Proteins of the fibroblast growth factor (FGF) family play diverse roles in embryonic development, angiogenesis, and wound healing. The most well studied targets of FGF activity typically are cells of mesodermal and neuroectodermal origin; in addition, expression of FGF-1 (acidic FGF) is increased at several sites of chronic immunologic injury, and recent studies show that FGF-1 also may interact with cells of the immune system. In some human T cells, FGF-1 can induce signals necessary for production of interleukin-2, a key cytokine required for T cell proliferation. To better characterize the interaction of FGF-1 with FGF receptors on T cells, a fusion protein was constructed containing a portion of the constant region of human IgG1 (Fc) at the amino terminus of FGF-1. The Fc-FGF-1 fusion protein retained FGF function as determined by stimulation of tyrosine phosphorylation and DNA synthesis in NIH 3T3 cells. Binding of the intact fusion protein to FGF receptor 1 (FGFR1) on T cells was demonstrated by immunoprecipitation of the receptor bound to Fc-FGF-1 and by flow cytometry showing binding of fusion protein to T cells expressing FGFR1. This functional Fc-FGF-1 protein should prove useful in identifying FGFR-expressing cells. Proteins of the fibroblast growth factor (FGF) family play diverse roles in embryonic development, angiogenesis, and wound healing. The most well studied targets of FGF activity typically are cells of mesodermal and neuroectodermal origin; in addition, expression of FGF-1 (acidic FGF) is increased at several sites of chronic immunologic injury, and recent studies show that FGF-1 also may interact with cells of the immune system. In some human T cells, FGF-1 can induce signals necessary for production of interleukin-2, a key cytokine required for T cell proliferation. To better characterize the interaction of FGF-1 with FGF receptors on T cells, a fusion protein was constructed containing a portion of the constant region of human IgG1 (Fc) at the amino terminus of FGF-1. The Fc-FGF-1 fusion protein retained FGF function as determined by stimulation of tyrosine phosphorylation and DNA synthesis in NIH 3T3 cells. Binding of the intact fusion protein to FGF receptor 1 (FGFR1) on T cells was demonstrated by immunoprecipitation of the receptor bound to Fc-FGF-1 and by flow cytometry showing binding of fusion protein to T cells expressing FGFR1. This functional Fc-FGF-1 protein should prove useful in identifying FGFR-expressing cells. Classical targets for stimulation by heparin binding growth factors FGF-1 and FGF-2 (basic FGF) 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; IL, interleukin; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; BS3, bis(sulfosuccinimidyl) suberate; KGF, keratinocyte growth factor. include cells of mesodermal and neuroectodermal origin such as endothelial cells, smooth muscle cells, fibroblasts, and myoblasts (reviewed in Ref. 1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). FGF-1 and FGF-2 potently induce proliferation and migration of these cells as well as angiogenesis, events that are required during normal embryogenesis and tissue repair. Although expression of FGF-1 and FGF-2 at sites of injury is therefore beneficial, some evidence suggests these growth factors may also contribute to vascular pathology by promoting excessive intimal hyperplasia (2Banai S. Jaklitsch M.T. Casscells W. Shou M. Shrivastav S. Correa R. Epstein S.E. Unger E.F. Circ. Res. 1991; 69: 76-85Crossref PubMed Scopus (136) Google Scholar, 3Flugelman M.Y. Virmani R. Correa R. Yu Z. Farb A. Leon M.B. Elami A. Fu Y. Casscells W. Epstein S.E. Circulation. 1993; 88: 2493-2500Crossref PubMed Scopus (150) Google Scholar, 4Broji E. Winkles J.A. Underwood R. Clinton S.K. Alberts G.F. Libby P. J. Clin. Invest. 1993; 92: 2408-2418Crossref PubMed Scopus (192) Google Scholar, 5Wagner C.R. Morris T.E. Shipley G.D. Hosenpud J.D. J. Clin. Invest. 1993; 92: 1269-1277Crossref PubMed Scopus (27) Google Scholar). FGF-1 expression is increased at several sites of chronic immune injury, including the synovium in rheumatoid arthritis (6Sano H. Forough R. Maier J.A.M. Case J.P. Jackson A. Engleka K. Maciag T. Wilder R.L. J. Cell Biol. 1990; 110: 1417-1426Crossref PubMed Scopus (161) Google Scholar, 7Sano H. Engleka K. Mathern P. Hla T. Crofford L.J. Remmers E.F. Jelsema C.L. Goldmuntz E. Maciag T. Wilder R.L. J. Clin. Invest. 1993; 91: 553-576Crossref PubMed Scopus (79) Google Scholar, 8Byrd V. Zhao X.M. McKeehan W.L. Miller G.G. Thomas J.W. Arthritis Rheum. 