The Role of CD47 in Neutrophil Transmigration
CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response. CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response. polymorphonuclear leukocyte immunoglobulin variable domain myeloperoxidase Hanks' balanced salt buffer devoid of Ca2+ and Mg2+ formylmethionylleucylphenylalanine phosphatidylinositol 3-kinase monoclonal antibody Dulbecco's modified Eagle's medium junction adhesion molecule thrombospondin-1 fluorescence-activated cell sorting protein kinase C mitogen-activated protein kinase/extracellular signal-regulated kinase kinase fluorescein isothiocyanate polymerase chain reaction alkaline phosphatase In many inflammatory conditions, large numbers of PMN1migrate across vascular endothelium, cell matrix, and mucosal surfaces in response to chemotactic signals. Under these conditions, PMN release active oxygen species, proteolytic enzymes, and inflammatory mediators that result in tissue injury. Thus, it is intuitive that large scale PMN migration and accumulation in tissues correlates with disease activity and patient symptoms (1Stockley R.A. Am. J. Med. 1995; 99: 8S-13SAbstract Full Text PDF PubMed Scopus (47) Google Scholar,2Kumar N.B. Nostrant T.T. Appelman H.D. Am. J. Surg. Pathol. 1982; 6: 523-529Crossref PubMed Scopus (142) Google Scholar). The process of PMN movement from the vasculature, through the interstitial matrix, and across mucosal surfaces requires sequential interactions between neutrophil surface molecules and counter-receptors present on tissues or cell surfaces (3Parkos C.A. Bioessays. 1997; 19: 865-873Crossref PubMed Scopus (67) Google Scholar, 4Lawrence M.B. Springer T.A. Cell. 1991; 65: 859-873Abstract Full Text PDF PubMed Scopus (1887) Google Scholar, 5Muller W.A. Weigl S.A. Deng X. Phillips D.M. J. Exp. Med. 1993; 178: 449-460Crossref PubMed Scopus (990) Google Scholar, 6Spertini O. Kansas G.S. Munro J.M. Griffin J.D. Tedder T.F. 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Despite the of studies on CD47, the of CD47 Although we have anti-CD47 inhibition of PMN transmigration C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google the of this is The of the was to investigate the of CD47 in PMN migration across epithelial cell monolayers and collagen-coated a transmigration we that PMN migration across epithelial monolayers and collagen-coated filters is significantly delayed after cell surface CD47 is blocked by anti-CD47 mAbs. delayed transmigration by anti-CD47 mAbs, the of PMN that is the same as that with Cell surface labeling and immunoprecipitation increased CD47 on the PMN cell surface after with fMLP and after PMN fractionation revealed that increased cell surface CD47 is from intracellular that co-sediment with the secondary Experiments performed to investigate potential signaling pathways revealed that inhibition of partially reversed anti-CD47 inhibition of migration the rate of PMN migration in a that a rapid increase of PMN cell surface CD47. studies revealed that tyrosine phosphorylation inhibition by genistein reversed anti-CD47 in a near complete In of the that cell surface CD47 the rate of we that transfection of CD47 epithelial cells (CaCO2) results in PMN these results suggest that of CD47 to the cell surface and tyrosine signaling events that the rate of PMN migration in an inflammatory response. T84 cells were in a of Dulbecco's modified Eagle's medium and medium with buffer and were or with and in Ca2+ and transmigration T84 cells were on filters with a surface of as C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). cells were in with cells and were in the same medium PMN were from of as C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). PMN were in modified Hanks' balanced salt devoid of or a of and of a was in as C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). anti-CD47 M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google was from of cells from the CD47 and were in by with CD47 from of cells C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). from were with cells and in were with CD47 by in with cell surface by and on T84 epithelial cells and were by from and the of were and antibody was to by the and immunoprecipitation of CD47. antibody was by with to a of the C of CD47 H.D. J. Cell Biol. 1993; PubMed Scopus Google Scholar). a non-inhibitory with junction adhesion molecule was A. T.A. S. Parkos C.A. J. Cell Sci. PubMed Google Scholar). to CD47, is a on the surfaces of PMN and epithelial cells A. T.A. S. Parkos C.A. J. Cell Sci. PubMed Google Scholar). a was Parkos C.A. J. Google Scholar). integrin and were from was by was from to was by thrombospondin-1 and antibody were by J.M. