Btk Plays a Crucial Role in the Amplification of FcϵRI-mediated Mast Cell Activation by Kit
2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês
10.1074/jbc.m506063200
ISSN1083-351X
AutoresShoko Iwaki, Christine Tkaczyk, Anne B. Satterthwaite, Kristina E. Halcomb, Michael A. Beaven, Dean D. Metcalfe, Alasdair M. Gilfillan,
Tópico(s)Immune Cell Function and Interaction
ResumoStem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk-/- BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk-/- BMMCs. The impairment of responses in Btk-/- cells correlated with an inability of SCF to augment phospholipase Cγ1 activation and calcium mobilization, and to phosphorylate NFκB and NFAT for cytokine gene transcription in these cells. Similar studies with Lyn-/- and Btk-/-/Lyn-/- BMMCs indicated that Lyn was a regulator of Btk for these responses. These data demonstrate, for the first time, that Btk is a key regulator of a Kit-mediated amplification pathway that augments FcϵRI-mediated mast cell activation. Stem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk-/- BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk-/- BMMCs. The impairment of responses in Btk-/- cells correlated with an inability of SCF to augment phospholipase Cγ1 activation and calcium mobilization, and to phosphorylate NFκB and NFAT for cytokine gene transcription in these cells. Similar studies with Lyn-/- and Btk-/-/Lyn-/- BMMCs indicated that Lyn was a regulator of Btk for these responses. These data demonstrate, for the first time, that Btk is a key regulator of a Kit-mediated amplification pathway that augments FcϵRI-mediated mast cell activation. Mast cell activation leads to the release of both preformed and de novo synthesized inflammatory mediators. The intracellular signaling cascade regulating these responses is initiated by aggregation of high affinity receptors for IgE (FcϵRI) 4The abbreviations used are: FcϵRIhigh affinity receptor for IgEAgantigenBtkBruton's tyrosine kinaseBtk-/-/Lyn-/-Btk, Lyn double knock-outBMMCbone marrow-derived mast cellILinterleukinpAbpolyclonal antibodyPI 3-kinasephosphoinositide 3-kinasePLCphospholipase CSCFstem cell factorTNFtumor necrosis factorWTwild typeDNPdinitrophenylHSAhuman serum albuminELISAenzyme-linked immunosorbent assayMES4-morpholineethanesulfonic acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolJNKc-Jun NH2-terminal kinaseRPARNase protection assaysMAPKmitogen-activated protein kinase. following antigen binding to receptor-bound IgE (1Turner H. Kinet J.P. Nature. 1999; 402: B24-B30Crossref PubMed Scopus (626) Google Scholar). However, antigen-induced triggering of mast cells in vivo is likely to occur with a background of stem cell factor (SCF)-mediated Kit activation, as SCF is essential for the growth, differentiation, homing, and survival of mast cells (2Galli S.J. Tsai M. Wershill B.K. Am. J. Pathol. 1993; 142: 965-974PubMed Google Scholar). By mimicking this situation in vitro, we have demonstrated that SCF dramatically augments both antigen-mediated degranulation and cytokine generation in these cells (3Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (136) Google Scholar, 4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar). Kitmediated signals are thus required for optimal mast cell degranulation and cytokine production induced by FcϵRI aggregation. high affinity receptor for IgE antigen Bruton's tyrosine kinase Btk, Lyn double knock-out bone marrow-derived mast cell interleukin polyclonal antibody phosphoinositide 3-kinase phospholipase C stem cell factor tumor necrosis factor wild type dinitrophenyl human serum albumin enzyme-linked immunosorbent assay 4-morpholineethanesulfonic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol c-Jun NH2-terminal kinase RNase protection assays mitogen-activated protein kinase. Antigen-mediated degranulation and cytokine production are thought to be initiated by the activation of the Src family tyrosine kinase, Lyn (5Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar). The resulting tyrosine phosphorylation of the β and γ chains of FcϵRI promotes the binding of the tyrosine kinase Syk to FcϵRI (6Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar). This permits the trans/auto-phosphorylation and activation of Syk (7Kimura T. Zhang J. Sagawa K. Sakaguchi K. Appella E. Siraganian R.P. J. Immunol. 