1996; 39: 914-922Crossref PubMed Scopus (28) Google Scholar), the myocardium in human hearts after transplantation (9Zhao X.M. Yeoh T.K. Hiebert M. Frist W.H. Miller G.G. Transplantation. 1993; 56: 1177-1182Crossref PubMed Scopus (43) Google Scholar, 10Zhao X.M. Yeoh T.K. Frist W.H. Porterfield D.L. Miller G.G. Circulation. 1994; 90: 677-685Crossref PubMed Scopus (65) Google Scholar, 11Zhao X.M. Frist W.H. Yeoh T.K. Miller G.G. J. Clin. Invest. 1994; 94: 992-1003Crossref PubMed Scopus (36) Google Scholar), and in transplanted kidneys undergoing chronic rejection (12Kerby J.D. Verran D.J. Luo K.L. Ding Q. Tagouri Y. Herrera G.A. Diethelm A.G. Thompson J.A. Transplantation. 1996; 62: 190-200Crossref PubMed Scopus (31) Google Scholar, 13Paul L.C. Saito K. Davidoff A.W. Benediktsson H. Am. J. Kidney Dis. 1996; 28: 441-450Abstract Full Text PDF PubMed Scopus (43) Google Scholar). The pathologic lesions in these sites demonstrate cellular responses typical of FGF effects on mesenchymal cells that result in vascular intimal hyperplasia, increased extracellular matrix deposition, and neoangiogenesis. In addition, these sites are characterized by chronic infiltration of T lymphocytes, suggesting there may be interactions between the immune system and fibroblast growth factors as demonstrated by the finding that T cells can produce FGF-2 (14Blotnick S. Peoples G.E. Freeman M.R. Eberlein T.J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1994; 4: 2890-2894Crossref Scopus (279) Google Scholar, 15Peoples G.E. Blotnick S. Takahashi K. Freeman M.R. Klagsbrun M. Eberlein T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6547-6551Crossref PubMed Scopus (135) Google Scholar). Evidence that FGFs may have immunoregulatory effects on T cells was first provided in 1985 by Johnson and Torres (16Johnson H.M. Torres B.A. J. Immunol. 1985; 134: 2824-2826PubMed Google Scholar), who showed that FGF, at physiologically relevant concentrations, could replace the requirement for IL-2 or helper cells in production of interferon-γ. Although FGF could activate intracellular signals necessary for interferon-γ production, FGF alone could not stimulate proliferation of T cells. More recent studies show directly that some human T cells express receptors for FGF-1 and that the normally small subpopulation of FGF-responsive T cells is expanded in the peripheral blood of patients with rheumatoid arthritis and in patients after heart transplantation (8Byrd V. Zhao X.M. McKeehan W.L. Miller G.G. Thomas J.W. Arthritis Rheum. 1996; 39: 914-922Crossref PubMed Scopus (28) Google Scholar, 17Zhao X.M. Byrd V.M. McKeehan W.L. Reich M.B. Miller G.G. Thomas J.W. J. Immunol. 1995; 155: 3904-3911PubMed Google Scholar). These data suggest that T cells bearing FGF receptors can be stimulated and expanded in the FGF-enriched environment at sites of immune injury and subsequently migrate to the peripheral blood. As found in the earlier studies (16Johnson H.M. Torres B.A. J. Immunol. 1985; 134: 2824-2826PubMed Google Scholar), FGF alone does not stimulate proliferation of T cells but together with engagement of the T cell antigen receptor induces production of IL-2 and proliferation (17Zhao X.M. Byrd V.M. McKeehan W.L. Reich M.B. Miller G.G. Thomas J.W. J. Immunol. 1995; 155: 3904-3911PubMed Google Scholar). In T cells, FGF thus functions in a manner analogous to other well described “costimulators” (18Fraser J.D. Weiss A. Mol. Cell. Biol. 1992; 12: 4357-4363Crossref PubMed Scopus (116) Google Scholar, 19Rudd C.E. Immunity. 1996; 4: 527-534Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) to activate a second signal transduction pathway necessary for T cell proliferation and effector function. These findings suggest that FGF and FGF receptors in T cells may function quite differently than in cells in which FGF alone can stimulate proliferation, migration, and secretion of effector molecules such as plasminogen activator (20Presta M. Moscatelli D. Joseph-Silverstien J. Rifkin D.B. Mol. Cell. Biol. 1986; 6: 4060-4066Crossref PubMed Scopus (226) Google Scholar). Our efforts to investigate T cells that express FGF receptors and the mechanisms by which FGF signals in T cells have been hampered by the lack of reagents that can conveniently identify these cells and allow us to examine the fate of FGF and its receptors in T cells. To address this difficulty, the experiments reported here describe the production and characterization of a fusion protein that includes a portion of the constant region (Fc) of human IgG1 at the amino terminus of full-length FGF-1. In contrast to a previously reported fusion protein of FGF-1 with diphtheria toxin A chain, Fc-FGF-1 retains full FGF function, including the ability to induce DNA synthesis, tyrosine phosphorylation, and FGFR1 binding as well as Fc-mediated binding to protein A and anti-human IgG antibodies. Plasmid BS-2 mutFc was kindly provided by Dr. Melanie Spriggs, Immunex Corp. This plasmid includes the murine IL-7 leader sequence, a nonamer FLAG epitope, CH2 and CH3 domains of human IgG1 (amino acids 242–470 (21Crowe J.S. Hall B.M. Smith M.A. Cooper J.H. Tite J.P. Clin. Exp. Immunol. 