CD40 Ligand Binds to α5β1 Integrin and Triggers Cell Signaling

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L–/– and CD40–/– mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an αIIbβ3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and αIIbβ3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-α5β1 monoclonal antibody P1D6, and soluble α5β1. The direct binding of sCD40L to purified α5β1 was confirmed in a solid phase binding assay. Binding of sCD40L to α5β1 was modulated by the form of α5β1 expressed on the cell surface as the activation of α5β1 by Mn2+ or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of α5β1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an α5β1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble α5β1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, α5β1, and αIIbβ3) for CD40L. It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L–/– and CD40–/– mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an αIIbβ3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and αIIbβ3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-α5β1 monoclonal antibody P1D6, and soluble α5β1. The direct binding of sCD40L to purified α5β1 was confirmed in a solid phase binding assay. Binding of sCD40L to α5β1 was modulated by the form of α5β1 expressed on the cell surface as the activation of α5β1 by Mn2+ or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of α5β1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an α5β1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble α5β1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, α5β1, and αIIbβ3) for CD40L. The CD40 ligand (CD40L), 4The abbreviations used are: CD40L, CD40 ligand; rsCD40L, recombinant soluble CD40L; rsCD40L-A, Alexa Fluor 488-labeled rsCD40L; avidin-A, Alexa Fluor 488-labeled avidin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; sα5β1, soluble α5β1; TNF, tumor necrosis factor; mAb, monoclonal antibody; IL-8, interleukin-8; HRP, horseradish peroxidase; BSA, bovine serum albumin; HBSS, Hanks’ balanced salt solution; DTT, dithiothreitol; PBS, phosphate-buffered saline. 4The abbreviations used are: CD40L, CD40 ligand; rsCD40L, recombinant soluble CD40L; rsCD40L-A, Alexa Fluor 488-labeled rsCD40L; avidin-A, Alexa Fluor 488-labeled avidin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; sα5β1, soluble α5β1; TNF, tumor necrosis factor; mAb, monoclonal antibody; IL-8, interleukin-8; HRP, horseradish peroxidase; BSA, bovine serum albumin; HBSS, Hanks’ balanced salt solution; DTT, dithiothreitol; PBS, phosphate-buffered saline. also known as CD154 or gp39, is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) superfamily and is expressed on a variety of hematopoietic and non-hematopoietic cells. The main sources of CD154 are activated platelets and CD4+ T cells (1Andre P. Nannizzi-Alaimo L. Prasad S.K. Phillips D.R. Circulation. 2002; 106: 896-899Crossref PubMed Scopus (490) Google Scholar). CD154 is also variably expressed on activated CD8+ T cells, activated B cells, eosinophils, mast cells, basophils, natural killer cells, dendritic cells, monocytes, and macrophages as well as endothelial cells, epithelial cells, and smooth muscle cells (reviewed in Ref. 2Schonbeck U. Libby P. Cell Mol. Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar). Expression of CD40L differs according to cell types and type of stimuli (2Schonbeck U. Libby P. Cell Mol. Life Sci. 2001; 58: 4-43Crossref PubMed Google Scholar). The expression of CD154 is inducible, and its expression on T cells is triggered primarily by T cell receptor signaling and is regulated by CD28-dependent and independent pathways (3Ding L. Green J. Thompson C. Shevach E. J. Immunol. 1995; 155: 5124-5132PubMed Google Scholar) CD40L is stored in platelets and a subpopulation of T cells and rapidly translocates to the cell membrane following T cell and platelet activation (4Casamayor-Palleja M. Khan M. MacLennan I.C. J. Exp. Med. 1995; 181: 1293-1301Crossref PubMed Scopus (224) Google Scholar, 5Henn V. Slupsky J.R. Grafe M. Anagnostopoulos I. Forster R. Muller-Berghaus G. Kroczek R.A. Nature. 