JAM2 Interacts with α4β1

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn2+-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and α4β1 in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an α4-specific inhibitor, to attenuate the Mn2+-enhanced component. Intriguingly, the engagement of α4β1 by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/α4β1 interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and α4β1counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on α4β1 interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin. We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn2+-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and α4β1 in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an α4-specific inhibitor, to attenuate the Mn2+-enhanced component. Intriguingly, the engagement of α4β1 by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/α4β1 interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and α4β1counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on α4β1 interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin. junctional adhesion molecule immunoglobulin superfamily Tris-buffered saline vascular cell adhesion molecule mucosal addressin cell adhesion molecule leukocyte function antigen alkaline phosphatase Convincing evidence suggests key roles for junctional adhesion molecules (JAMs)1 in leukocyte transmigration, although the mechanisms by which they may facilitate this process remain largely unresolved (1Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P.J. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1158) Google Scholar, 2Del Maschio A., De Luigi A. Martin-Padura I. Brockhaus M. Bartfai T. Fruscella P. Adorini L. Martino G. Furlan R., De Simoni M.G. Dejana E.J. Exp. Med. 1999; 190: 1351-1356Crossref PubMed Scopus (255) Google Scholar). While displaying differential tissue and cellular expression, all JAMs localize to endothelial sites of cell contact and as such are ideally situated to support leukocyte emigration (1Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P.J. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1158) Google Scholar, 3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 4Palmeri D. van Zante A. Huang C.C. Hemmerich S. Rosen S.D. J. Biol. Chem. 2000; 275: 19139-19145Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 5Aurrand-Lions M. Johnson-Leger C. Wong C., Du Pasquier L. Imhof B.A. Blood. 2001; 98: 3699-3707Crossref PubMed Scopus (220) Google Scholar, 6Aurrand-Lions M. Duncan L. Ballestrem C. Imhof B.A. J. Biol. Chem. 2001; 276: 2733-2741Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Most recently we demonstrated that JAM3 2Official nomenclature for junctional adhesion molecules can be viewed at LocusLink (www.ncbi.nlm.nih.gov/LocusLink). Other synonyms: human JAM1 protein is equivalent to human JAM, mouse JAM, and mouse JAM-1; human JAM2 protein is equivalent to VE-JAM and mouse JAM-3; human JAM3 protein is equivalent to mouse JAM-2. was the 43-kDa T cell-expressed JAM2 (VE-JAM) counter-receptor. Up-regulation of JAM3 following T cell activation revealed a mechanism by which selective adhesion and/or emigration of lymphocytes may occur (7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The observation that JAM3 is also expressed on natural killer and dendritic cells and is capable of adhering to JAM2 (VE-JAM) extends the role of the JAM2 (VE-JAM)/JAM3 heterotypic interaction in inflammation (8Liang T.W. Chiu H.H. Gurney A. Sidle A. Tumas D.B. Schow P. Foster J. Klassen T. Dennis K. DeMarco R.A. Pham T. Frantz G. Fong S. J. Immunol. 2002; 168: 1618-1626Crossref PubMed Scopus (123) Google Scholar). The importance of integrins in adhesion and transmigration is paramount and well established (9Worthylake R.A. Burridge K. Curr. Opin. Cell Biol. 2001; 13: 569-577Crossref PubMed Scopus (248) Google Scholar). Several key IgSF cell adhesion molecules engage integrin and in so doing impact on the multistep paradigm of leukocyte emigration (10Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar, 11Holness C.L. Simmons D.L. J. Cell Sci. 1994; 107: 2065-2070PubMed Google Scholar). To help define how JAM fits into this sequential cascade, we sought a relationship between the JAM and integrin families. In this study we report an interaction between JAM2 and α4β1 that is facilitated by prior engagement of JAM2 with T cell-expressed JAM3. The JAM nomenclature used throughout this report, and prior publications from this group, complies with the official names designated by the Human Genome Nomenclature Committee. JAM2-Fc adhesion to various calcein-acetoxymethyl ester (Molecular Probes Inc.)