Interleukin-4 Signaling in B Lymphocytes from Patients with X-linked Severe Combined Immunodeficiency
1997; Elsevier BV; Volume: 272; Issue: 11 Linguagem: Inglês
10.1074/jbc.272.11.7314
ISSN1083-351X
AutoresNaomi Taylor, Fabio Candotti, Susan M. Smith, Scott A. Oakes, Thomas Jähn, Judith Isakov, Jennifer M. Puck, John J. O'Shea, Kenneth I. Weinberg, James A. Johnston,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoInterleukin-4 (IL-4) is an important cytokine for B and T lymphocyte function and mediates its effects via a receptor that contains γc. B cells derived from patients with X-linked severe combined immunodeficiency (X-SCID) are deficient in γc and provide a useful model in which to dissect the role of this subunit in IL-4-mediated signaling. We found that although IL-4 stimulation of X-SCID B cells did not result in Janus tyrosine kinase-3 (JAK3) phosphorylation, other IL-4 substrates including JAK1 and IRS-1 were phosphorylated. Additionally, we detected signal transducers and activators of transcription 6 (STAT6) tyrosine phosphorylation and DNA binding activity in X-SCID B cells with a wide range of γc mutations. However, reconstitution of these X-SCID B cells with γc enhanced IL-4-mediated responses including STAT6 phosphorylation and DNA binding activity and resulted in increased CD23 expression. Thus, γc is not necessary to trigger IL-4-mediated responses in B cells, but its presence is important for optimal IL-4-signaling. These results suggest that two distinct IL-4 signaling pathways exist. Interleukin-4 (IL-4) is an important cytokine for B and T lymphocyte function and mediates its effects via a receptor that contains γc. B cells derived from patients with X-linked severe combined immunodeficiency (X-SCID) are deficient in γc and provide a useful model in which to dissect the role of this subunit in IL-4-mediated signaling. We found that although IL-4 stimulation of X-SCID B cells did not result in Janus tyrosine kinase-3 (JAK3) phosphorylation, other IL-4 substrates including JAK1 and IRS-1 were phosphorylated. Additionally, we detected signal transducers and activators of transcription 6 (STAT6) tyrosine phosphorylation and DNA binding activity in X-SCID B cells with a wide range of γc mutations. However, reconstitution of these X-SCID B cells with γc enhanced IL-4-mediated responses including STAT6 phosphorylation and DNA binding activity and resulted in increased CD23 expression. Thus, γc is not necessary to trigger IL-4-mediated responses in B cells, but its presence is important for optimal IL-4-signaling. These results suggest that two distinct IL-4 signaling pathways exist. INTRODUCTIONPatients with X-linked severe combined immunodeficiency (X-SCID) 1The abbreviations used are: X-SCIDX-linked severe combined immunodeficiencyILinterleukinJAKJanus tyrosine kinaseSTATsignal transducers and activators of transcriptionB-LCLB cell lymphoblastoid cell linesGASinterferon-γ activation siteRreceptorIRSinsulin receptor substrate. present with very few T cells and absent mitogenic responses (1WHO Scientific Group on Immunodeficiency. Immunodefic. Rev. 1992; 3: 195-236Google Scholar). Although B cells are present, immunoglobulin levels are low, and specific antibody production is lacking. The combination of these defects is fatal by 1-2 years of age unless the immune system is reconstituted by allogeneic bone marrow transplantation. These clinical manifestations are due to a wide range of mutations in the common gamma chain (γc) gene that result in either a lack of γc message, unstable γc proteins that are poorly expressed, or defective γc receptor subunits that are expressed but nonfunctional (2Noguchi M. Yi H. Rosenblatt H.M. Filipovich A.H. Adelstein S. Modi W.S. McBride O.W. Leonard W.J. Cell. 1993; 73: 147-157Abstract Full Text PDF PubMed Scopus (1154) Google Scholar, 3Puck J.M. Deschenes S.M. Porter J.C. Dutra A.S. Brown C.J. Willard H.F. Henthorn P.S. Hum. Mol. Genet. 