1992; 87: 105-110Crossref PubMed Scopus (71) Google Scholar)), and a (Gly4Ser)2 repeat to provide a flexible linker between the Fc domain and the amino terminus of the introduced protein (22Yao Z. Fanslow W.C. Seldin M.F. Rousseau A.M. Painter S.L. Comeau M.R. Cohen J.I. Spriggs M.K. Immunity. 1995; 3: 811-821Abstract Full Text PDF PubMed Scopus (806) Google Scholar). The Fc region of this construct includes several amino acid mutations introduced to diminish Fc receptor binding. SpeI and NotI sites were introduced at the 5′ and 3′ ends, respectively, of full-length FGF-1 cDNA by polymerase chain reaction, and the product was cloned into these sites downstream of The was and cloned into a the The fusion protein in could not be the and IL-7 leader were to containing the FLAG epitope, Fc and flexible linker by a 5′ and 3′ SpeI The and construct containing FGF downstream of were with and The a protein of FLAG and human IgG1 Fc to the amino terminus of FGF-1. for and were to the of fusion protein in the of the was to and were at for The were and in of and 1 and the containing was on experiments demonstrated some and of FGF-1 with at the protein was in at In some the protein was by protein with by the of to or A plasmid containing the human and region for expression in T cells, R. M. P. J. M. A. M. D. J. 1996; Scholar), was a Dr. cDNA J. M. J.W. E. Y. M. M. J. McKeehan W.L. J. Biol. 1992; Full Text PDF PubMed Google Scholar) was provided by Dr. was with and into the of A plasmid containing in the was and used to T cells as described To produce expressing a cDNA full-length FGF-1 (amino acids in was and into the of as A plasmid containing FGF-1 in the was and used to T cells as and with was to provide a were by of cells in with of or 1 of with was and were in containing with were by of cell with with were for FGFR1 by chain of for FGFR1. were with and with NIH 3T3 cells were in with and proliferation, NIH 3T3 cells were at in tissue in with was with for human FGF-1 or in were at the with and the were at for during which well 1 of for the was determined by are the of tyrosine phosphorylation, NIH 3T3 cells were in with serum on tissue with the was with cells were stimulated with human FGF-1 or heparin in for at The were with PBS, which the cells were with of containing and 1 cell were on to with in was with and and by T cells were in bovine and and were in the for of 1 for T cells or were and in BSA, was and the cells were on for 1 were by and in the and in with heparin and with The cells were with anti-human IgG (Fc) or anti-human IgG (Fc) for 1 on In some the cells were subsequently with (amino acids was in E. R. Engleka Thompson J.A. Jackson A. T. Maciag T. 1991; PubMed Scopus Google Scholar). was with T and on as described previously M. D. J. H. McKeehan W.L. J. Biol. Full Text PDF PubMed Google Scholar). was and in the manner that the with T was binding of FGF-1 or cells were with of FGF-1 or of in PBS, heparin in a of for 1 on The cells were by in PBS, in in containing and in The cells were in with of bis(sulfosuccinimidyl) as for at The was by and for at cells were in PBS, with with 1 and on with the cells were in PBS, with 1 were with protein directly or of anti-human IgG by the of protein immunoprecipitation of 1 of was used by binding to IgG to cells were of was in a and to the was and first with a cDNA and after with a were at protein was was was was in was of the on a of to the of the Fc-FGF-1 fusion protein 1 A portion of the fusion protein was with protein and was on the with to human and the FLAG 1 In the protein with the of the The was for of the fusion of the of a FLAG and binding by the with the and anti-human IgG antibodies. The on protein to and in of protein of of the fusion protein after with experiments were with on of at for than a to show to that with by at or was and showed of FGF by with cells in at 1 To that was NIH 3T3 cells were stimulated with or human and DNA synthesis was by stimulated DNA synthesis by NIH 3T3 cells in the of the required was on a than that required for to human FGF-1 or was in the of heparin not binding to FGF FGF-1 and FGF-2 stimulate tyrosine phosphorylation of several intracellular including a protein of R. W.H. Maciag T. Mol. Cell. Biol. 1989; PubMed Scopus Google L.J. J. Biol. Full Text PDF PubMed Google Scholar) that may be between and downstream effector protein J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) a protein that is in to FGF-2 and to the protein J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This protein is to a protein of function. More H. T. J. D. