Thy-1 (CD90) Is an Interacting Partner for CD97 on Activated Endothelial Cells Elke Wandel, Anja Saalbach, Doreen Sittig, Carl Gebhardt and Gabriela Aust This information is current as of September 28, 2021. J Immunol published online 30 December 2011 http://www.jimmunol.org/content/early/2011/12/30/jimmun ol.1003944 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription by guest on September 28, 2021 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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 30, 2011, doi:10.4049/jimmunol.1003944 The Journal of Immunology Thy-1 (CD90) Is an Interacting Partner for CD97 on Activated Endothelial Cells Elke Wandel,*,† Anja Saalbach,‡ Doreen Sittig,* Carl Gebhardt,‡ and Gabriela Aust* Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97+ myeloid cells colocalized with Thy-1+ EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1+–activated human dermal EC, Thy-1+ CHO cells, and immobilized Thy-1 protein. Binding of the CD97+ CHO clones correlated with their CD97 expression level. Soluble CD97 bound + specifically to immobilized Thy-1 protein, as well as Thy-1 –activated EC and CHO cells. In all assays, cellular adhesion or protein Downloaded from binding was blocked partially by CD97 and Thy-1–blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites. The Journal of Immunology, 2012, 188: 000– http://www.jimmunol.org/ 000. eukocyte extravasation into perivascular tissue plays a tating their subsequent migration into lesions of psoriatic skin (4– key role in inflammatory diseases. This recruitment re- 6). However, blocking of CD11b/CD18 did not result in complete L quires leukocyte interaction with vascular endothelium inhibition of Thy-1–mediated adhesion of myeloid cells to acti- and consists of multiple consecutive steps, including the capture vated EC (6). This suggests the presence of an additional inter- of circulating leukocytes, subsequent leukocyte rolling, arrest, acting partner of Thy-1 involved in Thy-1–mediated adhesion of firm adhesion, and ensuing diapedesis. The cascade occurs by myeloid cells to activated EC. by guest on September 28, 2021 sequential activation-dependent interactions between endothelial CD97, a member of the epidermal growth factor (EGF)–seven- cell (EC) adhesion molecules and their specific ligands on leu- span transmembrane (TM7) subfamily of adhesion (class B2) G kocytes. protein-coupled receptors (7), shows a hematopoietic expression Thy-1 (CD90), a highly glycosylated GPI-anchored cell surface profile that merits its consideration as a potential ligand for protein with a molecular mass ∼35 kDa, is a receptor on EC, Thy-1 on activated EC. CD97 is a cell surface receptor present belonging to the Ig superfamily, and is involved in arrest and firm in peripheral neutrophils, monocytes, and activated lymphocytes adhesion of leukocytes to the endothelium (1, 2). In humans, Thy- (8). CD97 is expressed as a heterodimer of a noncovalently 1 expression is restricted to activated EC, fibroblasts, neuronal bound extracellular a-chain, represented by tandemly arranged + cells, and a subset of peripheral CD34 stem cells (3). Adhesion of EGF domains and a stalk, and a b-chain, composed of the TM7 + neutrophils to activated Thy-1 EC, mediated by Thy-1/Mac-1 and a short intracellular portion. Both chains result from intra- (CD11b/CD18) interaction, is one attachment mechanism facili- cellular autocatalytic cleavage (9). The CD97 a-chain has been thought to be shed from the membrane of CD97-expressing cells. *Research Laboratories, Department of Surgery, University of Leipzig, 04103 Leip- It is very likely identical to soluble CD97 (sCD97), detected in zig, Germany; †Translational Center for Regenerative Medicine, University of Leip- zig, 04103 Leipzig, Germany; and ‡Department of Dermatology, Venerology, and synovial fluid of rheumatoid arthritis patients (10). As the result of Allergology, University of Leipzig, 04103 Leipzig, Germany alternative splicing in humans, three isoforms exist: CD97 Received for publication December 2, 2010. Accepted for publication November 