CD97 on Activated Endothelial Cells Thy-1 (CD90)

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 25, 2021. J Immunol 2012; 188:1442-1450; Prepublished online 30 December 2011; doi: 10.4049/jimmunol.1003944 http://www.jimmunol.org/content/188/3/1442 Downloaded from

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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: 1442– http://www.jimmunol.org/ 1450.

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 25, 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 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 of CD97 and demonstrated that PMNC interact specifically, via www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003944 The Journal of Immunology 1443

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). MAb directed to ICAM-1 (CD54; clone R6.5.D6), CD11b stimulated with 10 ng/ml PMA (Invitrogen, Karlsruhe, Germany) for 24 h. (clone X-5), and CD18 (clone IB4) were purchased from the American Type PMNC of normal subjects and psoriatic patients were isolated, as described Culture Collection (LGC Standards, Wesel, Germany), BMA Biomedicals (5). The purity was .95% CD15+ cells. PMNC were labeled with 0.1 mM (Augst, Switzerland), or Calbiochem (Darmstadt, Germany). carboxyfluorescein diacetate succinimidyl ester (CFDA; Invitrogen) for 15 http://www.jimmunol.org/

FIGURE 1. Expression of CD97 is elevated in PMNC of psoriatic patients. A, Upper left panel, CD97 was strongly expressed on infiltrating or existing my- by guest on September 25, 2021 eloid cells in psoriatic skin lesions (arrows). Smooth muscle cells are known to be CD97+ (arrowhead). Up- per middle and right panels, Compared with CD31, Thy-1 was found on EC (arrows) and, to some extent, on fibroblasts. Scale bar, 50 mm. Lower panels, Infil- trating leukocytes located in epidermal microabscesses of psoriatic skin lesions were CD97+. Microvascu- lar EC adjacent to these leukocytes expressed Thy-1 and CD31, as shown by double immunofluorescence staining. Scale bar, 30 mm. B, PMNC of psoriatic patients (n = 15) adhered more strongly to activated HDMEC compared with PMNC of healthy subjects (n = 12; median, 5th/95th percentile). **p , 0.01. C, PMNC of psoriatic patients (n = 15; pso) showed higher CD97 expression compared with PMNC of healthy controls (n = 12; ctr). Expression of EMR2 and CD11b on PMNC was comparable between both groups. Expression was quantified by flow cytometry as MFI (median: solid line, mean: dotted line; 5th/95th percentile). *p , 0.05. D, TNF-a increased the ex- pression of CD97 but not of EMR2 and CD11b on PMNC of healthy donors (n = 5, median: solid, mean: dotted; 5th/95th percentile). *p , 0.05. E, TNF-a– activated PMNC adhered more strongly to stimulated EC compared with untreated PMNC (n = 5). *p , 0.05, **p , 0.01. 1444 THY-1 IS AN INTERACTING PARTNER OF CD97 min on ice. In some experiments, CFDA-labeled PMNC were activated with In the sCD97-to-cell–binding assay, 1 3 105 Thy-1–transfected or 10 ng/ml TNF-a (Immunotools, Friesoythe, Germany) for 30 min. control CHO cells and unstimulated or activated HUVEC were incubated with 0.1 mg sCD97(EGF1,2,5), sCD97(EGF1–5), or mFc-control protein Generation of stably transfected clones overexpressing Thy-1 for 60 min at room temperature, followed by PE-labeled goat anti-mouse or CD97 (Dako) to detect the Fc-tag in flow cytometry. Alternatively, biotinylated sCD97 could also be detected by PE-streptavidin (Dako). CD97(EGF1,2,5) and CD97(EGF1–5) cDNA cloned into the pcDNA3.1 To block binding of sCD97 to EC, cells were preincubated with 10 mg/ml Zeo(+) vector (22) were used as basic templates for the generation of new the respective Ab for 1 h at 37˚C prior to sCD97 addition. Nonbound constructs. pEGFP-N1 (BD Biosciences, Heidelberg, Germany) generates protein was washed away several times with PBS. EC were analyzed by a fusion protein consisting of CD97 with enhanced GFP, allowing the flow cytometry. direct monitoring of transfected cells without further labeling. Wild-type HT1080, WiDr, and CHO cells (American Type Culture Statistical analysis Collection) were stably transfected with 1 mg construct DNA by electro- poration (290 V, 1500 mF; Multiporator; Eppendorf, Hamburg, Germany). Statistical analysis was performed using the Student t test or Mann– A total of 7.5 mg/ml geneticin (Invitrogen) was added after 24 h to select Whitney U test; p values , 0.05 were regarded as significant. stable clones. From each transfection, 3 clones of .10 were randomly selected by high CD97 mean fluorescence intensity (MFI) in flow cy- Results tometry. Control cells contained either the empty plasmid (empty) or the inverted construct (mock). CHO clones stably expressing human Thy-1 PMNC of patients with psoriasis express elevated levels of were generated as described (18). CD97 + Preparation of sCD97 and soluble EMR2 Adhesion of PMNC to activated Thy-1 EC, mediated by the in- teraction of Thy-1 and Mac-1, facilitates their migration into d Downloaded from