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) a protein of growth factor receptor that is FGF-1 stimulation and also H. T. J. D. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). To tyrosine phosphorylation FGFR, with was on NIH 3T3 cells stimulated with FGF-1 or and human FGF-1 tyrosine phosphorylation of a protein and between the data that functional FGF-1 and The studies were to that the functional FGF activity of in the intact fusion protein and not result of FGF-1. studies demonstrated that a of human T cells and T cell can be stimulated to and produce IL-2 by the of FGF-1 and T cell antigen receptor engagement by (17Zhao X.M. Byrd V.M. McKeehan W.L. Reich M.B. Miller G.G. Thomas J.W. J. Immunol. 1995; 155: 3904-3911PubMed Google Scholar). were with the human T cell which was found to express FGFR1 by chain and with to FGFR1 experiments with T cells have that FGFR1 expression is in this cell the or of or of FGFR1 expression could not be to or in such as cell or of or of in To a system for of FGF-1 function and stimulation in T cells, T cells were with a plasmid expression of the of a T and In addition, T cells can produce FGF-2 (14Blotnick S. Peoples G.E. Freeman M.R. Eberlein T.J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1994; 4: 2890-2894Crossref Scopus (279) Google Scholar, 15Peoples G.E. Blotnick S. Takahashi K. Freeman M.R. Klagsbrun M. Eberlein T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6547-6551Crossref PubMed Scopus (135) Google Scholar) and of FGF-2 expression of FGFR1 A. S. P. Rifkin D.B. J. Cell Biol. 1995; PubMed Scopus Google Scholar, A. V. K. E. M. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), expressing FGF-1 were the expression demonstrated that with of for and with or for FGFR1. These cell were used first to examine binding of to demonstrate that the FGFR1 in a functional receptor that FGF and to that intact to FGFR1. and were by with BS3, a The cells were and FGFR1 binding of was by of cell and In were at and to the for to and or of the were also in with the of that was in these cells binding of was with with FGF-1. the was with were These findings several the FGFR1 in receptors of binding FGF as the binding of receptors were by the and by the used to of or with with was at the for of and FGFR1. This may not be the Fc domain bound to the receptor and the that other M. J.H. J. Cell Biol. 1995; PubMed Scopus Google Scholar). of to in these cells not demonstrate the at with of FGF-1. This suggests that there was not of FGF-1 to produce of FGFR1. To of to FGFR1 could be a of the was as described in the characterization of PubMed Scopus Google Scholar). In the of and of were of BS3, and of and at for of bound to FGFR1 as well as of on this immunoprecipitation experiments were in the and of at and were with and with As in immunoprecipitation of FGFR1 in the of of A to was also In immunoprecipitation of FGFR1 in the of of and with increased of fusion protein with with the of receptors by the As with the cell showed the binding of to The show that is with FGFR1. These experiments were also with and immunoprecipitation In to of bound to and are In and are also with fusion protein to FGFR, binding to is These binding of to FGFR1. As to demonstrate binding of intact fusion protein to the fusion protein was its Fc domain with protein by with to FGFR1 to identify FGFR1 that with was to at the were with protein A and the were with In that not been were at and The is at the for the of with The of the is not of FGFR1 are for and with the found in were with binding of at and the of the immunoprecipitation is demonstrated by the of a FGFR1 in the with fusion protein the cells were the showed at and with the of and In the data show that immunoprecipitation of FGFR1 intact and immunoprecipitation of the fusion protein its Fc domain that is bound to FGFR1. demonstrated that FGF with receptors on The constant for FGF-1 was with for not These are to findings reported for a fusion protein of with M. J.H. J. Cell Biol. 1995; PubMed Scopus Google Scholar). A of the fusion protein