NF-(Kappa)B Activation in Adhesion-Mediated TEC Survival

Journal of Cell Science 113, 169-177 (2000) 169 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0310 Lymphoid adhesion promotes human thymic epithelial cell survival via NF-κB activation Maria T. Scupoli1,*, Emma Fiorini1, Pier C. Marchisio2,3, Ornella Poffe1, Elda Tagliabue4, Monica Brentegani5, Giuseppe Tridente1,5 and Dunia Ramarli1,5 1Department of Pathology, Section of Immunology, University of Verona, 37134 Verona, Italy 2DIBIT, Department of Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milano, Italy 3Department of Biomedical Sciences and Human Oncology, University of Torino, 10126 Torino, Italy 4Oncologia Sperimentale E-Istituto Nazionale Tumori, Milano, Italy 5Clinical Immunology, Azienda Ospedaliera Verona, 37134 Verona, Italy *Author for correspondence (e-mail: [email protected]) Accepted 27 October; published on WWW 9 December 1999 SUMMARY Inside the thymus, thymic epithelial cells and thymocytes analysis. The activation of NF-κB was necessary to promote show an interdependent relationship for their functional survival, since its inhibition by acetyl salicylic acid differentiation and development. As regards possible prevented the promoting effect. The mAb-mediated interdependency for their mutual survival, it is clear that crosslinking of α3β1 was considered as a potential inducer lympho-epithelial adhesion can control the survival of of TEC survival, since we have previously demonstrated developing thymocytes whereas the effects of lymphoid that the engagement of this integrin was able to induce NF- adhesion on epithelial cell survival have never been κB activation in TEC. The crosslinking of α3β1, which described. To address this issue, we performed co-cultures clustered at the lympho-epithelial contact sites, partially between normal human thymic epithelial cells (TEC) and reproduced the promoting activity of cell adhesion. These a mature lymphoid T cell line (H9) or unfractionated results highlight that lympho-epithelial adhesion can thymocytes. TEC were induced to apoptosis by growth control the survival of thymic epithelial cells through an factor deprivation and the level of cell death was measured intracellular pathway which requires the activation of NF- by flow cytometry. TEC stimulated by cell adhesion showed κB and is triggered by integrins of the β1 family. a significant reduced apoptosis when compared to the control and this phenomenon was associated with increased Key words: Thymus, Apoptosis, Cell interaction, Adhesion binding activity of NF-κB, as measured by gel shift molecule, Transcription factor INTRODUCTION proteins and increased IL-6 mRNA transcription (Couture et al., 1992; Meilin et al., 1995; Cohen-Kaminski et al., 1993). The thymic epithelium is composed of various subsets of Moreover, adhesion with mature lymphoid T cells results in epithelial cells characterized by tonofilaments, interconnected activation of NF-κB (Ramarli et al., 1996). Previously our by desmosomal junctions, and surrounded by extracellular results have also provided evidence that integrin recruitment at matrix, to form an intralobular meshwork filled with the TEC surface during their adhesion to thymocytes initiates developing T lymphocytes. It is known that thymic epithelial an intracellular signaling leading to increased IL-6 production cells (TEC) play a pivotal role in the homing, intrathymic via activation of the IL-6 gene transcription factors, NF-κB and migration, and differentiation of developing T lymphocytes NF-IL6 (Ramarli et al., 1998). through the release of cytokines, the secretion of extracellular Adhesion to extracellular matrix (ECM) components as well matrix components, and the establishment of adhesive as cell-to-cell contact are known to regulate cell survival. In interactions (Crisa et al., 1996; Schluns et al., 1997). On the this processes the major deliverers of the adhesion-derived other hand, it is now apparent that these adhesive interactions signals are members of the integrin family. In fact, in epithelial are also required by TEC to modulate their own functions and and endothelial cells the disruption of integrin-mediated development. In mouse models, the adhesion of thymocytes is interactions with the ECM induces a form of apoptosis termed necessary for the epithelial component of the thymus to anoikis (Frish and Francis, 1994; Montgomery et al., 1994; undergo compartmental differentiation and to properly develop Brooks et al., 1994; Boudreau et al., 1995). Intercellular during embryogenesis (for a review, see Boyd et al., 1993). In adhesion has also been shown to protect from apoptosis and addition, in vitro experiments indicate that thymocyte adhesion there is evidence to suggest that these processes are also to TEC results in tyrosine