-loaded leukocyte cell lines was performed by capture of fusion protein onto 96-well plates by either goat anti-mouse IgG or chicken anti-Myc antibodies as described previously (3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Adhesion was performed in Tris-buffered saline (TBS) with various combinations of 1 mm EDTA, 1 mm CaCl2, 1 mm MgCl2, and 1 mmMnCl2 for 90 min at 37 °C in 5% CO2. Adhered cells were lysed, and fluorescence was quantified in a CytoFluor plate reader with excitation at 485 ± 20 nm and emission at 530 ± 25 nm. For inhibitor studies, the JAM3 ectodomain was cleaved from the JAM3-Fc by thrombin and purified as described previously (7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The JAM3 ectodomain, neutralizing JAM3 serum, integrin antibodies, or the compounds TBC 772 (C*WLDVC*) and TBC 1194 (C*DLVWC*) were preincubated for 30 min at 37 °C with calcein-loaded cells prior to their incorporation into the adhesion assay. For generation of the secreted JAM2-Fc-Myc fusion, sense 5′-GGGAAGCTTACTATCATAAGGCCTATGGGTTTTC-3′ and antisense 5′-GGAAGATCT TTTACCCGGAGTCCGGGAGAAGCTC-3′ oligonucleotides incorporating HindIII and BglII sites, respectively, were used to amplify the JAM2-Fc, minus the signal peptide and stop codon, from a previously generated construct (3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Cycling was achieved with Pfu DNA polymerase (Stratagene) as follows: one cycle at 95 °C for 45 s; 25 cycles at 95 °C for 45 s, 59 °C for 45 s, and 72 °C for 120 s; one cycle at 72 °C for 600 s. The product was inserted into the APtag-5 vector (GenHunter Corp.) usingHindIII and BglII to generate the JAM2-Fc with further C-terminal tags of AP, Myc, and His combined with the Ig κ-chain secretion signal peptide. For the JAM2 Ig fold domain 1 constructs, sense 5′-GCCGCGGATCCAAGATGGCGAGGAGG-3′ and antisense 5′-GGTACCTGCTGGAGCCACTAATAC-3′ primers that incorporatedBamHI and KpnI sites, respectively, were used. Cycling was with Takara Ex Taq DNA Polymerase (Panvera) as follows: one cycle at 95 °C for 120 s; 20 cycles at 95 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s; and one cycle at 72 °C for 300 s. For JAM2 Ig fold domain 2 constructs, sense 5′-GTTCCATCATGTGAAGTACC-3′ and antisense 5′-GGCCTATGGGTTTTCTGCC-3′ oligonucleotides were used to loop out the N-terminal Ig fold using Pfu DNA polymerase (Stratagene) and cycling as follows: one cycle at 94 °C for 240 s, 50 °C for 120 s, 72 °C for 600 s; 12 cycles at 94 °C for 60 s, 55 °C for 120 s, and 68 °C for 600s. Individual domains were subcloned into pFastBac1 vector (Invitrogen) possessing the constant region of mouse IgG2a (3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The QuikChange site-directed mutagenesis kit (Stratagene) was used for mutagenesis. Primers for JAM2-D82A were: sense, 5′-CAGACTCTTCAAGGTGCTTTTAAAAATCGAGCTG-3′and antisense 5′-CAGCTCGATTTTTAAAAGCACCTTGAAGAGTCTG-3′. JAM-Fc fusion proteins were generated as secreted proteins in Sf21 cells as previously described or in COS cells (3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). For the latter, cells were transfected with 6 μg of the various pcDNA6 JAM-Fc constructs and 18 μl of FuGENE 6 reagent (Roche Molecular Biochemicals). Serum-free media from either cell type was harvested on day 3 and purified over HiTrap Protein A HP columns (Amersham Biosciences). HSB cells (1 × 106) were labeled with primary monoclonal antibodies in phosphate-buffered saline for 45 min followed by subsequent incubation with fluorescein isothiocyanate-conjugated secondary antibodies. Cells were analyzed in a Beckman Coulter Epics XL. The neutralizing integrin antibodies against α4 (clone P4C2), β1 (clone P5D2), and β2 (clone YFC118.3) were from Chemicon, and anti-β7 (clone R35-95) was from BD PharMingen. Chicken anti-Myc was purchased from Aves Labs, Inc. TBC 772 and TBC 1194 are drugs generated by Texas Biotechnology Corp. Neutralizing anti-JAM3 polyclonal serum was generated in female BALB/c mice. The purified JAM3 ectodomain was used as immunogen using procedures described previously (3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In characterizing the JAM2 interaction with T cell-expressed JAM3, we routinely performed adhesion in TBS plus all three of the cations Ca2+, Mg2+, and Mn2+ (binding buffer). An examination of the divalent cation dependence of adhesion revealed that JAM2 binding to HSB cells occurred independently of cation additions (Fig. 1 a). Thus, binding in the presence of TBS plus 1 mm EDTA was comparable with that obtained in binding buffer. In