1993; 2: 1099-1104Crossref PubMed Scopus (253) Google Scholar, 4DiSanto J.P. Dautry-Varsat A. Certain S. Fischer A. de Saint Basile G. Eur. J. Immunol. 1994; 24: 475-479Crossref PubMed Scopus (63) Google Scholar). γc was originally identified as a component of the IL-2 cytokine receptor (IL-2Rγ), but as it has been shown to be shared by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, it is now designated γc (5Noguchi M. Nakamura Y. Russell S.M. Ziegler S.F. Tsang M. Cao X. Leonard W.J. Science. 1993; 262: 1877-1880Crossref PubMed Scopus (781) Google Scholar, 6Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (740) Google Scholar, 7Kondo M. Takeshita T. Higuchi M. Nakamura M. Sudo T. Nishikawa S. Sugamura K. Science. 1994; 263: 1453-1454Crossref PubMed Scopus (346) Google Scholar, 8Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (964) Google Scholar, 9Kimura Y. Takeshita T. Kondo M. Ishii N. Nakamura M. Van Snick J. Sugamura K. Int. Immunol. 1995; 7: 115-120Crossref PubMed Scopus (193) Google Scholar).IL-4 is thought to be important for mature B cell functions including immunoglobulin class switching to IgG4 and IgE as well as expression of CD23 and major histocompatibility complex class II genes (10Paul W.E. Blood. 1991; 77: 1859-1870Crossref PubMed Google Scholar). Because IL-4 regulates B lymphocyte function, it is important to determine the response of X-SCID B cells to this cytokine. The functional IL-4 receptor (IL-4R) consists of at least two components, IL-4Rα and γc subunits (6Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (740) Google Scholar, 11Kondo M. Takeshita T. Ishii N. Nakamura M. Watanabe S. Arai K. Sugamura K. Science. 1993; 262: 1874-1877Crossref PubMed Scopus (733) Google Scholar). Signal transduction through the IL-4R, as well as through other hematopoietic receptors, is initiated by activation of Janus family tyrosine kinases (JAKs) (12Ihle J. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1139) Google Scholar, 13Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). IL-4 elicits tyrosine phosphorylation of the JAK family members JAK1 and JAK3, which interact with the IL-4Rα and γc subunits, respectively (14Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z- J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (498) Google Scholar, 15Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (503) Google Scholar, 16Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalsteig F.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (583) Google Scholar, 17Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (532) Google Scholar). The current model of cytokine signaling proposes that upon cytokine binding, members of the JAK family are rapidly activated and subsequently tyrosine-phosphorylate the receptor, forming a docking site for signal transducers and activators of transcription (STATs) that are also phosphorylated by JAKs (12Ihle J. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1139) Google Scholar, 13Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). The STAT proteins then dimerize and translocate to the nucleus where they bind DNA sequences on target genes. One important STAT that is activated in response to IL-4, STAT6 (IL-4 STAT), has been shown to bind to promoter sequences of IL-4-inducible genes (18Kotanides H. Reich N.C. Science. 1993; 262: 1265-1267Crossref PubMed Scopus (230) Google Scholar, 19Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar, 20Schindler C. Kashleva H. Pernis A. Pine R. Rothman P. EMBO J. 1994; 13: 1350-1356Crossref PubMed Scopus (169) Google Scholar, 21Fenghao X. Saxon A. Nguyen A. Ke Z. Diaz-Sanchez D. Nel A. J. Clin. Invest. 