1997; 159: 4426-4434PubMed Google Scholar, 8El-Hilal O. Kurosaki T. Yamamura H. Kinet J.P. Scharenberg A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1919-1924Crossref PubMed Scopus (109) Google Scholar), which in turn phosphorylates the transmembrane adaptor molecules LAT (9Saitoh S. Arudchandran R. Manetz T.S. Zhang W. Sommers C.L. Love P.E. Rivera J. Samelson L.E. Immunity. 2000; 12: 525-535Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and NTAL (3Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (136) Google Scholar, 10Brdicka T. Imrich M. Angelisova P. Brdickova N. Horvath O. Spicka J. Hilgert I. Luskova P. Draber P. Novak P. Engels N. Wienands J. Simeoni L. Osterreicher J. Aguado E. Malissen M. Schraven B. Horejsi V. J. Exp. Med. 2002; 196: 1617-1626Crossref PubMed Scopus (180) Google Scholar). These adaptor molecules orchestrate the recruitment of downstream signaling molecules to the receptor-signaling molecular complex by providing docking sites for cytosolic adaptor molecules, including SLP-76, Vav, Gads, Grb2, Gab1, and Gab2 (11Rivera J. Curr. Opin. Immunol. 2002; 14: 688-693Crossref PubMed Scopus (158) Google Scholar) and signaling enzymes such as phospholipase (PL)Cγ1, PLCγ2, and phosphoinositide (PI) 3-kinase (12Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 6: 1145-1158Crossref Scopus (124) Google Scholar, 13Tkaczyk C. Beaven M.A. Brachman S.M. Metcalfe D.D. Gilfillan A.M. J. Biol. Chem. 2003; 278: 48474-48484Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The subsequent elevation of intracellular calcium levels and activation of protein kinase C (PKC) leads to degranulation (14Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 266: 1749-1756Abstract Full Text PDF Google Scholar), whereas activation of the Ras-Raf-MAPK pathway induces arachidonic acid metabolite release (15Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar) and downstream phosphorylation and activation of specific cytokine gene-related transcription factors (16Zhang C. Baumgartner R.A. Yamada K. Beaven M.A. J. Biol. Chem. 1997; 272: 13397-13402Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). A parallel pathway controlled by the Src kinase, Fyn, also appears to help regulate FcϵRI-dependent mast cell activation (17Parravicini V. Gadina M. Kovarova M. Odom S. Gonzales-Espinosa C. Furumoto Y. Saitoh S. Samelson L.E. O'Shea J. Rivera J. Nature Immun. 2002; 3: 741-748Crossref Scopus (402) Google Scholar). Many of these same signaling events are initiated upon binding of SCF to Kit (18Linnekin D. J. Biochem. Cell Biol. 1999; 31: 1053-1074Crossref PubMed Scopus (313) Google Scholar) but are insufficient on their own to induce degranulation (4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar). Our previous studies have suggested that this may be related to the inability of SCF to induce phosphorylation of LAT (3Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (136) Google Scholar) and downstream activation of PKC (4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar). Nevertheless, SCF can potentiate FcϵRI-mediated degranulation and phosphorylation of NTAL as well as enhance calcium mobilization (3Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (136) Google Scholar). How SCF augments these responses, however, was unclear. Given that the tyrosine kinase, Btk, is thought to play a role in the regulation of PLCγ-mediated calcium mobilization for both the B cell receptor (19Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar) and the FcϵRI (20Kawakami Y. Kitaura J. Satterthwaite A.B. Kato R.M. Asai K. Hartman S.E. Maeda-Yamamoto M. Lowell C.A. Rawlings D.J. Witte O.N. Kawakami T. J. Immunol. 