17, (EGF1,2,5), CD97(EGF1,2,3,5), and CD97(EGF1–5). CD97 shows 2011. remarkable homology to the EGF-TM7 receptor EMR2 (CD312) This work was supported in part by a grant from the German Federal Ministry of (11), especially within the EGF-like domains. Although both re- Education and Research (BMBF, Projekttra¨ger Ju¨lich, 0315883, to E.W.) and by grants from the German Research Foundation (AU132/7-1 to G.A. and SA863/2-1 ceptors are present at high levels in immune cells, the overall to A.S.). expression pattern, ligand binding, and, thus, function are dissim- Address correspondence and reprint requests to Prof. Gabriela Aust, Research Lab- ilar (12). oratories, Department of Surgery, University of Leipzig, Liebigstraße 20, 04103 Signal transduction through CD97 and EMR2 is still unknown. Leipzig, Germany. E-mail address: [email protected] Because truncation of the TM7 region disrupted CD97-increased Abbreviations used in this article: CFDA, carboxyfluorescein diacetate succinimidyl ester; EC, endothelial cell; EGF, epidermal growth factor; HDMEC, human dermal single random cell migration (13), and binding of a specific Ab microvascular endothelial cell; hFc, human Fc; mFC, murine Fc-tag; MFI, mean to EMR2 regulated human neutrophil function (14), signaling fluorescence intensity; PMNC, polymorphonuclear cell; sCD97, soluble CD97; through EGF-TM7 receptors seems very likely. sEMR2, soluble EMR2; TM7, seven-span transmembrane. In this study, we identified Thy-1 as a potential new ligand Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 of CD97 and demonstrated that PMNC interact specifically, via www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003944 2 THY-1 IS AN INTERACTING PARTNER OF CD97 CD97, with Thy-1 expressed in activated EC, thus mediating leu- Patients kocytic adhesion. Patients aged $18 y (n = 15; 11 males; mean age, 49.7 6 20 y) with plaque-type psoriasis that had been refractory to topical treatment with Materials and Methods external glucocorticoids or vitamin D3 analogs within the last 4 wk were Ab included in the study. Age- and sex-matched normal subjects (n = 12; 10 males; mean age, 47.7 6 14 y) were used as controls. The study was The CD97EGF mAb (clone BL-Ac/F2 (8) detects a glycosylation-dependent approved by the University of Leipzig Ethics committee. All persons gave epitope within the first two EGF-like domains of CD97 and EMR2 (15). The their written consent prior to their enrollment into the study. The study was CD97stalk mAb (clone CLB-CD97/3) (16) binds to the stalk region of the conducted in accordance with the guidelines of the World Medical Asso- CD97 a-chain. The rabbit polyclonal CD97 Ab was purchased from Sigma- ciation’s Declaration of Helsinki. Aldrich Chemie (Munich, Germany). The mAb 1B5 binds to the fourth EGF-like domain present only in the largest isoform of CD97 and EMR2 Immunohistology (17). The Thy-1 mAb clone AS02 does not block Thy-1–dependent cell Cryostat sections of patients with psoriasis were stained for double im- adhesion, whereas clone BC9 does (3, 18). The CD55 mAb (clone BRIC 216), binding to the short consensus repeat 3 domain of CD55, was pur- munofluorescence and analyzed by laser-scanning microscopy or for simple chased from the International Blood Reference Group laboratory (Bristol, immunohistology, as described (20). U.K.). The CLB-CD97L/1 mAb, binding to the short consensus repeat 1 do- Cell separation and cell culture main of CD55 (19), and the EMR2 mAb (clone 1A2) (11) were kind gifts of J. Hamann (University of Amsterdam, Amsterdam, The Netherlands). Both Human dermal microvascular EC (HDMEC) and HUVEC were prepared, as CD55 mAb inhibit binding of erythrocytes to COS-7 cells transfected with described (21). HDMEC were cultured in EGM-MV media (Promocell, CD97(1,2,5) cDNA (19). The mAb to avb3 integrin (clone LM609) and the Heidelberg, Germany). Only preparations with .95% CD31+ EC were used a b polyclonal Ab to 5 1 integrin were purchased from Millipore (Schwal- (21). For induction of Thy-1, EC in the first or second passage were Downloaded from bach, Germany).