is of cells in therefore was for its in flow cytometry with and were with IgG directly or first with heparin to sites on the cell showed binding of by that was by with binding to a of sites and a small of was binding of to than to and this binding was by with binding to receptors on The requirement for with heparin to binding not with of other cell as for with after for the fusion protein of the cell well with the heparin The than in in by flow cytometry with expression found by immunoprecipitation and The experiments here show that a fusion protein of human IgG1 Fc at the amino terminus of FGF-1 retains FGF function and IgG constant region function, by stimulation of DNA synthesis and tyrosine phosphorylation in NIH 3T3 cells, binding to heparin and and binding to protein A and to human These in a a previously reported FGF-1 fusion a construct with diphtheria toxin A chain at the terminus of FGF-1 A. K. S. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, A. A. R. S. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). to induce cell proliferation or DNA synthesis in NIH 3T3 cells, induce tyrosine phosphorylation, suggesting bound to on NIH 3T3 cells A. K. S. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). In contrast to was found in the and to to the the fusion protein in the was in the or bound to was not In cells that express diphtheria toxin receptors but are to toxin of with in to the toxin receptors and DNA synthesis, but tyrosine phosphorylation was these cells lack FGF receptors A. K. S. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). experiments in cells with showed that this FGF receptor could FGF-1 to the but not binding of FGF-1 and the fusion These data were to that tyrosine phosphorylation and of FGF-1 to induce cell proliferation A. A. R. S. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). this is the the experiments here suggest that Fc-FGF-1 does to the as well as tyrosine phosphorylation FGF receptors and retains the functions of FGF-1. The that be for the functional of the fusion are at this A with fusion or at the amino terminus of FGF-1 is that at the amino terminus is during of to the of functional molecules to amino acids P. A. D. J. 1985; 4: PubMed Scopus (136) Google Scholar, W.H. T. B.A. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1986; PubMed Scopus Google Scholar, W.L. J.W. Biochem. PubMed Scopus Google Scholar). The of during of FGF-1 tissue increased the of full-length FGF-1 W.L. J.W. Biochem. 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The fusion protein and IgG Fc functions as of DNA synthesis in cells, binding to NIH 3T3 cells with receptor and not FGFR1 or by flow immunoprecipitation with and of receptor in As with the protein functional required of the fusion and there was a in receptor for on these data with the fusion protein and FGF-1 described the of IgG Fc domains to FGF family to function and receptor binding the of the Fc region with the The reported toxin of FGF-1 Epstein S. E. Hla T. Forough R. Maciag T. D.J. J. 1991; PubMed Scopus (27) Google Scholar) and FGF-2 W. D. M. A. J. Biol. 1994; Full Text PDF PubMed Google Scholar) are at the of the FGFs and to In the fusion at the terminus of FGF-2 to be required for the toxin in the after to or with FGF family are most for the may be may have a of useful for studies of FGF function and cells expressing cytometry demonstrated binding with expressing as as the is and at the of expressing FGFR1 are the suggest that cells expressing receptors should be by flow cytometry with in which of were with human peripheral blood cells that as as could be of as of the cell was not This could thus be useful for to cells expressing or of the of T cells in human peripheral blood to T cells by functional (17Zhao X.M. Byrd V.M. McKeehan W.L. Reich M.B. Miller G.G. Thomas J.W. J. Immunol. 1995; 155: 3904-3911PubMed Google Scholar), is that flow cytometry with be useful in these cells in peripheral blood are in to the fusion protein can be used for to identify cells in tissue that may be useful in studies of and A second of is in the intracellular fate of FGF and its studies show that the of cells are to the fusion protein at after with at suggesting that of the receptors can be directly by not intact fusion protein bound by receptors after this may allow of the of FGF and its and of the factors with which by immunoprecipitation the Fc The Dr. Maciag for the Dr. Melanie Spriggs and Immunex Corp. for fusion Dr. for McKeehan and for FGFR1 and and for on protein with
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