (1) involves interactions with collagen fibers at the leading edge and uropod likewise, (2) occurs independent of the clustering of pI integrins, F-actin, focal adhesion kinase, and protein kinase C at interaction sites to collagen fibrils, and (3) cannot be blocked by a panel of adhesion-perturbing anti-pl, -P2, -P2, and -av antibodies. Adhesion blocking of pI integrins did not alter cell polarity, directed interaction with fibers, migration velocity, path structure, nor the percentage of locomoting non-activated T cells as well as CD4+ Con-A-blasts. Furthermore, Pi integrin redistribution as well as intracellular tyrosine phosphorylation of FAK remained unaffected by blocking antibodies.

304 . 28th Annual Meeting 1997 The concept of ~1 integrin-independent migration in leukocytes was confirmed using spontaneously migrating in vitro generated human dendritic cells from peripheral blood as well as promononcytic U937 cells the migration of which remained equally active after ~1 integrin blocking. In summary, T cell migration in a 3-D collagen matrix, a most efficient, rapid, and adaptive process lacking specific llq, -~2, -~2, and -av integrin-mediated adhesion events might exemplify a specialized leukocytic type of cell migration. Consequently, leukocytic migration stategies could be fundamentally distinct from less rapid and focal adhesion-dependent migratory action of other cell types including fibroblasts and tumor cells.

Genzentrum Miinchen, Miinchen, Germany

L. 6 Functional map~ing of the CD 18 cyh?plasmic domain and the role of the cytoskeleton in the Cytohesin-1 regulated adhesion of LFA-1 to ICAM-1

C. GEIGER and W. KOLANUS We recently identified an intracellular protein, cytohesin-l, which specifically interacts with the cytoplasmic domain of the integrin ~2 chain and which apparently is involved in the intracellular control of LFA-l mediated adhesion to ICAM-l. In an attempt to gain a more precise understanding of cytohesin-l function, we first mapped its binding domain with the CD18 cytoplasmic sequence using the yeast two-hybrid technique. Deletion analysis as well as site directed mutagenesis of the CD18 cytoplasmic tail yielded two point mutants which were either completely or largely compromised with respect to their interaction with cytohesin-l. We then used recombinant vaccinia viruses to express these mutants in the context of the full length LFA-1 protein in K562 cells, a cell type which usually does not express LFA-l. Cell adhesion analysis revealed that both mutants were strongly affected in their ability to bind ICAM-l. These findings underscore the importance of the regulatory function of a region within the CD18 cytoplasmic domain which is responsible for the interaction with cytohesin-l. Additionally, we examined the role of the cytoskeleton according to the cytohesin-l-mediated adhesion of LFA-l to ICAM-l. The adhesion of Jurkat cells (E6) to ICAM-l after overexpression of cytohesin-l via the vaccinia virus expression system was almost unaltered when the cells were incubated with Cytochalasin D, a reagent which inhibits actin polymerization. This result suggests that under the conditions used cytohesin-l may influence the affinity rather than the avidity of the integrin LFA-l to ICAM-l.

Institute of Immunology, University of Witten/Herdecke, lDepartment of Dermatology, University of Wiirzburg, Germany

L. 7 Tissue source and maturation stote determine the spontaneous and

cytokine driven migration pattern of murine dendritic cells and Langerhans cells within a 3-D collagen lattice

M. GUNZER, P. FRIEDL', B. NIGGEMANN, K. ZANKER, E.-B. BROCKER', and E. KAMPGEN 1 Active migration enables dendritic cells (DC) to transport antigen from the periphery into lymphatic organs to stimulate T cells. Numerous studies have addressed DC migration, however the migratory capacity of different DC subsets and properties of individual migrating cells are unknown. We embedded highly enriched DC from spleen or freshly purified epidermal Langerhans cells (fLC) within 3-D collagen lattices as substratum for migration. The migration of indi-