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). antibody was by with a to the C of to the cell domain of shown to to cells M.B. W.A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, W.A. Biol. Cell. PubMed Scopus Google was and to by the PMN migration were performed epithelial monolayers as C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). T84 or monolayers were with PMN in of with or antibody was to the of the was by of fMLP the by PMN migration across monolayers the was by myeloperoxidase as C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). the of PMN the of PMN and monolayers were to of fMLP in as in PMN migration the the was by the PMN with epithelial transmigration was and monolayers were with and with were to PMN migration was performed that as many PMN were the transmigration were performed collagen-coated filters devoid of epithelial these of a PMN cells was to from the to the of were the to the effects of anti-CD47 on PMN these PMN were with the to in migration or and genistein phosphorylation and from phosphorylation tyrosine kinase protein kinase C kinase migration was in the presence of the same of in inhibition PMN were with in migration CD47 labeling was performed by of of PMN with anti-CD47 in after with PMN were with secondary antibody in by CD47 labeling in this condition was to that with PMN surface CD47 during PMN that in the or the of were on after migration by antibody labeling and Cell surface CD47 on PMN was after PMN were with In these PMN in of were to in or fMLP was by were on and labeling performed as PMN in or to were with fMLP by cell surface with of in of on was and with were with buffer and Cell of were with control with of of of were in of buffer and to by to was blocked by in cell surface CD47 was with by CD47 was by with anti-CD47 antibody and was with secondary and and PMN were in of buffer by by The were and were to fractionation on in a as C.A. J. Biol. Full Text PDF PubMed Google Scholar). fractions were from and plasma granules and secondary granules as C.A. J. Biol. Full Text PDF PubMed Google Scholar). The protein of CD47 was from with and the and was with and with the same cells were with or as by the were by in Cell surface of CD47 was by labeling and The domain of CD47, the was by and from T84 The was the Cell. Full Text PDF PubMed Scopus Google the domain of alkaline phosphatase the C and were and the was by of were performed a The of and of Scholar). and was by activity in cell medium Cell. Full Text PDF PubMed Scopus Google Scholar). protein in medium was by and with buffer by and we demonstrated that anti-CD47 PMN migration across T84 monolayers C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). we that the of inhibition by or anti-CD47 M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google was variable in the transmigration of a that the variable inhibition the result of in PMN after CD47 this and investigate the of we modified the in migration in PMN migration was this we the of anti-CD47 and on the of PMN migration across T84 monolayers. were performed with non-inhibitory A. T.A. S. Parkos C.A. J. Cell Sci. PubMed Google and an control antibody with Parkos C.A. J. Google shown in in the presence of control non-inhibitory mAbs, of the PMN across T84 monolayers during the antibody and PMN migration across T84 monolayers during the with of the PMN migrating the antibody and In the PMN migration the to antibody and In PMN migration across the monolayers to to in the and to in the and migration of PMN the as a of migration is shown in non-inhibitory conditions, of the PMN across T84 monolayers after the of the by to control the of anti-CD47 antibodies or in of PMN migrating the during the and In the PMN in the PMN migration the the and and and the and with of PMN in the after the shown in anti-CD47 in a delay in PMN migration across T84 monolayers. In PMN that across T84 monolayers after only of the cells with a migration of of the PMN control the delay in PMN migration by anti-CD47 mAbs, the of PMN that after was the same as that non-inhibitory we that the inhibition with anti-CD47 with those with there was inhibition of PMN migration the to inhibition of PMN In we the PMN the epithelial monolayers during PMN transmigration was by rapid of the monolayers on by PMN the and filters were by shown in in the of only a PMN the and this was by of PMN with T84 monolayers in the control and anti-CD47 PMN in the control by a result of migration the in the presence of the of PMN with the of PMN migration the These results with the of PMN with a migration was C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). Thus, inhibition of cell surface CD47 with antibodies results in a delayed a in the of PMN migration