2000; 165: 1210-1219Crossref PubMed Scopus (152) Google Scholar), we have examined whether Btk played a similar role in Kit-mediated responses. By use of bone marrow-derived mast cells (BMMCs) from gene-deficient mice, Btk was not only found to be essential for the ability of SCF to potentiate antigen-mediated degranulation but was also found to be required for the ability of Kit to regulate cytokine production in antigen-stimulated cells. Mast Cells—The Btk-/-, Lyn-/-, Btk-/-/Lyn-/- knock-out, and wild type (WT) mice used in this study have been described previously (20Kawakami Y. Kitaura J. Satterthwaite A.B. Kato R.M. Asai K. Hartman S.E. Maeda-Yamamoto M. Lowell C.A. Rawlings D.J. Witte O.N. Kawakami T. J. Immunol. 2000; 165: 1210-1219Crossref PubMed Scopus (152) Google Scholar). The mice were cross-bred on a C57BL/6 × 129/Sv genetic background. The wild type mice were derived from the same parental lines as the knock-out mice. Breeding pairs heterozygous for Lyn, Btk (males were either Btk-/Y or +/Y), or both were set up to generate both wild type and knock-out mice within the same litter. Whenever possible, littermates were compared directly. All animals were housed within the same room. The genotype of these mice was confirmed by reverse transcription-PCR of tail biopsies and by immunoblot analysis of proteins extracted from the BMMCs derived from these mice. Bone marrow obtained by femur lavage was cultured in RPMI 1640 medium containing IL-3 as described (13Tkaczyk C. Beaven M.A. Brachman S.M. Metcalfe D.D. Gilfillan A.M. J. Biol. Chem. 2003; 278: 48474-48484Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The studies were then conducted on BMMCs after 4-6 weeks in culture. Cell Activation—For degranulation and signaling studies, cultured BMMCs were sensitized overnight with anti-mouse monoclonal dinitrophenyl (DNP) IgE (100 ng/ml) (Sigma) in IL-3-free RPMI medium and then rinsed with HEPES buffer (21Lin P. Fung S.J. Chen T. Repetto B. Huang K.S. Gilfillan A.M. Biochem. J. 1994; 299: 109-114Crossref PubMed Scopus (22) Google Scholar) containing 0.04% bovine serum albumin (Sigma). The cells were triggered in the same buffer with DNP-human serum albumin (HSA) (0-100 ng/ml) and/or murine SCF (0-100 ng/ml; PeproTech, Rocky Hill, NJ) for 30 min for the degranulation studies or for the indicated periods for the signaling studies. For cytokine mRNA and release studies, cells were similarly sensitized but triggered for 4 or 10 h, respectively, in RPMI. Degranulation Assay—Degranulation was monitored by the release of β-hexosaminidase into the supernatants (22Chaves-Dias C. Hundley T.R. Gilfillan A.M. Kirshenbaum A.S. Cunha-Melo J.R. Metcalfe D.D. Beaven M.A. J. Immunol. 2001; 166: 6647-6656Crossref PubMed Scopus (29) Google Scholar). Briefly, BMMCs, sensitized as above, were triggered in 96-well plates (5 × 105 cells per well, 100 μl final volumes). The reactions were terminated by centrifugation (3000 rpm) at 4 °C, and the supernatants were aliquoted to 96-well plates for β-hexosaminidase assay. The remaining cells were lysed by adding distilled water and freeze-thawing, and then aliquots were similarly assayed for β-hexosaminidase content. Degranulation was then calculated as the percentage of total (cells and supernatants) β-hexosaminidase content found in the supernatants following challenge. Cytokine Production—RNase protection assays (RPA) were utilized to measure mRNA levels for multiple cytokines and chemokines following cell activation. Cells were sensitized and then triggered as above at a concentration of 10 × 106 cells/ml. Messenger RNA was extracted by lysing the cells with 1 ml of TRIzol (Invitrogen) for 5 min at room temperature. Chloroform (200 μl) was added to the lysates, and the mixtures were centrifuged for 15 min at 14,000 rpm. Isopropyl alcohol (500 μl) was then added to the aqueous phases, and the mixture was incubated for 10 min to precipitate RNA. Ten μg of RNA was used in the mRNA assay by using an in vitro transcription kit and pre-designed or custom-designed RPA templates (BD Biosciences). RPA was conducted according to the manufacturer's instructions; however, the synthesized radioactive probes labeled with [α-33P]UTP were purified with a probequant G-50 microcolumn (Amersham Biosciences) instead of ethanol precipitation, and the protected mRNA was precipitated with ethanol and ammonium acetate containing Glyco-blue (Ambion, Austin, TX). The gels were prepared with 80 ml of SequaGel-6 (National Diagnostics, Inc.), 20 ml of SequaGel-complete (National Diagnostics, Inc., Atlanta, GA), and 10% ammonium persulfate (Sigma). Levels of the secreted cytokines IL-4, IL-6, IL-13, and TNF-α were measured in the supernatants of activated BMMCs by ELISA (BIOSOURCE, Camarillo, CA). Cell Extraction, Immunoprecipitation, and Immunoblotting—Cell lysates and/or immunoprecipitates were prepared as described (23Tkaczyk C. Metcalfe D.D. Gilfillan A.M. J. Immunol. Methods. 