The Neutrophil-specific Antigen CD177 Is a Counter-receptor for Platelet Endothelial Cell Adhesion Molecule-1 (CD31)
2007; Elsevier BV; Volume: 282; Issue: 32 Linguagem: Inglês
10.1074/jbc.m701120200
ISSN1083-351X
AutoresUlrich J. Sachs, Cornelia Andrei‐Selmer, Amudhan Maniar, Timo Weiß, Cathy Paddock, Valeria V. Orlova, Eun Young Choi, Peter J. Newman, Klaus T. Preissner, Triantafyllos Chavakis, Sentot Santoso,
Tópico(s)Blood disorders and treatments
ResumoHuman neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration. Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration. CD177 (NB1 and PRV-1) is a 58- to 64-kDa glycosylphosphatidylinositol-anchored glycoprotein expressed exclusively by neutrophils, neutrophilic metamyelocytes, and myelocytes, but not by any other blood cells (1Stroncek D.F. Skubitz K.M. McCullough J.J. Blood. 1990; 75: 744-755Crossref PubMed Google Scholar, 2Stroncek D.F. Shankar R.A. Noren P.A. Herr G.P. Clement L.T. Transfusion. 1996; 36: 168-174Crossref PubMed Scopus (39) Google Scholar). We and others elucidated its primary structure by sequencing the NB1 and PRV-1 genes, which later turned out to be two alleles of a single CD177 gene (3Kissel K. Santoso S. Hofmann C. Stroncek D. Bux J. Eur. J. Immunol. 2001; 31: 1301-1309Crossref PubMed Scopus (75) Google Scholar, 4Temerinac S. Klippel S. Strunck E. Roder S. Lubbert M. Lange W. Azemar M. Meinhardt G. Schaefer H.E. Pahl H.L. Blood. 2000; 95: 2569-2576Crossref PubMed Google Scholar, 5Caruccio L. Bettinotti M. rector-Myska A.E. Arthur D.C. Stroncek D. Transfusion. 2006; 46: 441-447Crossref PubMed Scopus (30) Google Scholar). The surface expression of CD177 is unique in that only a subpopulation of neutrophils expresses this protein on the cell surface, with the mean size of the CD177-positive subpopulation ranging from 45% to 65% (2Stroncek D.F. Shankar R.A. Noren P.A. Herr G.P. Clement L.T. Transfusion. 1996; 36: 168-174Crossref PubMed Scopus (39) Google Scholar, 6Matsuo K. Lin A. Procter J.L. Clement L. Stroncek D. Transfusion. 2000; 40: 654-662Crossref PubMed Scopus (63) Google Scholar). CD177 has been well studied as a target antigen in immunemediated disorders. During pregnancy, women with a CD177 null phenotype are prone to produce alloantibodies against CD177 that cross the placenta, react with fetal neutrophils, and cause neutropenia of the newborn. This mechanism let to the initial discovery of the NB1 antigen in 1971 (7Lalezari P. Murphy G.B. Allen Jr., F.H. J. Clin. Invest. 1971; 50: 1108-1115Crossref PubMed Scopus (116) Google Scholar). Alloantibodies to CD177, present in blood products obtained from immunized donors, have also been implicated as mediators of transfusion-related acute lung injury (8Sachs U.J. Hattar K. Weissmann N. Bohle R.M. Weiss T. Sibelius U. Bux J. Blood. 2006; 107: 1217-1219Crossref PubMed Scopus (133) Google Scholar). Although well characterized as an immunotarget, the function of CD177 is largely unknown. It has been reported that CD177 is up-regulated on the neutrophil surface upon stimulation, including during severe bacterial infections, and following granulocyte-colony-stimulating factor treatment (9Gohring K. Wolff J. Doppl W. Schmidt K.L. Fenchel K. Pralle H. Sibelius U. Bux J. Br. J. Haematol. 