across epithelial with T84 monolayers during migration with migration across T84 monolayers to was and PMN monolayers were by in a PMN migration across T84 monolayers to was and PMN monolayers were by CD47 and of these to CD47 and T84 epithelial CD47 and PMN Thus, these to in the domain of CD47. shown in of these delayed PMN migration across T84 monolayers in the transmigration with the In to PMN migration across T84 epithelial we the effects of anti-CD47 on PMN transmigration across collagen-coated similar of delayed PMN migration by anti-CD47 antibodies and in these These results that the of PMN migration of PMN CD47 interactions with cell surface CD47 or on T84 cells on the role of CD47 in PMN we PMN cell surface CD47 as a result of shown in in the of anti-CD47 cell surface CD47 on PMN from the of only the transmigration In cell surface CD47 was significantly on the with the that of cells of cell surface CD47 during PMN transmigration in the presence of and delayed this antibody an increase of CD47 on PMN surfaces These results suggest that the delayed migration by anti-CD47 to of cell surface CD47 of CD47 on PMN cell surface after transmigration by in the in the PMN were to across collagen-coated filters by PMN in the or those that the were cell surface CD47 by as as of in a PMN were to across collagen-coated filters by PMN in the or those that the were cell surface CD47 by as as of Experiments were performed to investigate the increase of PMN cell surface CD47 is to fMLP stimulation. 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These results suggest that a of CD47, is by cell surface labeling in is to the cell surface after PMN to the rapid of cell surface after PMN after the increase of CD47 on the PMN cell surface was a and process The in the kinetics of between these cell surface that in intracellular with the intracellular of CD47 in we performed fractionation In these PMN were by by on Analysis of as shown in revealed that the between fractions and with plasma between fractions and secondary granules between fractions and and granules between fractions and similar to C.A. J. Biol. Full Text PDF PubMed Google Scholar). of CD47 in these fractions the of CD47 with and secondary granules in fractions a of CD47 was in plasma fractions with of intracellular CD47 the plasma fractions was similar to that to the CD47 a shown in a of CD47 with the granules after PMN stimulation. CD47 was in fractions protein in either condition shown to an important role in PMN and S. S. R. 1995; PubMed Scopus Google Scholar, C. 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Thus, increased cell surface CD47 correlates with PMN migration after inhibition of In an to the of anti-CD47-mediated transmigration delay, we a large of to signaling pathways in transmigration epithelial we collagen-coated filters in these that C and and and and and and the mitogen-activated protein kinase and were or in with anti-CD47 in migration Although of these effects on there was of anti-CD47 effects kinase PMN migration from anti-CD47 whereas the tyrosine kinase PMN transmigration and anti-CD47-mediated inhibition of PMN with the tyrosine kinase genistein J. S. S. J. Biol. Full Text PDF PubMed Google PMN transmigration an early in a similar to or shown in genistein in an increase in PMN transmigration to after the with of PMN in the control condition In to the with of PMN with genistein result in rapid increase of CD47 on PMN cell surface this reversed anti-CD47 inhibition in PMN migration in a near complete and the transmigration delay was reversed by PMN with genistein and in increased PMN transmigration to after the in the presence of the same of antibody and In genistein reversed inhibition of PMN transmigration in a shown in genistein PMN transmigration as as it reversed the that the of genistein was tyrosine we the of genistein with is a similar that many effects of genistein the inhibition of tyrosine phosphorylation J. S. S. J. Biol. Full Text PDF PubMed Google Scholar, J. Biol. PubMed Scopus Google Scholar). shown in PMN migration to the anti-CD47-mediated migration to tyrosine kinase and either PMN migration or the of genistein was that genistein have kinase to the results suggest that protein tyrosine phosphorylation by as downstream of the epithelial of CD47 C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google and results that cell surface CD47 PMN we epithelial of CD47 PMN In these T84 monolayers were with anti-CD47 or by in PMN transmigration was as a control in these to after the same antibodies were to PMN transmigration by to shown in of cell surface CD47 on T84 epithelial cells in a delay of PMN migration across the monolayers. in the presence of or PMN were