2002; 268: 239-243Crossref PubMed Scopus (46) Google Scholar) and aliquots loaded onto 4-12% NuPAGE BisTris gels (Invitrogen). The proteins were then separated by electrophoresis in MES buffer (Invitrogen) as described in the manufacturer's protocol. Following transfer onto nitrocellulose membranes, the proteins were probed for immunoreactive proteins utilizing the following antibodies: goat anti-Btk pAb, goat anti-Tec pAb, anti-Itk pAb, anti-Lyn pAb, anti-Fyn pAb, anti-Blk pAb, anti-Yes pAb, antic-Fos pAb, anti-c-Jun pAb, anti-Syk pAb (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Hck pAb and anti-Src pAb (Upstate Biotechnology, Inc., Lake Placid, NY); anti-actin monoclonal antibody (clone AC-15) (Sigma); anti-phospho-Btk (Tyr(P)-223) pAb, anti-phospho-Src (Tyr(P)-416) pAb, anti-phospho-AKT (Ser(P)-473) pAb, anti-phospho-ERK (Thr(P)-202 and Tyr(P)-204), anti-phospho-JNK (Thr(P)-183 and Tyr(P)-185) pAb, anti-phospho-p38 (Thr(P)-180 and Tyr(P)-182) pAb, anti-phospho-c-Jun (Ser(P)-73) pAb, and anti-phospho NFκB (Ser(P)-536) pAb (Cell Signaling, Beverly, MA); anti-phospho-NFAT (Ser(P)-54) pAb and antiphospho-PLCγ1 (Tyr(P)-783) pAb (BIOSOURCE). Unless specified, pAbs were of rabbit origin. The immunoreactive proteins were visualized by probing with horseradish peroxidase-conjugated anti-mouse, anti-goat (The Jackson Laboratories, West Grove, PA), or anti-rabbit IgG (Amersham Biosciences) and then by ECL (PerkinElmer Life Sciences). As described previously (20Kawakami Y. Kitaura J. Satterthwaite A.B. Kato R.M. Asai K. Hartman S.E. Maeda-Yamamoto M. Lowell C.A. Rawlings D.J. Witte O.N. Kawakami T. J. Immunol. 2000; 165: 1210-1219Crossref PubMed Scopus (152) Google Scholar), and as discussed below, there were no apparent differences in the expression of signaling molecules (apart from Btk and Lyn) in the knock-out BMMCs. Therefore, protein loading of the samples was normalized by stripping and then probing for actin or alternatively by probing identically loaded membranes for either actin or Syk. To quantitate changes in protein phosphorylation, the ECL films were scanned by using an ImageQuant 5.0 scanner (Amersham Biosciences). Intracellular Calcium Determination—Calcium flux was measured in the BMMCs following loading of the cells with Fura-2 AM ester (Molecular Probes, Eugene, OR) as described (13Tkaczyk C. Beaven M.A. Brachman S.M. Metcalfe D.D. Gilfillan A.M. J. Biol. Chem. 2003; 278: 48474-48484Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Cells were loaded with Fura-2 AM for 30 min at 37 °C, rinsed, and resuspended in HEPES buffer containing 0.04% bovine serum albumin and sulfinapyrazone (0.3 mm) (Sigma), and then placed in a 96-well black culture plate (20,000 cells/well) (CulturPlat-96 F, PerkinElmer Life Sciences). Fluorescence was measured at two excitation wavelengths (340 and 380 nm) and an emission wavelength of 510 nm. The ratio of the fluorescence readings was calculated following subtraction of the fluorescence of the cells that had not been loaded with Fura 2-AM. To investigate the role of Btk in Kit-mediated responses, we utilized BMMCs derived from the bone marrow of Btk-/- mice. Because previous studies had suggested that Btk and Lyn had both redundant and opposing functions in antigen-dependent mast cell (20Kawakami Y. Kitaura J. Satterthwaite A.B. Kato R.M. Asai K. Hartman S.E. Maeda-Yamamoto M. Lowell C.A. Rawlings D.J. Witte O.N. Kawakami T. J. Immunol. 