Constructs encoding sCD97(EGF1,2,5), sCD97(EGF1,2,5) stalk (without lesions of psoriatic skin (4–6). The presence of an additional the stalk), sCD97(EGF1–5), soluble EMR2 (sEMR2)(EGF1,2,5), sEMR2 (EGF1,2,3,5), and sEMR2(EGF1–5) fused to a murine Fc-tag (mFc) were interacting partner for Thy-1 in PMNC has been suggested, be- used (17, 23). mFc-sCD97 and mFc-control protein were biotinylated with cause blocking of Mac-1 did not result in complete inhibition of the RTS AviTag E. coli Biotinylation Kit from Roche Diagnostics (Mann- Thy-1–mediated adhesion of PMNC (6). In this study, we exam- heim, Germany). ined whether interaction of Thy-1 and CD97 is involved in the Cell-to-cell adhesion assays adhesion of PMNC to activated EC. Infiltrating leukocytes located in epidermal microabscesses and http://www.jimmunol.org/ In PMNC-to-cell–adhesion assays, EC or Thy-1– or mock-transfected in dermal inflammatory infiltrates of psoriatic skin lesions were CHO cells were cultured in a 96-well plate. Confluent EC were stimu- + lated for 24 h to induce stronger Thy-1 expression. A total of 2 3 104 CD97 . Microvascular EC adjacent to these leukocytes expressed CFDA-labeled unstimulated or TNF-a–activated PMNC were added to the Thy-1 (Fig. 1A). wells. After incubation for 45 min at 37˚C, unbound PMNC were removed According to Wetzel et al. (6), we confirmed that psoriatic by washing with PBS. Adherent PMNC were lysed by the addition of 50 PMNC adhered more to activated EC compared with PMNC ml 10% SDS to the wells. Fluorescence was quantified using a fluorimetric plate reader (SpectraMax; Molecular Devices, Sunnyvale, CA). Fluores- of normal subjects (Fig. 1B). However, expression of the Thy-1 cence and adherent cell number correlated in a linear fashion (data not counterreceptor Mac-1 was not different on psoriatic and healthy shown). PMNC (Fig. 1C). In contrast, psoriatic PMNC showed higher 5 by guest on September 25, 2021 In cell line to cell-adhesion assays, 2 3 10 HT1080, WiDr, or CHO expression of CD97 than did normal PMNC, whereas expression cells stably overexpressing CD97 as an enhanced GFP-fusion protein were of EMR2, the close subfamily member of CD97, was comparable added to a well of an eight-chamber slide precultured with Thy-1– or mock-transfected CHO cells to confluence. After incubation for 30 min at between psoriatic and healthy PMNC (Fig. 1C). 37˚C, unbound cells were removed by several washes with PBS. Adherent Stimulation of healthy PMNC with TNF-a, resulting in an cells were fixed with ice-cold methanol for 10 min at 220˚C, rinsed in enhanced adhesion of these PMNC to Thy-1–transfected cells, PBS, and mounted. The number of adherent cells was counted in 20 fields at 403 magnification under a fluorescence microscope. In blocking experiments, cells were preincubated with a specific or control IgG mAb (10 mg/ml) for 30 min at 37˚C in the case of endothelial or CHO monolayers or at 4˚C in the case of PMNC. EC were preincubated with Ab directed to Thy-1, CD55, avb3ora5b1 integrin, or ICAM-1. PMNC were preincubated with mAb to CD18 or CD97. Adhesion and binding assays with purified proteins In cell-to-protein adhesion assays, 96-well MaxiSorb plates (Fisher Sci- entific, Schwerte, Germany) were coated with 500 ng human Fc (hFc)– Thy-1 or hFc-control protein/well in TBS, 2 mM CaCl2, 0.1 mM MgCl2 overnight. Plates were washed with TBS and blocked with TBS/1% BSA for 1 h at room temperature. A total of 5 3 105 CFDA-labeled PMNC, preincubated with IgG ctr-mAb or mAb to CD97, were added to the coated wells. Fluorescence was measured as described above. In other experi- ments, cells of three CHO clones, differing in the expression level of CD97, were added to the coated wells. In protein-binding assays, 1 mg purified sCD97(EGF1,2,5), sCD97 (EGF1,2,5) d stalk, sCD97(EGF1–5), sEMR2(EGF1,2,5), sEMR2(1,2,3,5), or sEMR2(EGF1–5) was added to immobilized hFc–Thy-1 or hFc-control protein-coated wells and incubated for 2 h at room temperature. Plates were washed with TBS, 0.1% BSA, 0.05% Tween 20, 2 mM CaCl2, and 0.1 mM MgCl2. CD97 binding to immobilized Thy-1 was performed in the absence or presence of 10 mg/ml mAb to Thy-1 or CD97, respectively. Bound proteins were detected by addition of the biotinylated CD97EGF mAb and HRP-labeled streptavidin (DakoCytomation, Hamburg, Ger- many), followed by tetramethylbenzidine substrate (Fisher Scientific). FIGURE 2. Thy-1 expression is increased on activated HUVEC and Color reaction was measured at 450 nm. To evaluate whether binding HDMEC. Unstimulated HUVEC (A) and HDMEC (B) were slightly depends on the presence of Ca2+, binding was performed with buffers Thy-1+. Activation increased the percentage of Thy-1+ EC and the ex- containing Ca2+ or 1 mM EDTA without Ca2+. pression level of Thy-1 in both EC types. **p , 0.01, ***p , 0.001. The Journal of Immunology 1445 thus imitating characteristics of psoriatic PMNC, increased the Because adhesion of PMNC to activated Thy-1+ HDMEC and to expression of CD97 1.5-fold. The expression