2Sth Annual Meeting 1997 . 305 vidual cells and cell populations were monitored for up to 120 h using videomicroscopy and computer assisted cell tracking. In the absence of exogenous cytokines, both spleen DC and fLC showed immediate and vigorous spontaneous migration. Spleen DC migrated at high initial activity (>SO% migrating cells) for at least 10 h which was not further enhanced or prolonged by cytokine effectors (GM-CSF, IL-I0). Unstimulated fLC showed an initial migratory onset of 15-45% migrating cells after 10 h steadily decreasing thereafter. In contrast to DC, addition of GM-CSF or TNF-a prolongued detectable migratory activity in fLC (up to 120 h), accompanied by morphological transition from initially round cells with small filopodia to a highly dendritic morphology with long veiled pseudopods. In conclusion, the spontaneous migration of spleen DC and freshly isolated LC is an inherent property of the cells after contact with a collagen environment, which, in the case of fLC, is maintained and further modulated by a proinflammatory environment.

IMolecular Immunology, Robert Koch-Institute, Berlin, 2Haemostasis Research Unit, MaxPlanck-Institut fur Physiologische und Klinische Forschung, Bad Nauheim, and 3Deutsches Herzzentrum, Berlin, Germany

L. 8 TRAP (C040 ligand) on activated platelets triggers a pro-inflammatory reaction of endothelial cells

v. HENN1,J. R. SWPSKy2, M. GRAFE 3, G. MULLER-BERGHAUS2, R. A. KROCZEK 1 TNF-related activation protein (TRAP, CD154) was originally identified on stimulated CD4+ T cells. Interaction of TRAP on T cells with CD40 on B cells has been shown to be of paramount importance for the development and function of the humoral immune system. Recent work has revealed that CD40 is not only constitutively present on B cells but also on monocytes, macrophages and endothelial cells, suggesting a broader function of TRAP in vivo. We now report that platelets carry preformed TRAP molecules which are expressed on the platelet surface within seconds of activation. Like TNF-a and IL-l, TRAP on platelets induces endothelial cells to secrete chemokines (IL-S, MCP-l) and to express adhesion molecules (VCAM-l, ICAM-l, E-selectin), thereby generating signals for the recruitment and extravasation of leukocytes at the site of vessel injury. Thus our data indicate for the first time that platelets are not only involved in haemostatis but also directly initiate an inflammatory response of the vessel wall. This observation contributes no the understanding of the local inflammatory reaction and is relevant for the pathogenesis of atherosclerosis.

Departments of IClinical Immunology, 2Anaesthesiologie, and 3Clinical Psychiatry, Hannover Medical School, Hannover, Germany

L. 9 Opioid-induced effects on cells of the immune system R. JACOBS!, M. KARST2, D. SCHEINICHEN2, SCHEDLOWSKI3, R. E. SCHMIDT l

c.

BEVILACQUA\ U. SCHNEIDERJ, J. HEINE 2, M.

Opioids are commonly used anaesthetic substances. These drugs act via specific 0, K, and \l receptors on cells of the nervous system. It has also been clearly shown that cells of the human hematopoietic system express 0 and K opioid receptors. However, the presence of \l receptors on white blood cells is still under discussion. The role of opioid receptors on lymphocytes is not well understood until now. In this study seven healthy male individuals were treated intra-

306 . 28th Annual Meeting 1997 venously (0.2 pg/kg bw) with a single dose of the opioid fentanyl, five subjects received placebo (saline). Blood samples were drawn at three time points: before (Tl), 15 (T2) and 30 (T3) min after application. Respiratory burst of PMNL and phenotypic properties of peripherallymphocytes were analysed by FACS. In vitro effects of fentanyl on NK activity was assessed. Fentanyl injection did not affect superoxide production of PMNL ex vivo. In contrast an increased number of NK cells could be observed in response to fentanyl. NK cells more than double in the Fentanyl group from 184 cells/pI at T1 to 410 cells/pI at T2 decreasing to 340 cells/pI at T3, whereas NK cell numbers remained unaffected in placebo treated subjects (205, 118 and 129 cells/ml respectively). In contrast, fentanyl did not influence NK activity in vitro. In conclusion, fentanyl affects in vivo circulation of NK cells but not their function.