2004; 126: 252-254Crossref PubMed Scopus (58) Google Scholar). In addition, antibody-mediated clustering of CD177 primes the N-formyl-methionyl-leucyl-phenylalanine (fMLP) 3The abbreviations used are: fMLP, N-formyl-methionyl-leucyl-phenylalanine; HUVEC, human umbilical vein endothelial cell; mAb, monoclonal antibody; sr, soluble recombinant; SPR, surface plasmon resonance; IL, interleukin; BCECF, 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein; JAM, junctional adhesion molecule; PECAM-1, platelet endothelial cell adhesion molecule-1; PBS, phosphate-buffered saline; BSA, bovine serum albumin. 3The abbreviations used are: fMLP, N-formyl-methionyl-leucyl-phenylalanine; HUVEC, human umbilical vein endothelial cell; mAb, monoclonal antibody; sr, soluble recombinant; SPR, surface plasmon resonance; IL, interleukin; BCECF, 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein; JAM, junctional adhesion molecule; PECAM-1, platelet endothelial cell adhesion molecule-1; PBS, phosphate-buffered saline; BSA, bovine serum albumin.-activated respiratory burst reaction of the neutrophil (8Sachs U.J. Hattar K. Weissmann N. Bohle R.M. Weiss T. Sibelius U. Bux J. Blood. 2006; 107: 1217-1219Crossref PubMed Scopus (133) Google Scholar). Taken together, these observations make it reasonable to suppose that CD177 may be involved in processes of neutrophil-mediated host defense. One preliminary study suggests a participation of CD177 in neutrophil-endothelial cell interaction (10Stroncek D.F. Herr G.P. Plachta L.B. J. Lab. Clin. Med. 1994; 123: 247-255PubMed Google Scholar). The latter observation is in line with the fact that CD177, as a member of the leukocyte antigen-6 superfamily, shares a similar structure with the urokinase plasminogen activator receptor (11Plesner T. Behrendt N. Ploug M. Stem Cells. 1997; 15: 398-408Crossref PubMed Scopus (145) Google Scholar). Urokinase plasminogen activator receptor is expressed on numerous cell types and plays an important role in cell-extra-cellular matrix and cell-cell interaction by binding of vitronectin, and by regulating β1 and β2 integrin-dependent adhesion of leukocytes (12Chavakis T. Kanse S.M. May A.E. Preissner K.T. Biochem. Soc. Trans. 2002; 30: 168-173Crossref PubMed Google Scholar). Our understanding of the role that CD177 might similarly play in mediating neutrophil-endothelial cell interactions is limited, however, by lack of an identifiable counter-receptor on the endothelial cell surface. A complex molecular crosstalk is known to be responsible for the interaction between neutrophils and endothelial cells (13McIntyre T.M. Prescott S.M. Weyrich A.S. Zimmerman G.A. Curr. Opin. Hematol. 2003; 10: 150-158Crossref PubMed Scopus (139) Google Scholar, 14Kakkar A.K. Lefer D.J. Curr. Opin. Pharmacol. 2004; 4: 154-158Crossref PubMed Scopus (82) Google Scholar). Whereas the initiating step of rolling and subsequent firm leukocyte adhesion have been well characterized (15McEver R.P. Thromb. Haemost. 2001; 86: 746-756Crossref PubMed Scopus (360) Google Scholar), less is known about the mechanisms that mediate the migration of leukocytes through the endothelium. A number of adhesion molecules have been implicated in this process, including β2 integrins, ICAM-1 (intercellular adhesion molecule-1), junctional adhesion molecules (JAMs), CD99, and platelet endothelial cell adhesion molecule-1 (PECAM-1) (16Muller W.A. Trends Immunol. 