Ephrin-A1 Suppresses Th2 Cell Activation and Provides a Regulatory Link to Lung Epithelial Cells This information is current as Jan G. Wohlfahrt, Christian Karagiannidis, Steffen of October 2, 2021. Kunzmann, Michelle M. Epstein, Werner Kempf, Kurt Blaser and Carsten B. Schmidt-Weber J Immunol 2004; 172:843-850; ; doi: 10.4049/jimmunol.172.2.843 http://www.jimmunol.org/content/172/2/843 Downloaded from References This article cites 42 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/172/2/843.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Ephrin-A1 Suppresses Th2 Cell Activation and Provides a Regulatory Link to Lung Epithelial Cells1 Jan G. Wohlfahrt,* Christian Karagiannidis,* Steffen Kunzmann,* Michelle M. Epstein,† Werner Kempf,‡ Kurt Blaser,* and Carsten B. Schmidt-Weber2* Gene expression screening showed decreased ephrin-A1 expression in CD4؉ T cells of asthma patients. Ephrin-A1 is the ligand of the Eph receptor family of tyrosine kinases, forming the largest family of receptor tyrosine kinases. Their immune regulatory properties are largely unknown. This study demonstrates significantly reduced ephrin-A1 expression in T cells of asthma patients using real time-PCR. Immunohistological analyses revealed strong ephrin-A1 expression in lung tissue and low expression in cortical areas of lymph nodes. It is absent in T cell/B cell areas of the spleen. Colocalization of ephrin-A1 and its receptors was found only in the lung, but not in lymphoid tissues. In vitro activation of T cells reduced ephrin-A1 at mRNA and protein levels. T cell proliferation, activation-induced, and IL-2-dependent cell death were inhibited by cross-linking ephrin-A1, and not by Downloaded from engagement of Eph receptors. However, anti-EphA1 receptor slightly enhances Ag-specific and polyclonal proliferation of PBMC cultures. Furthermore, activation-induced CD25 up-regulation was diminished by ephrin-A1 engagement. Ephrin-A1 engagement reduced IL-2 expression by 82% and IL-4 reduced it by 69%; the IFN-␥ expression remained unaffected. These results demon- strate that ephrin-A1 suppresses T cell activation and Th2 cytokine expression, while preventing activation-induced cell death. The reduced ephrin-A1 expression in asthma patients may reflect the increased frequency of activated T cells in peripheral blood. That the natural ligands of ephrin-A1 are most abundantly expressed in the lung may be relevant for Th2 cell regulation in asthma and http://www.jimmunol.org/ Th2 cell generation by mucosal allergens. The Journal of Immunology, 2004, 172: 843–850. NA array-based screening of T cell cDNA of asthmatic group can bind several ephrins beside EphA1, which can bind only patients revealed reduced ephrin-A1 expression com- ephrin-A1 (2, 7). The ligands of the Eph receptor family have to be D pared with those of healthy donors (1). This finding is membrane bound to be active. Therefore, cell-cell interaction is interesting because ephrin-A1 expression was previously not re- necessary for Eph receptor-ephrin signaling, and soluble ephrins ϩ ported in peripheral CD4 T cells and in addition Eph receptors are active only if they are artificially clustered (9–11). are expressed on epithelial cells, which are important cells for the Characteristic for the ephrin biology is that not only is a forward lung function. Therefore, ephrins may represent a regulatory in- signaling pathway by the Eph receptors induced upon ligand bind- by guest on October 2, 2021 terface of epithelial surfaces in the lung and the immune system. ing but also the ligand itself transmits a reverse signals into the cell The ephrins are the ligands of the Eph receptors, which form (4). Interestingly, even the GPI-anchored ephrin-A ligands can in- with at least 14 members the largest group of tyrosine-kinase re- duce signals due to lipid raft microdomain-associated uncharac- ceptors (2–4). Ephrins are divided into two subclasses. The A terized receptors, which are specifically phosphorylated after eph- subclass (ephrin-A1–A6) consists of proteins tethered to the cell rin engagement (12–15). Involvement of Eph receptors and ephrins membrane via a GPI anchor, whereas the B subclass (ephrin-B1– is currently discussed in developmental processes (16–19), as well B3) consist of a transmembrane domain and a short cytoplasmic as their role in carcinogenesis (20). region (5, 6). Similarly, the Eph receptors are divided in an A Human ephrin-A1 is a 25-kDa glycoprotein with high homology (EphA1–9) and a B group (EphB1–6), dependent on the group