Diabetes Research Institute at the Heinrich Heine University Dusseldorf, Clinical Department, Dusseldorf, Germany

L. 10 Autoimmune insulitis and diabetes are suppressed in NOD mice with deficient intercellular adhesion molecule (ICAM)-l gene S. MARTIN, E. HEIDENTHAL, B. SCHULTE, A. VINKE, H. KOLB The pathogenesis of autoimmune (type 1) diabetes is characterized by infiltration of pancreatic islets with mononuclear cells and destruction of insulin-producing ~-cells. The pathophysiological role of ICAM-1 during the pathogenesis of type 1 diabetes is not known. Expression of ICAM-1 in pancreatic islets can be induced by cytokines (CAMPBELL, Proc. Nat!. Acad. Sci. USA, 86: 4282, 1989). However, we observed ICAM-1 expression in prediabetic islets only on infiltrated cells, but not on ~ cells. (Martin, J. Autoimmun., 9: 637, 1996.) To analyse the particular role of ICAM-1 during, chronic islet in inflammation we have bred a deficient ICAM-21 gene (Xu, J. Exp. Med., 180: 95, 1994) into NOD mice, a spontaneous animal model for human type 1 diabetes. The diabetes-specific genes Idd1, Idd3 and Idd4 were monitored during breeding. Animals of the 3,d and 4th back-cross generation were intercrossed and 30 ICAM-1-expressing(o =9, 'l' =21)and 14 ICAM-1-deficient(o =6, 'l' =8)NODmice were obtained. All animals were observed until an age of 360 days. 11 female ICAM-1expressing NOD mice (52%) developed diabetes while no case of hyperglycemia appeared in ICAM-1-deficient NOD mice. Histological examinations revealed severely infiltrated pancreatic islets in ICAM-1-expressing NOD mice. In contrast, no infiltrations were observed in ICAMI-deficient NOD mice (p < 0.001). In conclusion, these results demonstrate a key role of ICAM-1 during infiltration of pancreatic islets and the pathogenesis of type 1 diabetes in NOD mice. Because ICAM-1 is located on the 9th chromosome near to the unidentified diabetes-specific gene Idd2, these data indicate a possible identity of the Idd2- and ICAM-1 gene.

Laboratory for Molecular Biology, Genzentrum der Universitat Munchen, Munchen, Germany

L. 11 PI 3-kinase r~ulates membrane recruitment of ~hesin-1 via its PHdomain and tIlereby activates the aL~2 integrin adhesion pathway

W. NAGEL, L. ZEITLMANN, P. SCHILCHER, C. GEIGER, J. KOLANUS and W. KOLANUS Most recently we have identified cytohesin-1, a cytoplasmic mediator of LFA-1 inside-out signaling [KOLANUS, W., NAGEL, W., SCHILLER, B., ZEITLMANN, L., GODAR, S. STOCKINGER, H. & SEED, B. (1996) Cell 86, 233-242]. The molecule contains two distinct polypeptide

28th Annual Meeting 1997 . 307 motifs a) the central SEC7 domain showing homology to the yeast SEC7 gene product and b) the carboxy-terminal pleckstrin (PH) domain. Although the latter does not interact directly with the cytoplasmic tail of the ~2 chain, the expression of the isolated PH domain leads to inhibition of T cell receptor-stimulated adhesion. We show here that this dominant-negative inhibition on aL~2 integrin function was abolished by mutation of a residue which has previously been shown to be important for membrane recruitment and binding of phosphoinositides of other PH domains. In vitro binding studies revealed, that the cytohesin PH domain binds to inositol (1, 3, 4, 5) tetrakisphosphate with high specificity, whereas the mutant fails to interact with this compound. In concordance with these data the association of the isolated PH-domain mutant with the plasma membrane was abrogated. As was implied by the observed ligand specificity of the PH-domain, a constitutively active version of phosphoinositide 3-0H kinase (PI 3-kinase) induced aL~2 adhesion to intracellular adhesion molecule 1 ICAM-l) as well as enhanced membrane recruitment of endogenous cytohesin-l. Thus, cytohesin-I appears to be an intermediate regulatory molecule in the regulation of integrin adhesiveness by PI 3-kinase.