1995; 96: 907-914Crossref PubMed Scopus (57) Google Scholar). As STAT6 knockout animals parallel the IL-4 null phenotype and exhibit defects in Th2 helper T cell differentiation and immunoglobulin class switching (22Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, 23Shimoda K. Vandeursen J. Sangster M.Y. Srawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1104) Google Scholar, 24Takata K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S. Nakanishi K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1262) Google Scholar), STAT6 appears to be essential for many IL-4-mediated effects.It might be predicted that in the absence of a functional γc chain, X-SCID B cells would not be able to respond to IL-4. Indeed, two groups have reported that neither JAK1 phosphorylation nor STAT6 DNA binding activity is induced upon IL-4 stimulation of Epstein-Barr virus-transformed B cells (B-LCL) derived from X-SCID patients (25Izuhara K. Heike T. Otsuka T. Yamaoka K. Mayumi M. Imamura T. Niho Y. Harada N. J. Biol. Chem. 1996; 271: 619-622Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Russell S.M. Tayebi N. Nakajima H. Riedy M.C. Roberts J.L. Aman M.J. Migone T.S. Noguchi M. Markert M.L. Buckley R.H. O'Shea J.J. Leonard W.J. Science. 1995; 270: 797-800Crossref PubMed Scopus (675) Google Scholar). However, Matthews and colleagues (27Matthews D.J. Clark P.A. Herbert J. Morgan G. Armitage R.J. Kinnon C. Minty A. Grabstein K.H. Caput D. Ferrara P. Callard R.E. Blood. 1995; 85: 38-42Crossref PubMed Google Scholar) have recently demonstrated that although X-SCID B cells cannot undergo immunoglobulin class switching, they can proliferate in vitro in response to IL-4 when co-stimulated with CD40 ligand or anti-IgM. Although the underlying biochemical mechanisms are not clear, these data suggest that IL-4-mediated signaling in X-SCID B cells can occur.It was therefore important to analyze IL-4-signaling in a panel of B-LCL derived from X-SCID patients with a wide range of γc mutations in order to clarify these discrepancies. IL-4 failed to stimulate JAK3 tyrosine phosphorylation in X-SCID B cells, but we found that JAK1 and IRS-1 were phosphorylated. IL-4 also induced STAT6 phosphorylation as well as DNA binding activity in these cells. However, STAT6 activation and CD23 expression were significantly enhanced in X-SCID B-LCL reconstituted with wild type γc. Thus, we have demonstrated a γc-JAK3-independent pathway through which IL-4 activates JAK1 and STAT6 in B cells derived from X-SCID patients. These results have important implications for the understanding of IL-4 signal transduction and the lack of mature B cell function in patients with X-SCID.DISCUSSIONStimulation of B cells with IL-4 results in proliferation, immunoglobulin class switching, and regulation of cell surface proteins such as major histocompatibility complex class II molecules and the CD23 receptor. Previous reports have concluded that the JAK-STAT pathway is not activated by IL-4 in the absence of γc (25Izuhara K. Heike T. Otsuka T. Yamaoka K. Mayumi M. Imamura T. Niho Y. Harada N. J. Biol. Chem. 1996; 271: 619-622Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Russell S.M. Tayebi N. Nakajima H. Riedy M.C. Roberts J.L. Aman M.J. Migone T.S. Noguchi M. Markert M.L. Buckley R.H. O'Shea J.J. Leonard W.J. Science. 1995; 270: 797-800Crossref PubMed Scopus (675) Google Scholar). However, IL-4 can induce functional, although suboptimal, responses in X-SCID B cells (27Matthews D.J. Clark P.A. Herbert J. Morgan G. Armitage R.J. Kinnon C. Minty A. Grabstein K.H. Caput D. Ferrara P. Callard R.E. Blood. 1995; 85: 38-42Crossref PubMed Google Scholar) suggesting that the IL-4 signal transduction pathway is conserved in these cells. X-SCID B cells, therefore, provide an important model in which to examine the generation of γc-independent IL-4 responses. In this study, we determined that JAK1, IRS-1, and STAT6 were tyrosine-phosphorylated in response to IL-4 in B cell lines derived from X-SCID patients with a wide diversity of γc mutations and that IL-4 induced detectable levels of STAT6 DNA binding activity in these cells.The finding that JAK3 was not phosphorylated in X-SCID B-LCL stimulated with either IL-2 or IL-4 was not surprising as a number of recent studies have demonstrated a physical association between γc and JAK3 (14Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z- J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (498) Google Scholar, 16Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalsteig F.