contrast a marked enhancement of JAM2 adhesion, above and beyond that obtained in binding buffer, was observed in the presence of TBS plus Mn2+. Subsequent analysis using cation combinations revealed that calcium was responsible for masking the enhancement of Mn2+ (Fig.1 a). In HSB and other T cell lines, we routinely recorded up to a 10-fold enhancement of adhesion. The divalent cation dependence of integrin function is well established. Coordination of divalent cations by integrin, and in particular Mn2+, induces conformational changes within integrin extracellular domains resulting in exposure of epitopes required for ligand engagement (12Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 13Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (447) Google Scholar). Additionally, the ability of Ca2+ to counteract this effect is a general observation (11Holness C.L. Simmons D.L. J. Cell Sci. 1994; 107: 2065-2070PubMed Google Scholar, 12Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 13Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (447) Google Scholar, 14Dransfield I. Cabanas C. Craig A. Hogg N.J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (401) Google Scholar). Thus, our data suggested that JAM2 was not only able to adhere with T cell-expressed JAM3 but also possibly engage with an integrin counter-receptor on the same cell. As the first candidates to investigate, we considered the α4 integrins since they play a proven role in lymphocyte motility and appear particularly specialized to promote leukocyte migration (15Kassner P.D. Alon R. Springer T.A. Hemler M.E. Mol. Biol. Cell. 1995; 6: 661-674Crossref PubMed Scopus (96) Google Scholar, 16Liu S. Rose D.M. Han J. Ginsberg M.H. Trends Cardiovasc. Med. 2000; 10: 253-257Crossref PubMed Scopus (26) Google Scholar, 17Chan P.Y. Aruffo A. J. Biol. Chem. 1993; 268: 24655-24664Abstract Full Text PDF PubMed Google Scholar, 18Wu C. Fields A.J. Kapteijn B.A. McDonald J.A. J. Cell Sci. 1995; 108: 821-829PubMed Google Scholar, 19Szabo M.C. Teague T.K McIntyre B.W. J. Immunol. 1995; 154: 2112-2124PubMed Google Scholar). The α4 subunit associates with β1 and β7 to form α4β1 and α4β7. Analysis of HSB cells by flow cytometry revealed expression of both α4 and β1, whereas the β7 subunit was not detectable (Fig. 1 b). To probe for an interaction between JAM2 and α4β1, we used neutralizing antibodies raised against the individual α4 and β1 subunits to inhibit the JAM2 adhesion to HSB cells. Fig. 1 c unequivocally demonstrates that JAM2 binds α4β1 in HSB cells. As expected, both neutralizing antibodies and isotype controls were without effect when adhesion was tested in TBS, the component assigned to the binding of JAM2 with JAM3 (7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). In contrast both anti-α4 and anti-β1 greatly attenuated the Mn2+-enhanced component, reducing it to a level comparable to that obtained in TBS alone. Higher concentrations of antibody did not further attenuate adhesion (data not shown). The data thus demonstrate a mixed binding reaction in TBS + Mn2+ where molecules of JAM2 bind JAM3 and/or α4β1. VCAM-1, the classical IgSF binding partner for α4β1, also binds to α4β7 (20Ruegg C. Postigo A.A. Sikorski E.E. Butcher E.C. Pytela R. Erle D.J. J. Cell Biol. 1992; 117: 179-189Crossref PubMed Scopus (316) Google Scholar). Whether JAM2 can engage with α4β7 in other cell lines or under other conditions remains an open question. Just prior to completion of this study, Weber et al. (21Ostermann G. Weber K.S. Zernecke A. Schroder A. Weber C. Nat. Immunol. 2002; 3: 151-158Crossref PubMed Scopus (539) Google Scholar) demonstrated the interaction of JAM1 with LFA-1. A β2 integrin binding component is not apparent in our JAM2 adhesion assays; although the β2subunit is expressed on the HSB cell surface, the neutralizing antibody has no effect on JAM2 HSB cell adhesion (Fig. 1, b andc). To extend and further validate this interaction, we TBC 772, a and of prevent the engagement of JAM2 with α4β1 P. K. D. Bjercke R.J. P.J. J. Immunol. Google Scholar). The show that although TBC 772 is when in TBS, a is observed in the presence of Mn2+ (Fig.1 with adhesion a level that that obtained in TBS with an 60 nm over three The of is further demonstrated by the of TBC a to the Mn2+-enhanced component (Fig. 1 demonstrate that the is readily amenable to by molecules and thus for drug The selective adhesion of JAM2 to T cells was previously when binding in the presence of all three cations (3Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). While conditions are for the in this the that with the