Institute of Medical Immunology, Martin Luther University Halle, Germany

L. 12 Human lymphocyte adhesion is enhanced by expression of aminopeptidase N/CD13 D. RIEMANN, K. THIELE, A. KEHLEN, J. LANGNER T cells isolated from renal cell cancer [1] or synovial fluid of patients with various forms of arthritis [2] express the «myeloid» marker aminopeptidase N/CD13. Coincubation of tonsillar lymphocytes or lymphocytic cell lines (e.g. HUT78, Jurkat) with renal carcinoma cells or fibroblast-like synoviocytes results in a rapid induction of lymphocytic expression of CD13 protein and mRNA [3]. Since the membrane ectopeptidase CD13 has been shown to lay a role in adhesion and migration processes of melanoma cells [4, 5], we investigated whether CD13 expressing lymphocytes differ with respect to their adhesive properties. We used the new adhesion assay of GOODWIN et al. [6] that utilizes buoyancy to remove non-adherent cells. Lymphocytes were cocultured with ECV304 cells (no induction of CD13 expression on lymphocytes) or EDY.lds cells (transformed with an expression vector for CD13; kindly provided by LOTIE VOGEL, Panum Institute Copenhagen; lymphocytes become CD13 in coculture). After biotinylation, lymphocytes were cultured in wells coated with gelatine, fibronectin, collagen type I or Matrigel. For the detection of adherent lymphocytes treptavidin-peroxidase-labelling was used. Our results provide evidence that CDI3+ compared to CD13 negative lymphocytes show a significantly increased cell-to-substrate adhesion. Our results suggest a possible role of lymphocytic CD13 expression in adhesion to extracellular matrix. This work was supported by DFG (SFB 387). References [1] RIEMANN D et al Immunol Lett 42 (1994) 19-23. [2] RIEMANN D et al Immunobiol 187 (1993) 24-35. [3] RIEMANN D et al J Immunol158 (1997) 3425-3432. [4] FUJII H et al Clin Exp Metastasis 13 (1995) 337-344. [5] MENRAD A et al Cancer Res 53 (1993) 1450-1455. [6] GOODWIN AE et alJ Immunol Methods 187 (1995) 213-219

308 . 28th Annual Meeting 1997 IDept. of Tumor Progression and Immune Defense, DKFZ, Heidelberg, 2Dept. of Dermatology, University Homburg, Germany; lBasei Institute for Immunology, Basel, Switzerland

L. 13 Involvement of C044 variant isoforms in TH 1 and TH2 delayed type hypersensitive

Blockade or introduction of costimulatory molecules can be of clinical importance in transplantation, autoimmunity and cancer. Hence, there is ample interest in uncovering the whole range of molecules possibly being involved as well their preferential target and pathways of signal transduction. CD44 has been described to be one of these accessory molecules, Yet, the question, which of the multiple CD44 isoforms are involved and whether different isoforms mediate distinct functions has not been answered. We here addressed the question of CD44v3, CD44v6, CD44v6, CD44v7 and CD44v10 being involved in T H1 and T H2 reactions using as model systems for T H1 activation a DNFB-induced DTH reaction and for T H2 activation a FITC-induced allergic dermatitis. The variant exons CD44v3, CD44v6, CD44v7 and CD44v10 were not detected on resting lymphocytes. During activation, subpopulations of T cells expressed CD44v6 and CD44v7 whereas expression of CD44v3 and CD44v10 was mainly detected intradermally and not restricted to leukocyte subpopulations. In vitro studies revealed that antibodies against both CD44v6 and CD44v7 inhibited T lymphocyte proliferation, anti-CD44v10 interfered predominantly with B cell activation, while anti-CD44v3 provided a proliferative stimulus, In vivo, anti-CD44v6 as well as anti-CD44v7 significantly mitigated the DNFB-induced, T H1-mediated DTH reactions, while only anti-CD44v7 interfered with a FITC-induced, T H2-mediated contact dermatitis. The in vitro analysis of cytokine producing cells supported the assumption that anti-CD44v7 interfered with T H1 and T H2 expansion/activation, whereas CD44v6 appeared to be involved only in T H1 reactions. Anti-CD44v10 was most efficient in suppressing T H1-mediated DTH reactions by interfering with monocyte recruitment and activation, without exerting a major influence on T H activation. Anti-CD44v3 strongly reduced T H1 and THZ DTH reactions. In conclusion, it could be demonstrated that 4 distinct CD44 variant isoforms are differentially involved in DTH reactions by either interfering with T H1 and T H2 activation, with monocyte activation or with leukocyte recruitment. Regarding the efficient blockade of DTH reactions by antibodies recognizing these accessory molecules, which are exclusively expressed during lymphocyte activation, side effects in clinical application should be minimal.