2003; 24: 327-334PubMed Scopus (0) Google Scholar, 17Shaw S.K. Ma S. Kim M.B. Rao R.M. Hartman C.U. Froio R.M. Yang L. Jones T. Liu Y. Nusrat A. Parkos C.A. Luscinskas F.W. J. Exp. Med. 2004; 200: 1571-1580Crossref PubMed Scopus (189) Google Scholar). PECAM-1 is a constitutively expressed, abundant component of endothelial cell junctions at all levels of the vascular tree (18Muller W.A. Ratti C.M. McDonnell S.L. Cohn Z.A. J. Exp. Med. 1989; 170: 399-414Crossref PubMed Scopus (289) Google Scholar, 19Albelda S.M. Oliver P.D. Romer L.H. Buck C.A. J. Cell Biol. 1990; 110: 1227-1237Crossref PubMed Scopus (330) Google Scholar). Homophilic PECAM-1-PECAM-1 interaction as part of a sensing and activating process of neutrophils plays a central role in leukocyte migration (20Dangerfield J. Larbi K.Y. Huang M.T. Dewar A. Nourshargh S. J. Exp. Med. 2002; 196: 1201-1211Crossref PubMed Scopus (180) Google Scholar). In addition, a number of heterophilic binding partners for PECAM-1 have been described, including CD38, αvβ3, and glycosaminoglycans (21Deaglio S. Morra M. Mallone R. Ausiello C.M. Prager E. Garbarino G. Dianzani U. Stockinger H. Malavasi F. J. Immunol. 1998; 160: 395-402PubMed Google Scholar, 22Piali L. Hammel P. Uherek C. Bachmann F. Gisler R.H. Dunon D. Imhof B.A. J. Cell Biol. 1995; 130: 451-460Crossref PubMed Scopus (340) Google Scholar, 23DeLisser H.M. Yan H.C. Newman P.J. Muller W.A. Buck C.A. Albelda S.M. J. Biol. Chem. 1993; 268: 16037-16046Abstract Full Text PDF PubMed Google Scholar), but the relevance of these partners in leukocyte transmigration is currently not well established (24Sun Q.H. Paddock C. Visentin G.P. Zukowski M.M. Muller W.A. Newman P.J. J. Biol. Chem. 1998; 273: 11483-11490Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 25Sun Q.H. DeLisser H.M. Zukowski M.M. Paddock C. Albelda S.M. Newman P.J. J. Biol. Chem. 1996; 271: 11090-11098Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 26Thompson R.D. Wakelin M.W. Larbi K.Y. Dewar A. Asimakopoulos G. Horton M.A. Nakada M.T. Nourshargh S. J. Immunol. 2000; 165: 426-434Crossref PubMed Scopus (75) Google Scholar, 27Luu N.T. Rainger G.E. Buckley C.D. Nash G.B. J. Vasc. Res. 2003; 40: 467-479Crossref PubMed Scopus (40) Google Scholar). In this study, we demonstrate that neutrophil-specific CD177 can directly bind PECAM-1 and that CD177 and PECAM-1 constitute a heterophilic ligand pair with contribution to neutrophil-endothelial cell interactions. Cells—U937 cells were obtained from DSMZ, Braunschweig, Germany, and maintained in α-minimal essential medium and RPMI 1640 medium (Invitrogen). HEK cell lines were kindly provided from Dr. B. Nieswandt (Wuörzburg, Germany) and grown in Dulbecco's modified Eagle's medium/high glucose medium (PAA Cell Culture Co., Coölbe, Germany). All media were supplemented with 10% fetal calf serum (Seromed, Berlin, Germany) and 1% penicillin/streptomycin (PAA Cell Culture Co.). Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cord and cultured as previously described (28Chavakis T. Keiper T. Matz-Westphal R. Hersemeyer K. Sachs U.J. Nawroth P.P. Preissner K.T. Santoso S. J. Biol. Chem. 2004; 279: 55602-55608Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). HUVECs were cultured in MCDB131 medium (Invitrogen) supplemented with 1% penicillin/streptomycin (PAA Cell Culture Co.). Monoclonal Antibodies—Hybridoma producing mAb 7D8 against CD177 was kindly provided by Dr. D. Stroncek (Dept. of Transfusion Medicine, NIH, Bethesda, MD). mAb MEM166 specific for CD177 was purchased from Serotec (Duösseldorf, Germany). mAb Gi18 directed against the first two domains of PECAM-1 was produced and characterized in our laboratory (29Kroll H. Sun Q.H. Santoso S. Blood. 2000; 96: 1409-1414Crossref PubMed Google Scholar). mAbs PECAM 1.1 and PECAM 1.2 recognizing domains 5 and 6 of PECAM-1, respectively, have been previously described (24Sun Q.H. Paddock C. Visentin G.P. Zukowski M.M. Muller W.A. Newman P.J. J. Biol. Chem. 1998; 273: 11483-11490Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 30Yan H.C. Pilewski J.M. Zhang Q. DeLisser H.M. Romer L. Albelda S.M. Cell Adhes. Commun. 