of EMR2 and Thy-1–transfected cells can be blocked only partially with Ab to CD11b was unchanged (Fig. 1D). Psoriatic PMNC showed no CD18 (6), we suggested additional interaction partners for endo- additional increase in CD97 levels after TNF-a treatment (data thelial Thy-1 on PMNC. To examine whether CD97 is involved in not shown). Accordingly, TNF-a–activated PMNC adhered more binding of PMNC to Thy-1+ cells, the adhesion of PMNC to strongly to stimulated micro- and macrovascular EC compared HUVEC and HDMEC was determined upon blocking by specific with control PMNC (Fig. 1E). In summary, our data suggested mAb. the possible involvement of CD97 in adhesion of PMNC to acti- First, PMNC were pretreated with different mAb to CD97 (Fig. vated EC. 3A,3C). As a positive control, PMNC were preincubated with mAb to CD18, which indeed partially blocked the binding of MAb to CD97 and Thy-1 inhibit adhesion of PMNC to PMNC to HUVEC (Fig. 3A) and HDMEC (Fig. 3C). Interestingly, activated EC both CD97 mAb directed to the stalk (CD97stalk) or to the EGF- PMNC showed stronger adhesion to activated, compared with like domains (CD97EGF) blocked PMNC binding. The CD97stalk unstimulated, micro- and macrovascular EC, indicating the up- mAb showed a stronger blocking compared with the CD97EGF regulation of an endothelial receptor that mediates PMNC adhesion mAb in both EC. In a parallel assay, adhesion of CD55+ eryth- (Fig. 2). Unstimulated EC slightly expressed Thy-1. Activation rocytes to CD97-transfected HT1080 cells, performed as de- increased the percentage of Thy-1+ HUVEC (Fig. 2A) and scribed by Hamann et al. (24), was strongly inhibited by the HDMEC (Fig. 2B), as well as the expression level of Thy-1 in CD97EGF mAb, thus demonstrating the blocking function of this these cells. mAb (data not shown). Consequently, our data indicated that the Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Adherence of PMNC to activated HUVEC (A, B) and HDMEC (C–E) is mediated, in part, by CD97 and Thy-1. Either PMNC (A, C, E)orEC (B, D) were pretreated with the various Abs, as indicated, and incubated together. The number of adherent PMNC was determined. PMNC adhered more strongly to activated EC compared with unstimulated EC. Binding of PMNC could be inhibited, in part, by mAb directed to CD97 on PMNC and to Thy-1 on activated EC. The combination of mAb to CD18 and CD97 for blocking PMNC (C) and the combination of mAb to Thy-1 and ICAM-1 for blocking EC (D) caused additional inhibition. E, The increased adhesion of TNF-a–stimulated PMNC to activated HDMEC was prevented by mAb to CD18 and CD97. The CD97stalk mAb decreased the adhesion of TNF-a–activated PMNC compared with the level of unstimulated PMNC, which were set as 100%. Data are shown as mean 6 SD (n = 5). *p , 0.05, **p , 0.01, ***p , 0.001, compared with ctr; #p , 0.05, compared with the respective mAb alone. bl, blocking; ctr, control; mAb CD55/1, clone BRIC 216; mAb CD55/2, clone CLB-CD97L1; nbl, nonblocking. 1446 THY-1 IS AN INTERACTING PARTNER OF CD97 stalk of CD97 is involved in the interaction of CD97 with Thy-1. The combination of mAb to CD18 and CD97stalk caused a further significant decrease in adhesion of PMNC compared with block- ing with either mAb alone (Fig. 3C). This indicated that both molecules mediate the adhesion of PMNC to activated EC. Second, EC were pretreated with mAb directed to EC-specific Ags (Fig. 3B,3D). As expected, adherence of PMNC to HUVEC was decreased by the Thy-1–blocking mAb (Fig. 3B). Thus, blocking of HDMEC (Fig. 3D) was expanded to ICAM-1, a known ligand of Mac-1 (CD11b/CD18), as well as to the other potential interacting partners of CD97 on EC, such as CD55 and avb3 and a5b1 integrin. Strongest blocking was demonstrated with the Thy- 1–blocking and the ICAM-1 mAb. Using two CD55 mAb, we demonstrated slight blocking with the mAb BRIC 216. In a pre- assay, we confirmed that both CD55 mAb used, BRIC 216 and CLB-CD97/L1, nearly completely blocked adhesion of erythro- FIGURE 4. CD97+ cells adhere to Thy-1–overexpressing CHO cells. A, cytes to CD97-transfected HT1080 cells (data not shown). Adhe- CD97-overexpressing clones of various cell lines were strongly CD97+ in sion of PMNC to activated HDMEC was also blocked by avb3 flow cytometry. CD97 MFI of one typical experiment of one clone is and a5b1 integrin Ab, but not as strongly as by the Thy-1 shown. B, Adhesion of CD97(EGF1,2,5) or mock-expressing CHO, WiDr, Downloaded from blocking mAb. The combination of the Thy-1–blocking mAb and HT1080 clones to Thy-1–overexpressing or vector-transfected CHO and the ICAM-1 mAb caused an additive effect, whereas the clones was quantified. The number of adherent cells was counted/obser- vation field (mean 6 SEM, n = 4). Strongest binding was seen between combination of the Thy-1–blocking mAb and the CD55 mAb + (BRIC 216) did not. CD97 clones of the various cell types and Thy-1–overexpressing CHO cells. **p , 0.01, ***p , 0.001. The slight adhesion of PMNC to unstimulated EC could not be