phosphorylation of cytoplasmic mediated by integrins. Homotypic cell adhesion mediated by 170 M. T. Scupoli and others β1 integrins is required by mammary epithelial cells for F12 medium (3:1 mixture), 10% FCS (fetal calf serum), 5 µg/ml survival on the basement membrane (Pullan et al., 1996) and insulin, 5 µg/ml transferrin, 0.18 µM adenine, 0.4 µg/ml survival of bronchial epithelial cells is enhanced by homotypic hydrocortisone, 0.1 nM cholera toxin, 2 nM triiodothyronine, 10 cell contact mediated at least partially by αv integrins (Aoshiba ng/ml EGF (epidermal growth factor), 4 mM glutamine, and 50 IU/ml et al., 1997). As regards heterotypic cell adhesion, it has been penicillin-streptomycin. Confluent TEC primary cultures were shown that engagement of α β and/or α β integrins on detached by trypsin-EDTA treatment, plated on a 3T3-J2 feeder layer L 2 4 1 and expanded to confluent secondary cultures in growth medium. TEC germinal center B cells by their ligand on follicular dendritic destined for co-culture experiments were derived from secondary cells can protect B cells from apoptosis (Koopman et al., 1994). cultures, already devoid of 3T3-J2 cells, and grown to confluence in Moreover, rescue from apoptosis was described in early medium containing one third of the concentration of insulin, hematopoietic cells by α4β1 integrin-dependent adhesion to transferrin, adenine, hydrocortisone, cholera toxin, triiodothyronine, bone marrow stromal cells (Wang et al., 1998). So far, the and EGF, as we have previously observed that this culture condition effects of heterotypic cell adhesion in modulating the life/death minimizes the NF-κB basal activation in unstimulated cells, thus balance of epithelial cells have never been described. giving the optimal NF-κB gel-shift signal following cell adhesion Recently, it has been demonstrated that integrin-mediated (Ramarli et al., 1998). This medium composition is referred to survival requires the activation of the transcription factor NF- throughout the text as ‘medium’. κB, which has been indicated to have a key role in preventing Media were purchased from Seromed (Berlin, FRG), EGF was from Austral Biological (San Ramon, CA), and supplements were apoptosis in many cell types (Beg and Baltimore, 1996; Wang from Sigma-Aldrich (Milan, Italy). et al., 1996; Van Antwerp et al., 1996; Scatena et al., 1998). The H9 human T cell line (kindly provided by Dr A. De Rossi, NF-κB is a pleiotropic regulator of many genes involved in Istituto di Oncologia, Università di Padova, Italy) was maintained in immune and inflammatory responses. The NF-κB family of culture in RPMI 1640 medium (Seromed) supplemented with 10% proteins comprises homo- or heterodimers of the Rel family, FCS, 4 mM glutamine, and antibiotics. including p50 and p65 (Rel A) subunits. In unstimulated cells, Human thymocytes were prepared by mechanical disruption of NF-κB is localized in the cytosol bound to inhibitors of the thymus specimens. At least 95% viable cells were isolated from the IκBs family which mask its nuclear localization sequence (for cell suspension by Ficoll-Hypaque gradient centrifugation, washed, a review, see Baldwin, 1996). Various extracellular stimuli and used immediately after preparation. are able to induce the serine hyperphosphorylation and Adhesion assay and mAb treatment proteosomal degradation of inhibitor (Palombella et al., 1994), Co-culture experiments were performed by adding H9 cells or or the tyrosine phosphorylation and subsequent dissociation of thymocytes to TEC monolayer at 1:1 H9/TEC or 5:1 thymocyte/TEC inhibitor (Imbert et al., 1996), thus allowing NF-κB to move ratios, respectively. Co-cultures were carried on for 24 hours in into the nucleus, bind to promoter regions, and transactivate medium devoid of insulin, transferrin, adenine, hydrocortisone, genes. cholera toxin, triiodothyronine, and EGF (minimal medium). When In the present study, we explored whether the increased NF- indicated, H9 cells or thymocytes were replaced with supernatatants κB activity we have previously observed in TEC upon adhesion derived from the same cells cultured for 24 hours in minimal with H9 cells or thymocytes (Ramarli et al., 1996, 1998) could medium. At the end of co-culture, H9 cells as well as thymocytes also promote TEC survival. We observed that cell adhesion were removed by vigorous washings. Untreated and supernatant- with H9 cells or thymocytes partially inhibited TEC from treated TEC were equally subjected to vigorous washing, to prevent artifacts eventually created by washing. TEC were then cultured for apoptosis induced by growth factor deprivation and this κ 1 to 4 days in minimal medium. Blocking experiments were phenomenon was associated with increased NF- B binding

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