1995; 3: 45-66Crossref PubMed Scopus (61) Google Scholar). mAbs 68-5H11 and AK-4 against E- and P-selectin, respectively, were purchased from BD Pharmingen (Heidelberg, Germany). mAb Gi11 specific for JAM-C was produced in our laboratory (31Santoso S. Sachs U.J. Kroll H. Linder M. Ruf A. Preissner K.T. Chavakis T. J. Exp. Med. 2002; 196: 679-691Crossref PubMed Scopus (351) Google Scholar). All antibodies are of IgG1 isotype, except for PECAM1.1 (IgG2a). Recombinant Proteins—Soluble recombinant (sr) PECAM-1 derived from PECAM-1-stable transfected Chinese hamster ovary cells has been previously described (32Goldberger A. Middleton K.A. Oliver J.A. Paddock C. Yan H.C. DeLisser H.M. Albelda S.M. Newman P.J. J. Biol. Chem. 1994; 269: 17183-17191Abstract Full Text PDF PubMed Google Scholar). All recombinant proteins were purified by affinity chromatography before use. Fc protein as control was purchased from R&D Systems (Wiesbaden, Germany). Generation of CD177-Fc Construct—Full-length CD177 cDNA (3Kissel K. Santoso S. Hofmann C. Stroncek D. Bux J. Eur. J. Immunol. 2001; 31: 1301-1309Crossref PubMed Scopus (75) Google Scholar) was amplified by PCR using forward primer 5′-92CTGCTCTGCCAGTTTGGGAC111-3′ and reverse primer 5′-1224CTGCACATCACGCTTCTCACG1204-3′. Aliquots of 1 μl of CD177 construct were diluted with 10× PCR buffer, 0.5 μm of each primer, 5 μm dNTP, 2.5 units of cloned Pfu DNA polymerase (Stratagene, Heidelberg, Germany) in a total volume of 50 μl. PCR amplification was performed for 32 cycles. Each cycle consisted of denaturation for 1 min at 95 °C, annealing for 1 min at 60 °C, and extension for 1 min at 72 °C. In the final cycle, the sample was kept at a temperature of 72 °C for 10 min and chilled to 4 °C. The PCR product was purified from a 1% agarose gel by using QIAquick (Qiagen), subcloned into the EcoRV cloning site of Signal pIgplus plasmid (kindly provided by Dr. B. Nieswandt, Wuörzburg, Germany), and then transformed into DH5α competent Escherichia coli (Invitrogen). Positive clones were screened by PCR using CD177 and T7 primer pairs as described above. A plasmid from selected positive clones was validated by nucleotide sequence analysis on an ABI Prism Genetic Analyzer 3100 (Applied Biosystems, Weiterstadt, Germany). Production of Recombinant CD177-Fc Fusion Protein—HEK cells were grown in 24-well plates and were transfected with 0.2 μg of CD177 construct in 350 μl of Opti-Mem medium (Invitrogen) by the use of Effectene (Qiagen). Transfected cells were selected with increasing concentration of Geneticin (40-800 μg/ml, PAA Cell Culture Co.). Culture supernatants from stable cell lines were then tested by enzyme-linked immunosorbent assay. In brief, microtiter wells were coated with 50 μl of donkey anti-human Fc (diluted 1:250, Dianova, Hamburg, Germany) overnight at 4 °C. After washing three times with 100 μl PBS, wells were blocked with 2% BSA in PBS for 30 min at 4 °C. An aliquot of 50 μl of supernatant was added, and the mixture was incubated for 30 min at 37 °C. Wells were washed twice, and bound Fc fusion protein was detected with 50 μl of peroxidase-labeled donkey anti-human Fc (diluted 1:3000, Dianova) and ortho-phenylenediamine (Dako, Hamburg, Germany) as substrate. Reaction was stopped after 15 min with 50 μl of H2SO4 and was read on an enzyme-linked immunosorbent assay reader at 405 nm. CD177-Fc