1998; 391: 591-594Crossref PubMed Scopus (1735) Google Scholar). A soluble form of biologically active CD40L trimer (sCD40L) is present in the supernatant of activated T cells (6Pietravalle F. Lecoanet-Henchoz S. Blasey H. Aubry J.P. Elson G. Edgerton M.D. Bonnefoy J.Y. Gauchat J.F. J. Biol. Chem. 1996; 271: 5965-5967Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and platelets (7Jin Y. Nonoyama S. Morio T. Imai K. Ochs H.D. Mizutani S. J. Med. Dent. Sci. 2001; 48: 23-27PubMed Google Scholar) and results from the proteolytic cleavage of the homotrimeric CD40L by a metalloproteinase (7Jin Y. Nonoyama S. Morio T. Imai K. Ochs H.D. Mizutani S. J. Med. Dent. Sci. 2001; 48: 23-27PubMed Google Scholar). It was originally thought that CD40L had only one receptor, CD40, which is a type I transmembrane protein that is a member of the TNF receptor superfamily. CD40 is expressed on the surface of many immune and non-immune cells, including B lymphocytes, monocytes/macrophages, and dendritic cells, as well as platelets, epithelial, and endothelial cells (8Grammer A.C. Lipsky P.E. Adv. Immunol. 2000; 76: 61-178Crossref PubMed Google Scholar). Most biological functions of CD40L have been attributed to its direct interaction with CD40. However, studies using CD40L–/– and CD40–/– mice have suggested that CD40L may also bind to one or more other receptors (9Mehlhop P.D. van de Rijn M. Brewer J.P. Kisselgof A.B. Geha R.S. Oettgen H.C. Martin T.R. Am. J. Respir. Cell Mol. Biol. 2000; 23: 646-651Crossref PubMed Scopus (38) Google Scholar). In support of this hypothesis, it has been elegantly demonstrated that sCD40L interacts with αIIbβ3 (GPIIb/IIIa), an integrin expressed on platelets (10Andre P. Prasad K.S. Denis C.V. He M. Papalia J.M. Hynes R.O. Phillips D.R. Wagner D.D. Nat. Med. 2002; 8: 247-252Crossref PubMed Scopus (646) Google Scholar), triggering outside-in signaling and inducing platelet activation and spreading (11Prasad K.S. Andre P. He M. Bao M. Manganello J. Phillips D.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12367-12371Crossref PubMed Scopus (184) Google Scholar). CD40L–/– mice exhibit increased bleeding time (12Crow A.R. Leytin V. Starkey A.F. Rand M.L. Lazarus A.H. J. Thromb. Haemost. 2003; 1: 850-852Crossref PubMed Scopus (30) Google Scholar) and reduced thrombus stability (10Andre P. Prasad K.S. Denis C.V. He M. Papalia J.M. Hynes R.O. Phillips D.R. Wagner D.D. Nat. Med. 2002; 8: 247-252Crossref PubMed Scopus (646) Google Scholar), showing that the interaction between sCD40L and the integrin αIIbβ3 is physiologically relevant. Based on the above observations, we hypothesized that CD40L may bind to another still unknown receptor on human monocytes and induce cellular activation. Our results show that sCD40L binds to CD40-negative human monocytic U937 cells, an interaction that is inhibited by anti-α5β1 integrin (VLA-5) mAb as well as by soluble α5β1 integrin (sα5β1). The binding of sCD40L to immobilized purified α5β1 confirms the direct interaction of sCD40L with this integrin. sCD40L induces the translocation of α5β1 to the Triton X-100-insoluble fraction, the rapid activation of MAPKs ERK1/2 in U937 cells, and IL-8 gene expression, confirming the existence of a third functional receptor for CD40L on α5β1. Cells—The myelomonocytic cell line U937 (ATCC, and the B cell cell line BJAB J. in bovine and and soluble CD40L R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was by was from Alexa of and was according to the and from the The mAb and mAb in mAb and mAb by mAb has been A. H. C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The following antibody mAb and antibody and antibody and antibody and mAb α5β1 and as J. Cell Sci. 2002; PubMed Scopus Google Scholar, K. 2003; PubMed Scopus Google Scholar). soluble was from the binding cells in