in the migration after the of The of this is demonstrated by the of inhibition by a control with migration in the of antibody to the domain of adhesion molecule a is on the of T84 cells similar to CD47 A. T.A. S. Parkos C.A. J. Cell Sci. PubMed Google Scholar). inhibition of PMN migration was monolayers with In studies of the role of epithelial CD47 in PMN we that the epithelial cell CD47 in to the only CD47-deficient cell in H.D. J. Cell Biol. PubMed Scopus Google monolayers that to transmigration studies as shown in after CD47 was revealed a of CD47 on cells to demonstrated that the of CD47 the surfaces of monolayers as is in C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). of CD47 cells to the of these cells epithelial monolayers with of was from that with monolayers. The of epithelial CD47 on PMN migration was by migration across monolayers. shown in PMN migration across monolayers was increased to migration across In these of PMN across monolayers after the with of PMN that across control monolayers. In the PMN across whereas PMN across the of monolayers with anti-CD47 mAbs, by in PMN migration to an to that across These suggest it is PMN migration in PMN CD47 on epithelial cell surfaces PMN In of these we a increase in PMN migration across filters that with a CD47 protein of the domain to alkaline phosphatase These to the that PMN migration is by interactions by cell surface CD47. Previous studies have a role of CD47 in PMN migration across endothelial and epithelial monolayers C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar, M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). these effects anti-CD47 PMN migration by a of as and S. J. and C. A. this of PMN migration across epithelial monolayers and and collagen-coated filters the of of PMN The of PMN transmigration was to the effects of anti-CD47 mAbs. In these we the of PMN migration across T84 epithelial monolayers the between and cell surface CD47 with antibodies, PMN migration the was significantly delayed and was by increased PMN accumulation in epithelial monolayers and inhibition of CD47 and the of PMN that was the same as that with these it that CD47 the rate of PMN The anti-CD47-mediated delay in PMN migration we is secondary to antibody in of increased of antibodies to in or with antibody during the same results In the by anti-CD47 antibodies was through delayed PMN migration to the same as that CD47 from and epithelial cells is to PMN inhibition of CD47 in either cell in delayed PMN transmigration and C.A. A. Madara J.L. J. Cell Biol. PubMed Scopus Google Scholar). of CD47 on a CD47-deficient epithelial cell PMN migration across the monolayers CD47 is on cell H.D. J. Cell Biol. 1993; PubMed Scopus Google S. J. 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PubMed Scopus Google CD47 is to the PMN cell surface with a that is and the kinetics of cell migration in these In of on the PMN cell surface early after the of intracellular CD47 fractionation results that CD47 with of secondary granules and in and is to plasma and fractions stimulation. is only partially to plasma from the secondary granules after fMLP stimulation. The between these suggest that CD47 in a that is from that with a role of cell surface CD47 in migration the results of inhibition of PMN with or in rapid of CD47 on the PMN cell surfaces that correlated with increased of Previous studies have shown that have PMN migration C. O. S. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. A. A. A. J.M. PubMed Scopus Google Scholar). results suggest that as and important in modulating PMN on these we a of CD47 in PMN PMN CD47 is to the PMN cell it through in with or in In CD47 downstream signaling result in an rate of PMN on the is in a of CD47 to the cell surface and PMN Thus, inhibition of with or results in rapid in cell surface CD47 with an increased rate of The of inhibition is a of the inhibition with anti-CD47 antibodies many studies have shown that is after PMN S. S. R. 1995; PubMed Scopus Google Scholar, S. S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. PubMed Scopus Google results suggest that this results in the of a pathways in the of the rate of PMN tyrosine on the as a downstream that after fMLP and CD47 that of CD47 a of signaling from a to a in and PMN of CD47 by antibodies this in a rate of PMN Under this inhibition of tyrosine kinase by genistein results in a tyrosine in PMN migration and anti-CD47-mediated inhibition of Although the to studies to the intracellular signaling events through protein tyrosine and to the of these tyrosine with CD47 in the process of PMN to protein and the downstream that to the inflammatory response. these events that PMN migration and to and tissue and