fusion protein was isolated from 1 liter of culture supernatant and purified by the use of a protein G column. Purified protein was analyzed on 7.5% SDS-PAGE by silver staining and verified by immunoblotting (see below). Analysis of CD177-Fc Binding to Endothelial Cells by Flow Cytometry—Aliquots of 4 × 106 HUVECs were fixed with 1% formaldehyde for 5 min and washed twice with PBS. Subsequently, cells were incubated with 4 μg of CD177-Fc or Fc alone for 1 h at 37 °C, washed with 0.02% BSA in PBS buffer, and then labeled with 40 μl of fluorescein-conjugated donkey anti-human IgG (diluted 1:80, Dianova). After washing, cells were resuspended in 500 μl of 0.2% BSA for flow cytometry analysis (FACSCalibur, BD Biosciences, Heidelberg, Germany). Antigen Capture Assay—Microtiter wells were coated with 100 μl of donkey anti-human Fc (see above) overnight at 4 °C. Wells were washed extensively three times with 0.2% BSA and blocked with 2% BSA for 30 min at 4 °C. Aliquots of 250 ng of CD177-Fc fusion protein or Fc alone (as control) were added, and the mixture was incubated for 1 h at 37 °C. Wells were washed twice and then incubated with 100 μl of endothelial cell lysates (1 μg) for 30 min at 37 °C. Bound protein was detected by the addition of mAbs against PECAM-1, E-selectin, and P-selectin (1:500 dilutions) at 37 °C for 30 min. After washings, 100 μl of peroxidase-labeled donkey anti-mouse IgG (diluted 1:8000, Dianova) was added for 1 h at 37 °C. Bound antibodies were measured on an enzyme-linked immunosorbent assay reader as described above. Affinity Isolation of the CD177 Binding Partner—HUVECs were labeled with 2 ml of 5 mm NHS-LC-Biotin (Pierce) and lysed in 1 ml of lysis buffer containing protease inhibitors as previously described (33Santoso S. Kiefel V. Richter I.G. Sachs U.J. Rahman A. Carl B. Kroll H. Blood. 2002; 99: 1205-1214Crossref PubMed Scopus (56) Google Scholar). After preclearing with protein-G beads (Amersham Biosciences), cell lysates were stored at -70 °C until use. Aliquots of 100 μl of Protein-G beads were coupled with 2.5 μg of CD177-Fc fusion protein or Fc control for 1 h at 4 °C. 300 μl of labeled cell lysates were added to the beads and were rotated overnight at 4 °C. After washings with immunoprecipitation buffer (50 mm Tris, 150 mm NaCl, 1% Triton X-100), bound proteins were eluted by adding SDS buffer for 5 min at 100 °C. Eluates were analyzed on 7.5% SDS-PAGE under reducing conditions. Separated proteins were transferred onto nitrocellulose membranes and developed with peroxidase-labeled streptavidin and a chemiluminescence system (ECL, Amersham Biosciences). After stripping with Restore™ Western blot (Pierce), the membrane was redeveloped with selected mAbs against endothelial cell markers (20 μg/ml). Bound mAbs were visualized with peroxidase labeled rabbit anti-mouse IgG (diluted 1:100,000, Dianova) and the ECL system. Expression of CD177 in U937 Monocyte Cell Line—U937 cells were grown in RPMI medium containing 10% fetal calf serum and 0.5% penicillin/streptomycin. An aliquot of 250 μl of cell suspension (2 × 107cells/ml) was transfected with 20 μg of CD177 in pcDNA3 vector (3Kissel K. Santoso S. Hofmann C. Stroncek D. Bux J. Eur. J. Immunol. 2001; 31: 1301-1309Crossref PubMed Scopus (75) Google Scholar) using electroporation technique for 40 ms (Bio-Rad GenePulser). After 10-min incubation on 4 °C, cells were seeded in 10 ml of RPMI medium. The next day, cells were washed and selected for stable expression by the use of RPMI medium containing Geneticin (1 mg/ml). Transfectants expressing recombinant CD177 were identified by flow cytometry analysis using mAb 7D8 as described above. Phenotyping of Neutrophils—Neutrophils were phenotyped for CD177 expression by flow cytometry as previously described (9Gohring K. Wolff J. Doppl W. Schmidt K.L. Fenchel K. Pralle H. Sibelius U. Bux J. Br. J. Haematol. 