binding of or for at in a and a binding with cell surface and cells with of human cells in for at with mAb for at to with binding with or soluble and with or for at to the of the cells. Cell surface with as C. F. R. J. Immunol. PubMed Scopus Google Scholar). cells on a with Mn2+ in or (10Andre P. Prasad K.S. Denis C.V. He M. Papalia J.M. Hynes R.O. Phillips D.R. Wagner D.D. Nat. Med. 2002; 8: 247-252Crossref PubMed Scopus (646) Google Scholar) in for at The cells in and in The cells used Cell Binding of with in for at was and the for at in PBS, was to the and the at The with and with in for at with and in to the and the for at cells and the with cells a and with a The with The cells with in for at and with in the with the cells with in and the at was Binding of with of purified sα5β1, or soluble recombinant in at with the with in for at with was to the at the and the was for at The with and was using at and at and with Cell and BJAB cells in for at and with for and at The was by the of and for cell by for to the and BJAB cells with for at in binding and in in Triton and Triton for on The cell at for at and by and by in in for at and with antibody at by antibody or with antibody at by The of ERK1/2 was by using according to the and with antibody with of IL-8 cells with of in bovine serum at for the time by and cells by at at was from using the for a was from of using a and the by the was at for and the on for the from for IL-8 and was by using The of the as and for of for for and the for of was at the of the The on a The of the for IL-8 and and not we the in this by on of at and on using a and from are for on at time and on the of expression of gene of as time The was as of IL-8 time of the to CD40-negative U937 an to a novel receptor for CD40L, we the expression of CD40 by and using cell and CD40-negative cell that bind Alexa The human BJAB B cell line was used as a The undifferentiated monocytic U937 cell line not CD40, as by and not was to bind at a to that on BJAB cells The of this binding was confirmed by a of or by with the anti-CD40L mAb results with the CD40-negative and cell not Based on we for evidence of binding to U937 cells in a manner. that of U937 cells with mAb not with the binding of rsCD40L-A, the the binding of to BJAB cells. These results that U937 cells at least one that is from CD40 and that as a receptor for also confirmed that CD40 is the receptor for CD40L on BJAB cells. to on the above and sCD40L also binds to αIIbβ3 (10Andre P. Prasad K.S. Denis C.V. He M. Papalia J.M. Hynes R.O. Phillips D.R. Wagner D.D. Nat. Med. 2002; 8: 247-252Crossref PubMed Scopus (646) Google Scholar, K.S. Andre P. He M. Bao M. Manganello J. Phillips D.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12367-12371Crossref PubMed Scopus (184) Google Scholar), which is expressed on platelets PubMed Scopus Google Scholar), hematopoietic J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), and mast cells T. J. K. Y. M. H. Y. H. H. T. J. Immunol. 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Cell 2003; PubMed Scopus Google Scholar) not on BJAB cells bind to α5β1, we a binding using soluble α5β1 as for that of with inhibited the binding of to U937 cells. of with not binding to CD40 on B cells, suggesting that sCD40L bind concomitantly to both CD40 and α5β1. provide support for the that α5β1 is in the binding of to U937 cells, cells with mAb or mAb and with that mAb inhibited the interaction of to U937 cells as not the binding of to α5β1-negative BJAB cells. the results support that α5β1 is the third receptor for to that sCD40L binds to α5β1, a solid phase binding was using immobilized soluble as a receptor The results in that binds to purified α5β1 in a manner. In not bind to other purified and confirming the of the α5β1, CD40 and is a receptor for the of α5β1 for the Binding of to U937 is expressed on the cell surface in an form that bind (reviewed in Ref. 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