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (583) Google Scholar). This interaction is thought to be critical for IL-2, IL-4, IL-7, IL-9, and IL-15 signaling in lymphocytes (14Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z- J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (498) Google Scholar, 16Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalsteig F.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (583) Google Scholar, 43Johnston J.A. Bacon C.M. Reidy M.C. O Shea J.J. J. Leukocyte Biol. 1996; 60: 411-452Crossref Scopus (96) Google Scholar). The data described here show that IL-4-induced JAK1 and IRS-1 phosphorylation as well as STAT6 activation can occur via mechanisms that are independent of γc and JAK3. IRS-1 is thought to be important for mediating IL-4 proliferative effects, but the mechanism by which IL-4 stimulates IRS-1 phosphorylation is unclear (44Wang L.M. Keegan A.D. Frankel M. Paul W.E. Pierce J.H. Stem Cells. 1995; 13: 360-368Crossref PubMed Scopus (21) Google Scholar). Janus kinases likely play an important role (45Johnston J.A. Wang L.-M. Hanson E.P. Sun X.-J. White M.F. Oakes S.A. Pierce J.H. O'Shea J.J. J. Biol. Chem. 1995; 270: 28527-28530Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and our data suggest that in the absence of JAK3, JAK1 may mediate IRS-1 tyrosine phosphorylation in response to IL-4. It remains to be determined whether other tyrosine kinases known to be induced by IL-4, including JAK2, Tyk2, and Fes (46Murata T. Noguchi P.D. Puri R.K. J. Biol. Chem. 1995; 270: 30829-30836Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 47Palmer-Crocker R.L. Hughes C.C.W. Pober J.S. J. Clin. Invest. 1996; 98: 604-609Crossref PubMed Scopus (95) Google Scholar, 48Izuhara K. Feldman R.A. Greer P. Harada N. J. Biol. Chem. 1994; 269: 18623-18629Abstract Full Text PDF PubMed Google Scholar), play a role in IL-4-mediated responses in X-SCID B cells.Matthews et al. (27Matthews D.J. Clark P.A. Herbert J. Morgan G. Armitage R.J. Kinnon C. Minty A. Grabstein K.H. Caput D. Ferrara P. Callard R.E. Blood. 1995; 85: 38-42Crossref PubMed Google Scholar) showed that functional responses such as proliferation, IgE secretion, and CD23 expression could occur in X-SCID B cells when co-stimulated in vitro by IL-4 or IL-13 together with CD40L or IgM. However, in our studies, we did not detect an increase in CD23 expression in X-SCID B-LCL even though CD23 expression did increase in normal and γc/reconstituted X-SCID B-LCL following stimulation with IL-4. Because of the already high level of CD23 expression on Epstein-Barr virus-transformed B cells, slight increases in CD23 expression may be difficult to detect. Nevertheless, even in the studies performed by Matthews et al. (27Matthews D.J. Clark P.A. Herbert J. Morgan G. Armitage R.J. Kinnon C. Minty A. Grabstein K.H. Caput D. Ferrara P. Callard R.E. Blood. 1995; 85: 38-42Crossref PubMed Google Scholar) in primary B cells, the increase in CD23 in X-SCID B cells was significantly lower than that detected in control B cells (27Matthews D.J. Clark P.A. Herbert J. Morgan G. Armitage R.J. Kinnon C. Minty A. Grabstein K.H. Caput D. Ferrara P. Callard R.E. Blood. 1995; 85: 38-42Crossref PubMed Google Scholar). Suboptimal CD23 activation in response to IL-4 suggests that the observed humoral deficiency in X-SCID patients may result, at least in part, from the inability to transduce the full set of IL-4-induced signals through non-γc containing receptors. Our finding that IL-4-induced STAT6 phosphorylation and CD23 expression were restored to wild type levels following introduction of γc into these cells indicates that γc potentiates the IL-4 signal transduction pathway. The importance of JAK3 in this γc-dependent pathway is further supported by the recent observation that IL-4-induced CD23 expression and STAT6 activation are also suboptimal in B cells that express γc but are deficient in JAK3 (49Oakes S.A. Candotti F. Johnston J.A. Chen Y.