2004; 126: 252-254Crossref PubMed Scopus (58) Google Scholar). Briefly, granulocytes were isolated from EDTA-anti-coagulated blood by dextran sedimentation and gradient centrifugation. Indirect immunofluorescence was performed by flow cytometry using mAb 7D8. Five thousand cells were analyzed. The size of the positive subpopulation was calculated from the histogram employing CellQuest software. To investigate the influence of fMLP (Sigma) or IL-8 (Immuno Tools, Friesoythe, Germany) on protein surface expression, granulocytes were isolated as described and incubated with 5 μg/ml cytochalasin B (Sigma) for 30 min at room temperature. Subsequently, cells were incubated for 30 min at room temperature in the absence or presence of 10 nm fMLP or 100 ng/ml IL-8, respectively. Indirect immunofluorescence was performed as outlined above using mAbs 7D8 and Gi18. Isolation of CD177 Proteins from Human Neutrophils—Granulocyte concentrates were obtained from healthy volunteers by a standard leukapheresis procedure (34Sachs U.J. Reiter A. Walter T. Bein G. Woessmann W. Transfusion. 2006; 46: 1909-1914Crossref PubMed Scopus (65) Google Scholar). Twelve hours prior to apheresis, all volunteers received 7.5 μg per kilogram bodyweight of human recombinant granulocyte-colony-stimulating factor (ChugaiPharma, Frankfurt am Main, Germany), as approved by the local ethics committee. Granulocyte concentrates (medium volume, 230 ml) were diluted in PBS (1:5). Aliquots of 15 ml were layered onto Ficoll gradient and then centrifuged for 20 min at 800 × g. Cell pellets were resuspended into 12 ml of ammonium chloride for 5 min on ice to lyse erythrocytes. After washing twice, 108 cells were solubilized in 1 ml of lysis buffer (20 mm Tris-buffered saline, pH 7.4, 0.25% Triton-X, 100 μl of protease inhibitor mixture, 100 μl of 5% EDTA) for 30 min. Cell lysates were then centrifuged for 30 min at 800 × g at 4 °C and pooled for purification by affinity chromatography as previously described (3Kissel K. Santoso S. Hofmann C. Stroncek D. Bux J. Eur. J. Immunol. 2001; 31: 1301-1309Crossref PubMed Scopus (75) Google Scholar). Isolated CD177 protein was then intensively dialyzed against PBS, and the remaining trace of Triton X-100 was removed by filtration through YM-10 column (Amicon, Witten, Germany). Platelet-derived αIIbβ3 protein as a control was isolated from outdated platelet concentrates by mAb Gi5 affinity column under identical conditions. The purity and identity of both proteins was proved by silver staining and immunoblotting analysis, and protein concentration was determined by BCA™ (Pierce). Aliquots of purified proteins were stored at -80 °C until use. Surface Plasmon Resonance—Direct protein interaction between PECAM-1 and CD177 was examined in real-time with a surface plasmon resonance (SPR) technique on a BIAcore 2000 (Biacore AB, Freiberg, Germany). Purified srPECAM-1 diluted in 10 mm acetate buffer (pH 4.0) to a concentration of 60 μg/ml was directly immobilized on a CM5-sensor chip via amino coupling as recommended by the manufacturer. Aliquots of 20 μl of purified CD177 or BSA (Pierce) as control were injected at a flow rate of 15 μl/min at different concentrations as indicated. In