inhibited by mAb directed to CD18 or CD97 on PMNC or by mAb http://www.jimmunol.org/ to the various Ags in EC (Fig. 3A–D). cell-to-protein adhesion assay. PMNC showed strong adhesion To clarify whether CD97 mediates the increased binding of to hFc–Thy-1–coated wells but only weak binding to an irrelevant + immobilized hFc-control protein (Fig. 5A,5B). This adhesion activated PMNC to Thy-1 EC, we performed blocking experi- stalk ments using TNF-a–stimulated PMNC (Fig. 3E). After preincu- could be partially inhibited by mAb to CD97. The CD97 mAb bation of activated PMNC with the CD18, CD97EGF, or CD97stalk showed the strongest inhibition. mAb, adhesion of activated PMNC was blocked significantly. To verify whether the number of adherent cells depends on the Blocking of CD97 by the CD97stalk mAb decreased adhesion of expression level of CD97, we examined three CHO clones with activated PMNC to the level of adhesion of unstimulated PMNC different CD97 expression levels (Fig. 5C). Clearly, the number of to activated EC. by guest on September 25, 2021 CD97-overexpressing clones adhere specifically to Thy-1–transfected cells Next, we examined whether CD97-overexpressing cells adhered to Thy-1+ CHO cells more strongly compared with the correspond- ing wild-type or CD97 mock-transfected cells. CHO, WiDr, and HT1080 clones stably overexpressing CD97 were generated to rule out that the measured effects depend on the parental cell line transfected. Wild-type WiDr and HT1080 cells showed basal ex- pression of CD97. However, the selected CD97-overexpressing clones showed higher levels of CD97 compared with wild-type or CD97 mock cells (Fig. 4A). Irrespective of the cell line, CD97-overexpressing clones adhered strongly to Thy-1–transfected CHO cells (Fig. 4B). The number of adherent cells was comparably low in all control combinations: adhesion of CD97 mock-transfected clones to vector-transfected CHO cells, CD97-overexpressing clones to vector-transfected CHO cells, and CD97 mock-transfected CHO or WiDr clones to Thy-1–transfected CHO cells. A greater number of wild-type HT1080 cells (data not shown) or CD97 mock-transfected HT1080 cells adhered to Thy-1–transfected CHO cells, because HT1080 wild-type cells express CD97 at a significant level. However, bind- + ing of CD97-overexpressing HT1080 clones to Thy-1–transfected FIGURE 5. CD97 cells adhere to Thy-1 protein. A and B, PMNC ad- CHO cells was significantly greater compared with binding of hered specifically to immobilized recombinant hFc–Thy-1 but only slightly to the unrelated hFc-control protein (Fc-ctr). MAb to CD97 inhibited this HT1080 mock-transfected cells (Fig. 4B). adherence; CD97stalk mAb showed the strongest blocking (n = 4, mean 6 PMNC and CD97-overexpressing CHO cells attach to SEM). Scale bar, 100 mm. *p , 0.05, ***p , 0.001. C, CD97 expression immobilized Thy-1 protein level of three CHO clones was determined by flow cytometry. CD97 MFI of one typical experiment is shown. D, The number of cells adhering to Next, we examined whether PMNC and CD97-overexpressing hFc–Thy-1 correlated with the CD97 expression level of these CHO clones CHO clones bind specifically to immobilized Thy-1 protein in a (n = 4, mean 6 SEM). *p , 0.05. The Journal of Immunology 1447 adherent CD97-overexpressing CHO cells correlated with the MFI Unstimulated EC, slightly expressing Thy-1, showed weak bind- of the different clones (Fig. 5D). ing of both sCD97(EGF1,2,5) and sCD97(EGF1–5). Activated EC showed an increase in sCD97 binding compared with unstimu- sCD97 binds to immobilized Thy-1 protein lated EC. To further examine the interaction between CD97 and Thy-1, we Both sCD97 isoforms also bound to Thy-1–transfected CHO analyzed binding of the purified proteins hFc–Thy-1 and sCD97 cells (Fig. 7B,7C). However, we observed significant binding of (EGF1,2,5) or sCD97(EGF1–5) in a cell-free in vitro ligand- sCD97(EGF1–5), but not sCD97(EGF1,2,5), to vector-transfected binding assay. Control binding to an irrelevant hFc-control pro- CHO cells. Because wild-type CHO cells express chondroitin tein was used to exclude unspecific binding of the Fc-tag. Both sulfate B, a potential ligand of the longest, but not the shortest and sCD97 isoforms bound to immobilized Thy-1 but not to the hFc- middle, CD97 isoform (17), it is very likely that sCD97(EGF1–5) control protein (Fig. 6A). Binding was concentration dependent bound to chondroitin sulfate B. We tested this hypothesis by and saturable. blocking the interaction site for chondroitin sulfate B on sCD97 The blocking, but not the nonblocking, Thy-1 mAb inhibited (EGF1–5). Pretreatment of sCD97(EGF1–5) with either chon- EGF stalk binding up to 20% (Fig. 6B). Both the CD97 and CD97 droitin sulfate B or the mAb 1B5 abolished the binding to vector- stalk mAb also disturbed Thy-1 binding. The CD97 mAb showed transfected CHO cells, whereas binding to Thy-1–transfected the strongest inhibition of Thy-1 binding, suggesting the involve- cells was diminished but still detectable (Fig. 7C). Binding of ment of the stalk of CD97 in Thy-1–CD97 interaction. sCD97(EGF1–5) to Thy-1+ EC could be partially inhibited by the To clarify this point in more detail, we compared binding of blocking mAb to Thy-1 and by the CD55 mAb BRIC 216 (Fig. sCD97(EGF1,2,5) and sCD97(EGF1,2,5) d stalk to hFc–Thy-1. 7D). Downloaded from sCD97 without the stalk did not bind hFc–Thy-1 (Fig. 6C), indi- cating that the stalk is essential for CD97 binding to Thy-1. Fur- thermore, we examined whether the three isoforms of sEMR2 bind Discussion to hFc–Thy-1. EMR2 shows 97% amino acid sequence identity to The adhesive interaction of PMNC with activated EC is an essential CD97 within the EGF-like domains but only 46% amino acid step in the process of PMNC accumulation at sites of inflammation in vivo. In this study, we demonstrated that CD97 is involved in the sequence identity in the stalk region. None of the sEMR2 isoforms http://www.jimmunol.org/ bound hFc–Thy-1 (Fig. 6C). Furthermore, withdrawal of Ca2+ adhesion of PMNC to activated EC. Binding of CD97 and Thy-1 did not disturb Thy-1–CD97 interaction (Fig. 6D). Because was shown at various levels. On the one hand, PMNC, CD97- binding to the EGF-like domains depends on Ca2+, this result overexpressing cells, and sCD97 bound to activated EC, Thy-1– also suggested the involvement of the CD97 stalk in binding overexpressing cells, as well as immobilized Thy-1 protein. On Thy-1. the other hand, adherence and binding could be blocked partially + and specifically with the corresponding Ab. sCD97 binds to Thy-1 cells CD97 belongs to the receptors that are able to interact with li- Finally, we examined binding of sCD97 to Thy-1+ cells. Binding gands different in structure and expression profiles. The long extra-