Pediatric Cardiology, Herzzentrum GmbH, University Leipzig, Germany, and ISemmelweis University of Medicine, 1st Institute of Pathology, Budapest, Hungary

L. 14 Is cardiopulmonary bypass-induced loss of lymphocytes due to their migration into the peripheral tissue? A. TARNOK,]. BOCSII,J. HAMBSCH, P. SCHNEIDER Cardiac surgery with cardiopulmonary bypass (CPB) can induce post-operative capillary leak syndrome (CLS) and multi organ failure. The reason for CPB induced CLS has not been clarified yet. During CPB the loss of activated leukocytes from the peripheral blood (PB) has been described (Faist: The Immune Consequences of Trauma, Shock and Sepsis. Monduzzi Editore, Italy, 1998, pp. 129). B-cell counts decreased by >50% due to the loss of 86% of early activated (CD69+) cells. The fraction of activated T-cells (CD69+, CD25+ or CD54+) decreased by 70%. Furthermore, a decrease in CD11a/CD18 and CD45RO expression was observed. The aim of this study was to investigate whether these cells get lost by binding to the CPB or by migration

28th Annual Meeting 1997 . 309 into the peripheral tissue and/or cell death. Six CPB open heart operations were studied. PB samples were collected at the beginning and the end of CPB (control). The various parts of CPB (filters and the oxygenator) were washed. The obtained leukocytes were immunophenotyped by three or four color flow cytometry. From the CPB of a high number of intact leukocytes (1-2xl0 9 cells) was recovered (at least 10% of all PB leukocytes). The filtration of PB cells was selective. The proportion of CD69+ T, Band NK cells was increased by up to 50% (p < 0.05). In contrast, the proportion of T cells, with the activation markers CD25, CD54 or HLA-DR were lower in the CPE. Measurements with Annexin V and propidium iodide did not yield detectable increase of damaged or apoptotic PB lymphocytes during CPB and no detectable increase of apoptosis in the CPE. Our data show that early activated T, NK cells and B cells adhere selectively to CPB filters. The loss of activated T cells could in part be due to selective migration into the peripheral tissue and not to cell death in the PB. Due to their increased ability to produce and secret cytokines these cells could in part be inducers of post-operative CLS. (Supported by the Sachsisches Ministerium fUr Wissenschaft und Kunst).

Department of Dermatology, Freiburg, Germany

L. 15 Presentation of the basic fibroblast growth factor (b-FGF) by C044

isoform containing exon v3 (C044v3) stimulates keratinocyte proliferation in normal skin and in epidermal skin cancers