some experiments, 2 mm CaCl2, 25 μm Zn2SO4, or 10 mm EDTA were added into CD177 analyte prior to analysis. For blocking studies, 50 μg of mAbs was added in selected experiments. Running buffer was PBS (10 mm sodium phosphate, 150 mm NaCl, pH 7.4) containing 0.005% surfactant P20 (Biacore AB). The sensor chip was regenerated with 25 mm NaOH. Cell Adhesion Assay—Cell adhesion to PECAM-1-coated wells was tested as described previously (35Chavakis T. Kanse S.M. Lupu F. Hammes H.P. Muller-Esterl W. Pixley R.A. Colman R.W. Preissner K.T. Blood. 2000; 96: 514-522Crossref PubMed Google Scholar). Briefly, microtiter plates were coated with 50 μl of Fc-PECAM-1 or Fc alone as control (10 μg/ml in PBS) and blocked with 3% BSA solution. Nontransfected or CD177-transfected U937 cells were incubated with 5 μl of 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF, Molecular Probes, Leiden, The Netherlands) for 30 min in the dark. Cells were washed twice in serum-free RPMI, and 105 cells/well were plated onto the precoated wells at 37 °C for 60 min. For inhibition studies, cells were incubated with 10 μg/ml mAbs specific for CD177 (MEM166) and PECAM-1 (Gi18, PECAM1.1, and PECAM1.2) or with 50 μl of purified CD177 (10 μg/ml) during the adhesion period. After the incubation period, the wells were washed gently, and adhesion was quantified using a fluorescence microplate reader Flx-800 (Biotek, Neufahrn, Germany). Transmigration Assay—Briefly, transmigration assays were performed using 6.5-mm Transwells with an 8-μm pore size (Costar, Bodenheim, Germany). Inserts were coated with gelatin (Sigma). HUVECs were seeded on Transwell filters 2 days prior to the assay and grown for 48 h in a humidified atmosphere (37 °C and 5% CO2). The integrity of HUVEC monolayers was assessed microscopically. At the beginning of the transmigration assay, HUVECs in the upper chamber were washed with serum-free RPMI, the lower chamber was filled with serum-free RPMI medium containing 10 nm fMLP (Sigma) or 100 ng/ml IL-8 (Immuno Tools). 1 ml of neutrophils (5 × 106 cells/ml) was labeled with 5 μl of BCECF as described above. An aliquot of 100 μl of untreated cells (5 × 106 cells/ml) or cells preincubated with mAbs (10 μg/ml, 15 min, room temperature) was added to the upper chamber on top of the endothelial monolayer. After incubation for 90 min h at 37 °C, the number of transmigrated cells in the lower chamber was measured using a fluorescence reader as above. Production and Characterization of CD177-Fc Fusion Protein—To determine the binding partner of CD177, we first established stably transfected mammalian HEK cells producing soluble recombinant CD177-Fc fusion protein. Supernatants were collected after 3 days of culture and purified by the use of a protein G column. The purified protein was analyzed by silver staining to document its purity (Fig. 1A, left panel) and by immunoblotting to prove the immunoreactivity of both CD177 and the Fc portion (Fig. 1A, right panel). Purified CD177-Fc showed positive reactions with mAb 7D8 specific for CD177, as well as with donkey anti-human Fc (lanes 1 and 2). In the control experiment, donkey anti-mouse Fc did not show any reaction (lane 3). These results demonstrate that both portions, CD177 and Fc, of our fusion protein are immunoreactive. Binding of CD177-Fc Fusion Protein to HUVECs—The reactivity of the CD177-Fc fusion protein with HUVECs was tested by flow cytometry (Fig. 1B). Isolated HUVECs were incubated with CD177-Fc or Fc alone as a control, and a specific reaction with CD177-Fc only