of sCD97(EGF1,2,5) and sCD97(EGF1–5), but not mFc-control cellular a-chain, probably identical to naturally occurring sCD97, by guest on September 25, 2021 protein, to EC and to Thy-1–transfected CHO cells was detected provides many possibilities for binding different cellular and ex- via the mFc-tag or the biotin-tag in flow cytometry (Fig. 7A). tracellular matrix ligands.

FIGURE 6. sCD97 binds to Thy-1 in a cell-free protein-binding assay. A, mFc-sCD97(EGF1,2,5) and mFc-sCD97(EGF1–5) bound, in a concentration- dependent manner, specifically to immobilized hFc– Thy-1. No binding was seen with the hFc-control pro- tein. B, Binding of 20 ng/ml mFc-sCD97(EGF1–5) to immobilized hFc–Thy-1 is shown in the presence of 10 mg/ml blocking and nonblocking Thy-1, CD97EGF, or CD97stalk mAb (n = 4, mean 6 SEM). C, Twenty nanograms per milliliter of sCD97(EGF1,2,5), but not sCD97(EGF1,2,5) d stalk, the various sEMR2 iso- forms, or the mFc-control protein bound to hFc–Thy-1 (n = 4, mean 6 SEM). D, Binding of 20 ng/ml sCD97 (EGF1,2,5) and sCD97(EGF1–5) to hFc–Thy-1 did not depend on Ca2+. ***p , 0.001. 1448 THY-1 IS AN INTERACTING PARTNER OF CD97