-Q. Ryan J.J. Taylor N. Lio X. Henninghausen L. Notarangelo L.D. Paul W.E. Blaese R.M. O'Shea J.J. Immunity. 1996; 5: 605-616Abstract Full Text PDF PubMed Scopus (111) Google Scholar).Although the precise mechanism by which this γc-independent signaling pathway is initiated is not clear, several groups have previously shown IL-4-induced proliferation and protein phosphorylation in endothelial, colon carcinoma, and plasmocytoma cell lines that lack γc (27Matthews D.J. Clark P.A. Herbert J. Morgan G. 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INTRODUCTIONPatients with X-linked severe combined immunodeficiency (X-SCID) 1The abbreviations used are: X-SCIDX-linked severe combined immunodeficiencyILinterleukinJAKJanus tyrosine kinaseSTATsignal transducers and activators of transcriptionB-LCLB cell lymphoblastoid cell linesGASinterferon-γ activation siteRreceptorIRSinsulin receptor substrate. present with very few T cells and absent mitogenic responses (1WHO Scientific Group on Immunodeficiency. Immunodefic. Rev. 1992; 3: 195-236Google Scholar). Although B cells are present, immunoglobulin levels are low, and specific antibody production is lacking. The combination of these defects is fatal by 1-2 years of age unless the immune system is reconstituted by allogeneic bone marrow transplantation. These clinical manifestations are due to a wide range of mutations in the common gamma chain (γc) gene that result in either a lack of γc message, unstable γc proteins that are poorly expressed, or defective γc receptor subunits that are expressed but nonfunctional (2Noguchi M. Yi H. Rosenblatt H.M. Filipovich A.H. Adelstein S. Modi W.S. McBride O.W. Leonard W.J. Cell. 1993; 73: 147-157Abstract Full Text PDF PubMed Scopus (1154) Google Scholar, 3Puck J.M. Deschenes S.M. Porter J.C. Dutra A.S. Brown C.J. Willard H.F. Henthorn P.S. Hum. Mol. Genet. 1993; 2: 1099-1104Crossref PubMed Scopus (253) Google Scholar, 4DiSanto J.P. Dautry-Varsat A. Certain S. Fischer A. de Saint Basile G. Eur. J. Immunol. 1994; 24: 475-479Crossref PubMed Scopus (63) Google Scholar). γc was originally identified as a component of the IL-2 cytokine receptor (IL-2Rγ), but as it has been shown to be shared by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, it is now designated γc (5Noguchi M. Nakamura Y. Russell S.M. Ziegler S.F. Tsang M. Cao X. Leonard W.J. Science. 1993; 262: 1877-1880Crossref PubMed Scopus (781) Google Scholar, 6Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (740) Google Scholar, 7Kondo M. Takeshita T. Higuchi M. Nakamura M. Sudo T. Nishikawa S. Sugamura K. Science. 1994; 263: 1453-1454Crossref PubMed Scopus (346) Google Scholar, 8Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (964) Google Scholar, 9Kimura Y. Takeshita T. Kondo M. Ishii N. Nakamura M. Van Snick J. Sugamura K. Int. Immunol. 1995; 7: 115-120Crossref PubMed Scopus (193) Google Scholar).IL-4 is thought to be important for mature B cell functions including immunoglobulin class switching to IgG4 and IgE as well as expression of CD23 and major histocompatibility complex class II genes (10Paul W.E. Blood. 1991; 77: 1859-1870Crossref PubMed Google Scholar). Because IL-4 regulates B lymphocyte function, it is important to determine the response of X-SCID B cells to this cytokine. The functional IL-4 receptor (IL-4R) consists of at least two components, IL-4Rα and γc subunits (6Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (740) Google Scholar, 11Kondo M. Takeshita T. Ishii N. Nakamura M. Watanabe S. Arai K. Sugamura K. Science. 