c. C. TERMEER, H. U. GRUMME, A. STINGL, TH. AHRENS,]. M. WEISS, E. SCHOPF, and J. C. SIMON In previous in vitro studies QCB 128: 687, 1995) we have shown CD44 containing the alternatively sliced exon v3 (CD44v3) to be modified with heparan sulfate (HS) and to bind HSbinding growth factors, e.g. basic-fibroblast growth factor (b-FGF). Here we demonstrate that also in vivo, exogenously added b-FGF is bound by CD44v3 on keratinocytes (KC) in normal skin (NS) or on tumor cells in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), two skin cancers of keratinocyte origin. Likewise, b-FGF-binding and CD44v3 expression colocalized on human cultured normal keratinocytes (HNK) and on the SCC-celiline A431. By contrast, benign or malignant tumors of melanocyte original failed to express CD44v3 and bound not b-FGF. The b-FGF-binding to normal or transformed KC in vivo and in vitro was dependent on the HS-modification of CD44v3 since it was completely eliminated by pretreatment with heparitinase or by blocking with free heparin, whereas chondroitin ABClyase had no effect. Furthermore, b-FGF bound to CD44vY HNK and A431 stimulated their proliferation in a dose-dependent fashion. This b-FGF effect was again completely abolished by heparitinase or free heparin, but not by chondroitinase. In aggregate, our results demonstrate that an important function of HS-modified CD44v on KC is to present the growth factor b-FGF, thereby stimulating the proliferation of normal or transformed keratinocytes. These findings may lead to new strategies for the therapeutic use of agents which block b-FGF presentation by CD44v3 in disease states associated with keratinocyte hyperproliferation.

310 . 28th Annual Meeting 1997 Chirurgische Universitatsklinik Freiburg; Institut fur Immunologie der Universitat Heidelberg, Germany

L, 16 Synthesis and surface expression by activated T lymphocytes of fibronectin C. WAGNER, M. RADSAK, R. RICHTER and G. M. HANSCH Fibronectin is an extracellular matrix protein which is synthesized by a variety of cells including fibroblasts, epithelial cells and hepatocytes. Though encoded by a single gene, due to alternative splicing, post-translational modifications or extracellular processing fibronectin is either released into the cell supernatant as a soluble protein or it is deposited as a non soluble extracellular matrix protein. Hematopoietic cells, particularly monocyte/macrophages have long been known to synthesize fibronectin. We now found that peripheral T lymphocytes after activation with either mitogen or cross-linked anti-CD3 synthesize fibronectin. FN-specific mRNA was found and 4 to 6 after stimulation considerable expression on the cell surface. No release into the cell supernatant was seen and also no deposits onto the culture plates. By monoclonal antibodies and by PCR the FN splice variants were analyzed. T lymphocytes preferentially synthesized the non-spliced form of FN; a minor portion of FN (± 5%) contained the extradomain A (EDA). The biological significance of fibronectin synthesis and surface expression by T lymphocytes is still elusive. Since fibronectin aside from being a structural matrix protein also mediates cell adhesion it is possible that surface-associated fibronectin contributes to cell-cell-interactions required for T cell activation and effector functions.

Centre of Anatomy, Medical School of Hannover, Germany, and Immunology Research Group, University of Manchester, UK

L,17 In vivo C04+ Tcells of both the naive and the memory phenotype enter rat lymph nodes and Peyer's patches via high endotllelial venules: within the tissue their migratory behavior differs

J. WESTERMANN, U. GEISMAR, U. PETERS, S. SPARSHOTT, E. BELL It is thought that naive T cells predominantly enter lymphoid organs such as lymph node (LNs) and Peyer's patches (PP) via high endothelial venules (HEVs), whereas memory T cells migrate into non-lymphoid organs such as the skin. However, direct evidence for the existence of these distinct migration pathways in vivo is incomplete, and nothing is known about their migration through the different compartments of lymphoid organs. In the present study we separated naive and memory CD4+ T cells from the rat thoracic duct according to the expression of the high and low molecular weight isoforms of CD45R, respectively. At various time points after injection into congenic animals, these cells were identified by quantitative immunohistology in HEVs, and T and B cell areas of different LNs and PP. Three major findings emerged. Firstly, both naive and memory CD4+ T cells enter lymphoid organs via the HEVs in comparable numbers. Secondly, naive and memory CD4+ T cells migrate into the B cell area, although in small numbers, and continuously enter established germinal centers (GCs) with a bias for memory CD4+ T cells. Thirdly, memory CD4+ T cells migrate faster through the T cell area of lymphoid organs than naive CD4+ T cells. Thus, «memory» might depend in part of the ability of T cells to specifically enter the B cell area and GCs, and to screen large quantities of lymphoid tissues in a short time.