of to murine and rat orthologs (21) and is abundantly expressed in ephrins they are binding. All Eph receptors of the A subclass can lung, but is also found in kidney, salivary gland, skin, and intestine bind ephrin-A1 (7), but the receptor EphA2 binds ephrin-A1 with (21, 22). It was found as an immediate-early response gene of ϭ the highest affinity (Kd 25 nM (8)). All receptors of the EphA endothelium induced by TNF-␣ (23) that possesses angiogenic and endothelial cell chemoattractant activity (24). EphA1 receptor is the least conserved member of the EphA receptor family and is * Swiss Institute of Allergy and Asthma Research, Davos, Switzerland; † Division of expressed in high levels in the liver, kidney, thymus, and lung but Immunology, Allergy and Infectious Disease, Department of Dermatology, Univer- sity of Vienna, Vienna, Austria; and ‡ Department of Dermatology, University Hos- not in the brain (25, 26). Eph receptors are also transcribed at high pital Zurich, Zurich, Switzerland. levels in tumor cells of epithelial origin (27, 28) and overproduc- Received for publication May 28, 2003. Accepted for publication November 4, 2003. tion of EphA1 induces tumorigenic ability of normal fibroblasts or The costs of publication of this article were defrayed in part by the payment of page melanoma progression (27, 29). This could support a role for charges. This article must therefore be hereby marked advertisement in accordance ephrins in the growth of the cells, but it could also indicate that the with 18 U.S.C. Section 1734 solely to indicate this fact. immune system recognizes the ephrin-overexpressing cells to a 1 This work was supported by Swiss National Foundation Grants 31.52986.97 and 31-65436.01, Roche Research Foundation Grant Mkl/stm 115-2001, the Gebert Ru¨f reduced degree. In fact, recent studies showed that immuno-com- Foundation Grant G-074/99, EMDO Foundation, Saurer Foundation, Zurich and OPO petent cells express Eph receptors and ephrin ligands. A soluble Foundation Zurich, Deutsche Forschungsgemeinschaft Grant KU 1403/1-1 (to splice variant of ephrin-A4 is secreted by activated human B lym- Z.S.K.). phocytes, and EphA2 receptors are present on dendritic cells/mac- 2 Address correspondence and reprint requests to Carsten B. Schmidt-Weber, Swiss Institute of Allergy and Asthma Research, Obere Strasse 22, CH-7270 Davos, Swit- rophages in tonsils (30) and peripheral blood (31). TCR engage- zerland. E-mail address: [email protected] ment along with EphB6 receptor cross-linking in the Jurkat cell Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 844 EPHRIN-A1 INHIBITS Th2 CELLS line results in FAS-mediated cell death (32). EphB6 receptor over- manufacturer. After hybridization, membranes were washed for 20 min with expression in this cell line suppresses TCR-mediated IL2 secretion 30 ml of a 0.1ϫ standard saline-citrate-phosphate/EDTA, 1% (w/v) SDS so- and CD25 expression by inhibition of the c-jun kinase pathway lution. Subsequently, moistened arrays were sealed in plastic bags and exposed for 24 h on phosphoimager screens (Fuji Film, Tokyo, Japan). The screens (33). Furthermore, plate-bound ephrin-A1 inhibits strong chemo- were analyzed using the FLA 3000 phosphoimager system (Fuji) and evalu- taxis in thymocytes and Jurkat cell line (34). These studies clearly ated with the AIDA program (Raytest, Urdorf, Switzerland). demonstrate that ephrins participate in immune regulation and pos- sibly convey an off signal to the immune system. T cell proliferation assay In this study, we describe ephrin-A1 expression by T cells and Triplicates of 1 ϫ 105 CD4ϩ T cells or PBMC were set up in 200 ␮lof the impact of ephrin-A1 cross-linking on T cell function. The RPMI 1640 in 96-well flat-bottom microtiter plates (Costar, Corning, NY). strong localized expression of Eph receptors on lung epithelia sug- Cells were activated by plate-bound mouse anti-human CD3 mAb (1 ␮g/ ml; clone CRL 8001; ATCC) and mouse anti-human CD28 mAb (2 ␮g/ml; gests that ephrins create microenvironmental conditions, which are clone 15E8; Netherlands Red Cross blood transfusion service). The bot- known to contribute to the pathogenesis of asthma such as hyper- toms of the wells were as well covered with rabbit Ig Fraction (DAKO- reactivity of the lung and airway remodeling. Cytomation, Zug, Switzerland), rabbit anti-human ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human EphA1 receptor (R&D Materials and Methods Systems), or rabbit anti-human EphA2 receptor Abs (Sigma-Aldrich, Buchs, Switzerland).