FIGURE 7. sCD97 binds to Thy-1+ cells. A, sCD97(EGF1,2,5) and sCD97 (EGF1–5), but not mFc-control protein, showed stronger binding to activated HUVEC compared with unstimulated HUVEC, measured as CD97 MFI in flow cytometry (n = 4, mean 6 SEM). B and C, sCD97(EGF1,2,5) and sCD97

(EGF1–5), but not mFc-control protein, Downloaded from bound more strongly to hFc–Thy-1 than to vector-transfected CHO cells. sCD97(EGF1–5) bound also to wild- type CHO cells expressing chondroitin sulfate B. C, Preincubation of sCD97 (EGF1–5) with chondroitin sulfate B or the mAb 1B5 prevented binding to http://www.jimmunol.org/ vector-transfected cells but not to Thy- 1–transfected CHO cells. One repre- sentative of three independent experi- ments is shown. D, Binding of sCD97 (EGF1,2,5) and sCD97(EGF1–5) to HUVEC was inhibited by the Thy-1– blocking (bl) and CD55 mAb (clone BRIC 216) but not by the Thy-1–non- a b

blocking (nbl) and 5 1 and ICAM-1 by guest on September 25, 2021 Ab (n = 5, mean 6 SEM). **p , 0.01, ***p , 0.001.