defined cation reported in Fig. 1 a have Thus, we JAM2 adhesion in the presence of Mn2+ to cells and cells that also α4β1 but not JAM3 (7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The cells that JAM3 α4β1 were as a Fig. 2 that JAM2 binding is to T cells. our conditions revealed a dependence for the JAM2/α4β1 interaction of JAM3 within the same cell. We to impact T cell-expressed JAM3 on the that binding of JAM2 to JAM3 facilitated interactions of JAM2 with α4β1. To this we the effect of soluble JAM3 ectodomain by thrombin from JAM3-Fc and which is an of JAM2 binding to cell JAM3 (7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Fig. 2 b that soluble JAM3 can prevent HSB cell adhesion to JAM2 of this our it be that JAM3 and α4β1 a binding on soluble JAM3 binding to JAM2-Fc, epitopes for engagement with α4β1 may be we used neutralizing anti-JAM3 serum to prevent the interaction JAM2 for adhesion (Fig. 2 The of JAM2 binding under conditions to that JAM2 bind HSB cell-expressed JAM3 as a to interactions with α4β1. To the mechanism in we out to the domain dependence of the The N-terminal and C-terminal Ig were generated as fusion proteins and into the adhesion assay. a that the first Ig fold is capable of both and Mn2+-enhanced adhesion and the primary sites of contact for both JAM3 and integrin The other well described IgSF molecules that engage α4 namely and also binding in the N-terminal Ig fold L. C. R. I. T. Cell Biol. 1994; PubMed Scopus Google Scholar, Springer T.A. J. Cell Biol. 1994; PubMed Scopus Google Scholar, S. Chiu H.H. M.E. D. S. Fong S. J. Immunol. 1996; Google Scholar, L. Butcher E.C. J. Immunol. 1996; Google Scholar). In the Ig fold of which is to the binding to (21Ostermann G. Weber K.S. Zernecke A. Schroder A. Weber C. Nat. Immunol. 2002; 3: 151-158Crossref PubMed Scopus (539) Google Scholar). IgSF molecules bind integrin through key within the the and of the Ig fold (11Holness C.L. Simmons D.L. J. Cell Sci. 1994; 107: 2065-2070PubMed Google Scholar). a has from within VCAM-1, adhesion and of the JAM2 only acidic and Asp-82, that within the and of the N-terminal Ig fold D. Brockhaus M. A. P. G. G. Dejana E. Bartfai T. M. J. 2001; PubMed Scopus Google Scholar). with is to be an of the namely between JAM2 D. Brockhaus M. A. P. G. G. Dejana E. Bartfai T. M. J. 2001; PubMed Scopus Google Scholar). well within the In is to within the While it not within the by other cell adhesion the is of the to extracellular To to JAM2 adhesion, we to and Fig. 3 b that JAM2 not play a in of the was the observation that of this not attenuate JAM3 JAM2 may an acidic residue in a loop or possibly a to bind α4β1. As it deviates somewhat from the classical The binding sites by JAM2 to α4β1 engagement may the apparent of this The for adhesion of JAM2 and with α4β1 under the same is capable of in the presence of 1 mm Ca2+, Mg2+, and Mn2+, and interaction is of JAM3. the other under the conditions defined the JAM2/α4β1 interaction not appear to capture of HSB cells in but the of JAM3. the Ca2+ cation be in the assay. Mn2+ is by the for the of the β1 subunit from the to our data that only the α4β1 for JAM2 binding A.P. J.A. S. Craig J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). this in is not but an by the JAM2 interaction with JAM3. While this may be of a interaction between JAM2 and α4β1 by between the other can be For it was recently from that JAM1 bind in both and (21Ostermann G. Weber K.S. Zernecke A. Schroder A. Weber C. Nat. Immunol. 2002; 3: 151-158Crossref PubMed Scopus (539) Google Scholar). JAM2 not appear to be a T cell-expressed a interaction be between α4 and JAM3 (7Arrate M.P. Rodriguez J.M. Tran T.M. Brock T.A. Cunningham S.A. J. Biol. Chem. 2001; 276: 45826-45832Abstract Full Text Full Text PDF PubMed Scopus (191) Google T.W. Chiu H.H. Gurney A. Sidle A. Tumas D.B. Schow P. Foster J. Klassen T. Dennis K. DeMarco R.A. Pham T. Frantz G. Fong S. J. Immunol. 2002; 168: 1618-1626Crossref PubMed Scopus (123) Google Scholar). using the same we have to such a relationship between the of α4 and JAM3 (data not In the multistep paradigm of leukocyte that JAM2 remains within the cell we the JAM2/α4β1 to occur to that of the interaction as the leukocyte it emigration between endothelial cells. endothelial molecules that facilitate leukocyte are required to form only and/or interactions with the cell. first report, the of JAM2 adhesion to α4β1, a interaction that may be particularly for this role and may a for of We are particularly to for of to for all leukocyte cell lines, and to for of Sf21 and COS cells. We and Vanderslice for of the and