1993; 262: 1874-1877Crossref PubMed Scopus (733) Google Scholar). Signal transduction through the IL-4R, as well as through other hematopoietic receptors, is initiated by activation of Janus family tyrosine kinases (JAKs) (12Ihle J. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1139) Google Scholar, 13Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). IL-4 elicits tyrosine phosphorylation of the JAK family members JAK1 and JAK3, which interact with the IL-4Rα and γc subunits, respectively (14Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z- J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (498) Google Scholar, 15Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (503) Google Scholar, 16Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. 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One important STAT that is activated in response to IL-4, STAT6 (IL-4 STAT), has been shown to bind to promoter sequences of IL-4-inducible genes (18Kotanides H. Reich N.C. Science. 1993; 262: 1265-1267Crossref PubMed Scopus (230) Google Scholar, 19Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar, 20Schindler C. Kashleva H. Pernis A. Pine R. Rothman P. EMBO J. 1994; 13: 1350-1356Crossref PubMed Scopus (169) Google Scholar, 21Fenghao X. Saxon A. Nguyen A. Ke Z. Diaz-Sanchez D. Nel A. J. Clin. Invest. 1995; 96: 907-914Crossref PubMed Scopus (57) Google Scholar). As STAT6 knockout animals parallel the IL-4 null phenotype and exhibit defects in Th2 helper T cell differentiation and immunoglobulin class switching (22Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, 23Shimoda K. Vandeursen J. Sangster M.Y. Srawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1104) Google Scholar, 24Takata K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S. Nakanishi K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1262) Google Scholar), STAT6 appears to be essential for many IL-4-mediated effects.It might be predicted that in the absence of a functional γc chain, X-SCID B cells would not be able to respond to IL-4. Indeed, two groups have reported that neither JAK1 phosphorylation nor STAT6 DNA binding activity is induced upon IL-4 stimulation of Epstein-Barr virus-transformed B cells (B-LCL) derived from X-SCID patients (25Izuhara K. Heike T. Otsuka T. Yamaoka K. Mayumi M. Imamura T. Niho Y. Harada N. J. Biol. Chem. 1996; 271: 619-622Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Russell S.M. Tayebi N. Nakajima H. Riedy M.C. Roberts J.L. Aman M.J. Migone T.S. Noguchi M. Markert M.L. Buckley R.H. O'Shea J.J. Leonard W.J. Science. 1995; 270: 797-800Crossref PubMed Scopus (675) Google Scholar). However, Matthews and colleagues (27Matthews D.J. Clark P.A. Herbert J. Morgan G. Armitage R.J. Kinnon C. Minty A. Grabstein K.H. Caput D. Ferrara P. Callard R.E. Blood. 1995; 85: 38-42Crossref PubMed Google Scholar) have recently demonstrated that although X-SCID B cells cannot undergo immunoglobulin class switching, they can proliferate in vitro in response to IL-4 when co-stimulated with CD40 ligand or anti-IgM. Although the underlying biochemical mechanisms are not clear, these data suggest that IL-4-mediated signaling in X-SCID B cells can occur.It was therefore important to analyze IL-4-signaling in a panel of B-LCL derived from X-SCID patients with a wide range of γc mutations in order to clarify these discrepancies. IL-4 failed to stimulate JAK3 tyrosine phosphorylation in X-SCID B cells, but we found that JAK1 and IRS-1 were phosphorylated. IL-4 also induced STAT6 phosphorylation as well as DNA binding activity in these cells. However, STAT6 activation and CD23 expression were significantly enhanced in X-SCID B-LCL reconstituted with wild type γc. Thus, we have demonstrated a γc-JAK3-independent pathway through which IL-4 activates JAK1 and STAT6 in B cells derived from X-SCID patients. These results have important implications for the understanding of IL-4 signal transduction and the lack of mature B cell function in patients with X-SCID.
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