The first identified ligand was CD55 (decay-accelerating factor) on HDMEC, it is unlikely that CD97 in myeloid cells interacts (19), which binds to the first two EGF domains of CD97 (24). with CD55 in EC in psoriatic lesions. CD55 is present in resting EC at a high level, whereas Thy-1 is Subsequently, a5b1 and avb3 integrins were identified as in- only slightly expressed on unstimulated EC but is increased after teracting proteins for human sCD97(1,2,5) and sCD97(EGF1–5) activation. In our study, CD97+ cells, either PMNC or CD97- on macrovascular EC (27). These sCD97 forms chemoattracted transfected cells, bound much more to activated, compared with HUVEC in a migration, as well as a Matrigel-based invasion, unstimulated, EC, which did not indicate binding via CD55. assay. The Arg-Gly-Asp tripeptide present only in the stalk region Moreover, pretreatment of activated EC with Thy-1– or CD55- of human, but not mouse, CD97 was partially involved in these blocking mAb resulted in stronger inhibition of adherence by the effects (27). avb3 integrin is present in EC in psoriatic lesions Thy-1–blocking mAb. Additionally, in normal human skin, CD55 (28). In our study, binding of CD97 to a5b1 and avb3 integrins is present in vascular structures, but its expression is decreased may be partially involved in the adhesion of PMNC to activated in nonlesional psoriatic skin and virtually abolished in lesional microvascular EC, because blocking Ab to these integrins slightly psoriatic skin (25). Furthermore, CD97–CD55 interaction is not inhibited the attachment of PMNC. We used the same Ab that involved in human leukocyte adhesion to porcine EC in trans- inhibited binding of sCD97 to HUVEC, as shown recently (27). In plantation models (26). All of the data indicated that, although one contrast to our study, Wang et al. (27) used only sCD97 to evaluate of the examined CD55 mAb slightly blocked adhesion of PMNC binding to a5b1 and avb3 integrins in EC. We performed cell- The Journal of Immunology 1449 to-cell adhesion assays to confirm binding of surface-associated structs, failed to bind Thy-1, although the EGF-like domains of CD97 to EC. Moreover, the investigators examined HUVEC for both proteins are almost identical. Moreover, binding did not need binding of sCD97, although microvascular EC are more relevant calcium that is necessary for binding to the CD97 EGF-like for migration of PMNC to inflammatory lesions. domains. However, both CD97 mAb used prevented binding of Furthermore, the fourth EGF domain of CD97 and EMR2 and, Thy-1 protein only partially, indicating that they did not bind to thus, only the longest isoforms of these receptors, interacts with the specific interaction site of CD97 and Thy-1. proteoglycans containing chondroitin sulfate B (17). Chondroitin Second, are there discrepancies between the binding affinities or sulfate B was identified as a ligand by the use of multivalent properties of CD97 expressed at the cell surface and in soluble fluorescent beads containing the EGF-like domains of EMR2 in forms of CD97? In our adhesion assays using PMNC in activated human tissue sections (17). The described binding pattern (i.e., EC, we observed a clear inhibiting effect of the Thy-1–blocking that of chondroitin sulfate B) did not resemble the expression mAb but only a weak effect of the CD55 mAb. However, binding pattern of human Thy-1. Although our data showed binding of of sCD97 to activated EC was equally well blocked by the Thy-1– sCD97(EGF1–5) to chondroitin sulfate B in wild-type and vector- blocking and CD55 mAb. Overall, our data agree with those transfected CHO cells, binding to Thy-1–transfected CHO cells of Hamann et al. (24) and Kwakkenbos et al. (34): cell surface- was much stronger and was not prevented by blocking the chon- associated CD97(EGF1–5) showed only a very weak binding to droitin sulfate B interaction site. This indicated a specific inter- CD55, whereas binding of sCD97(EGF1–5) to HEK293 cells was action between sCD97(EGF1–5) and Thy-1 and a minor role for clearly mediated by CD55, as shown by blocking of this inter- binding to chondroitin sulfate B at activated EC. action with a CD55-specific mAb.

The phenomenon that several ligands could bind to one recep- Third, does CD97–Thy-1 binding play a significant role in vivo? Downloaded from tor, and vice versa, is described as redundancy and also includes The interaction of CD97 with Thy-1 in EC is probably restricted interactions in which both partners are cellular receptors. Because to sites of inflammation, because Thy-1 is expressed in EC at the ligands of CD97 differ in expression pattern and structure, the a significant level only in regions in which cell activation and same is true for the known interacting proteins of Thy-1. Its in- inflammation occurred (35). Several murine experimental studies teraction with avb3 in melanoma is one mechanism whereby indicated the involvement of CD97 in PMNC accumulation at