was observed. These results indicate that endothelial cells carry a binding partner for CD177. To further characterize the binding partner of CD177, an antigen capture assay was performed using CD177-Fc fusion protein immobilized on microtiter wells. After adding HUVEC lysates, bound protein was screened with different mAbs against endothelial proteins. As shown in Fig. 2A, a positive reaction was observed with anti-PECAM-1, whereas no reaction was detectable with mAbs against E-selectin, P-selectin, and against JAM-C, indicating that PECAM-1 may represent the binding partner of CD177. Immunoprecipitation was then performed with surface biotin-labeled HUVECs to confirm this finding. Labeled cell lysates were precipitated with Fc alone, CD177-Fc, and mAb 7D8 coupled to protein G beads. Immunoprecipitates were analyzed by immunoblotting using a peroxidase-labeled streptavidin system (Fig. 2B, left panel). In comparison to control experiments with Fc alone and mAb 7D8, CD177-Fc precipitated a specific band with a mass of 120 kDa. The membrane was stripped extensively and stained with different mAbs against endothelial cell markers to identify the 120-kDa protein. Only when mAb Gi18 specific for PECAM-1 was applied, a positive staining was observed (Fig. 2B, right panel). In contrast, no reaction was detected with mAbs against P-selectin, E-selectin, and JAM-C (not shown). In the control experiments, pre-clearing of HUVEC lysates with anti-PECAM-1 mAb abrogated the specific reaction of CD177-Fc immunoprecipitates (data not shown). Real-time Analysis of CD177 and PECAM-1 Protein Interaction—To study the direct protein-protein interaction between CD177 and soluble recombinant PECAM-1 (srPECAM-1), a real-time analysis by SPR technique was performed. Both proteins were affinity-purified before use and verified by silver staining and immunoblotting (Fig. 3). srPECAM-1 was immobilized onto the sensor surface, and the binding of CD177 at various concentrations (0.5-2.0 μm) was measured. As shown in Fig. 4A, concentration-dependent binding of CD177 to immobilized PECAM-1 was observed. In the control experiment, no interaction between CD177 and immobilized BSA was detectable. According to the Langmuir 1:1 model, the association (KA) and dissociation constant (KD) of CD177 binding was 1.23 × 106 M and 8.15 × 10-7 m (χ2 = 1.85), respectively. To prove the accessibility of the homophilic binding region of PECAM-1, mAb Gi18 against domains 1 and 2 was injected. As shown in the insert, mAb Gi18, which was previously shown to interfere with the homophilic PECAM-1 binding (28Chavakis T. Keiper T. Matz-Westphal R. Hersemeyer K. Sachs U.J. Nawroth P.P. Preissner K.T. Santoso S. J. Biol. Chem. 2004; 279: 55602-55608Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), bound to immobilized PECAM-1 on the sensor surface.FIGURE 4Real-time analysis of immobilized PECAM-1 and CD177 by SPR on a BIAcore 2000. A, sensograms show the plot of relative response units (RU) in time (s) for buffer (PBS) and different concentrations of CD177. Purified srPECAM-1 was directly immobilized on a CM5-sensor chip via amino coupling. Analytes were injected with a flow rate of 15 μl/min at 25 °C. The inset shows the binding of mAb Gi18 against PECAM-1 in two different concentrations (10 and 100 μg/ml). B, SPR analysis of heterophilic PECAM-1-CD177 interaction in the presence of calcium and zinc cations and of a chelator, respectively. CD177 analytes in buffer containing 2 mm CaCl2, 25 μm ZnSO4, or 10 mm EDTA were injected as
Referência(s)