these tumor cells adhere to activated endothelium (18). Further- inflammatory sites. Targeting mouse CD97 by mAb inhibited the http://www.jimmunol.org/ more, Thy-1 mediates adhesion of PMNC to EC by interaction accumulation of neutrophils at sites of inflammation, thereby af- with Mac-1 (CD11b/CD18) (5). fecting antibacterial host defense (36) and inflammatory disorders In this article, we described CD97 as a new interaction partner of (37). Otherwise, accumulation of PMNC at sites of inflammation Thy-1, mediating the adhesion of PMNC to activated EC, whereby was not affected in CD97-deficient mice (20, 38) that display no activated, compared with unstimulated, PMNC adhered more overt phenotype at steady-state, except for a mild granulocyto- strongly. After blocking with the CD97stalk mAb, activated PMNC sis, which increases under inflammatory conditions. Interestingly, adhered only to the same extent as did unstimulated PMNC. In application of CD97 mAb blocked neutrophil trafficking after blocking experiments with combinations of mAb against CD97 thioglycollate-induced peritonitis in wild-type, but not in CD97 and CD18, we achieved a significant decrease in adhesion com- knockout, mice (20). Consequently, CD97 mAb induced an inhib- by guest on September 25, 2021 pared with blocking with each mAb alone. However, blocking was itory effect that disturbed normal granulocyte trafficking, which not complete. As we showed in protein–protein binding assays, was not perturbed in the absence of the molecule (20). Overall, the available CD97 mAb did not completely block the binding site comparison of the consequences of mAb treatment and gene tar- for Thy-1 on CD97. Moreover, in addition to CD97 and CD18, geting implied that CD97 mAb actively inhibited the innate re- other ligands for Thy-1 on PMNC may exist. sponse, presumably at the level of granulocyte or macrophage The physiological relevance of Thy-1 in activated EC for the recruitment to sites of inflammation in mice. recruitment of inflammatory cells was demonstrated very recently In summary, we identified Thy-1 as a potential new ligand of in thioglycollate-induced peritonitis and acute and chronic lung CD97. PMNC interact specifically via CD97 with Thy-1 upregu- inflammation using Thy-1–deficient mice (29). In this study, Thy-1 lated on activated EC. Thus, Thy-1–CD97 is probably involved, mediated the adhesion of granulocytes and monocytes to activated together with other receptor–ligand pairs, in mediating leukocyte EC. This interaction plays a pivotal role in the control of the em- adhesion at sites of inflammation. igration of granulocytes and monocytes from blood into peripheral tissue during inflammation. Consequently, the altered number and Acknowledgments composition of extravasated leukocytes affect the inflammatory The expression constructs for the various sCD97 and sEMR2 isoforms were tissue microenvironment, including the chemokine/cytokine and kindly provided by M. Stacey (Sir William Dunn School of Pathology, Ox- protease pattern (29). ford, U.K., now University of Leeds, Leeds, U.K.). The biological role of Thy-1, like that of CD97, is context dependent (30). Thy-1 distribution in mice differs from that in Disclosures humans. Thy-1 is also expressed in rat activated EC (31, 32). But The authors have no financial conflicts of interest. in contrast to humans, mouse Thy-1 is present on the surface of mouse thymocytes and peripheral T cells. The physiological li- gand or interacting molecule for mouse Thy-1 in the lymphoid References compartment has not been identified (33). 1. Crawford, J. M., and R. W. Barton. 1986. Thy-1 glycoprotein: structure, distri- bution, and ontogeny. Lab. Invest. 54: 122–135. Our data raise a number of interesting questions that must be 2. Dalchau, R., A. S. Daar, and J. W. Fabre. 1989. The human Thy-1 molecule. clarified in detail in future studies. First, which domain(s) of CD97 Immunol. Ser. 45: 185–196. 3. Saalbach, A., G. Aust, U. F. Haustein, K. Herrmann, and U. Anderegg. 1997. The mediate binding of Thy-1? Our results indicated that the stalk fibroblast-specific MAb AS02: a novel tool for detection and elimination of region of CD97 is involved in this interaction, because stronger human fibroblasts. Cell Tissue Res. 290: 593–599. blocking of CD97 binding to Thy-1+ cells or to Thy-1 protein was 4. Saalbach, A., U. F. Haustein, and U. Anderegg. 2000. A ligand of human thy-1 is stalk EGF localized on polymorphonuclear leukocytes and monocytes and mediates the seen with the CD97 compared with the CD97 mAb. sCD97 binding to activated thy-1-positive microvascular endothelial cells and fibro- (EGF1,2,5) without a stalk, as well as the different sEMR2 con- blasts. J. Invest. Dermatol. 115: 882–888. 1450 THY-1 IS AN INTERACTING PARTNER OF CD97

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