2000; 165: 1210-1219Crossref PubMed Scopus (152) Google Scholar) and cell activation (24Satterthwaite A.B. Lowell C.A. Khan W.N. Sideras P. Alt F.W. Witte O.N. J. Exp. Med. 1998; 188: 833-844Crossref PubMed Scopus (68) Google Scholar), we compared the responses in the Btk-/- BMMCs to those obtained in WT, Lyn-/-, and Btk-/-/Lyn-/- double knock-out BMMCs. The Btk-/-, Lyn-/-, and Btk-/-/Lyn-/- BMMC genotypes were confirmed by probing lysates from these cells for Btk and Lyn (data not shown). The levels of expression of the other Tec kinases, including Tec and Itk (as controls for Btk) and other Src kinases, including Blk, Fgr, Fyn, Hck, c-Src, and Yes (as controls for Lyn), were unaffected in these cells, apart from a slight reduction in the expression of Fgr in the Lyn-/- and Btk-/-/Lyn-/- BMMCs (data not shown). Both antigen and SCF induced the phosphorylation of Btk in WT mouse BMMCs (Fig. 1, a and b); however, maximum phosphorylation observed with SCF was of a lesser magnitude than that observed with antigen. Although there was little evidence of synergy in the responses at early time points (0-120 s), when cells were co-stimulated with SCF and antigen, Btk phosphorylation was more sustained than was observed with the individual stimulants. As expected, this phosphorylination was not detected in the Btk-/- BMMCs (Fig. 1, c and d). In addition, the phosphorylation of Btk was substantially reduced in the Lyn-/- BMMCs indicating that the phosphorylation of Btk was largely dependent on Lyn. Stimulation of BMMCs with antigen, but not SCF, resulted in an increase in the phosphorylation of the Src kinases (Fig. 1, e and f), although Src kinases were constitutively phosphorylated to some degree. In the Lyn-/- BMMCs there was virtually no phosphorylation of the Src kinases in both stimulated and non-stimulated BMMCs. Thus, the major Src kinase phosphorylated both constitutively and inducibly by antigen in the BMMCs was Lyn. However, overexposure of the gels revealed that SCF, but not antigen, also resulted in a lesser phosphorylation of another Src kinase that was not Lyn (Fig. 1e). There was little change in the phosphorylation of the Src kinases in the Btk-/- BMMCs, thus confirming that the phosphorylation of Btk is downstream of Lyn. To establish that SCF potentiated FcϵRI-dependent responses in WT mouse BMMCs as was the case in human mast cells (3Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (136) Google Scholar, 4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar), we examined degranulation and cytokine production in response to SCF, antigen, or both in combination. Fig. 2a shows that SCF, at concentrations up to 100 ng/ml, induced little degranulation. When added concurrently with antigen, however, SCF induced a marked concentration-dependent potentiation of antigen-mediated degranulation. Similarly, SCF and antigen acted in synergy to increase the message of multiple cytokines, including IL-1α, IL-1β, IL-4, IL-6, IL-13, TNF-α, and interferon-γ (Fig. 2b). To confirm that the potentiation of cytokine message levels translated into increases in cytokine protein, the release of TNF-α, IL-6, and IL-13 was examined by ELISA 10 h following challenge with SCF with or without antigen. Again, as in human mast cells (4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar), cytokine secretion was minimally elevated in response to either SCF or antigen alone, but when added in combination, there was a marked synergistic enhancement of cytokine production (Fig. 2, c-e). As reported (20Kawakami Y. Kitaura J. Satterthwaite A.B. Kato R.M. Asai K. Hartman S.E. Maeda-Yamamoto M. Lowell C.A. Rawlings D.J. Witte O.N. Kawakami T. J. Immunol. 2000; 165: 1210-1219Crossref PubMed Scopus (152) Google Scholar), antigen-mediated degranulation ∼50% in both the Btk-/- and Lyn-/- was reduced by BMMCs when compared with WT controls and was virtually abolished in the Btk-/-/Lyn-/- double knock-out BMMCs (Fig. 3a). SCF was unable to potentiate the residual antigen-mediated degranulation (i.e. 10-15%) in the Btk-/- and the Lyn-/- BMMCs and the minimal degranulation in the Btk-/-/Lyn-/- double knock-out BMMCs (Fig. 3b). This is in contrast to SCF-mediated potentiation of degranulation induced by minimally effective concentrations of antigen as shown previously in Fig. 2a. Production of TNF-α and IL-6 and IL-13 (Fig. 3, c-e, respectively) in response to SCF and antigen or both in combination was reduced by ∼50% in the Btk-/- BMMCs, potentiated in the Lyn-/- BMMCs, and virtually reduced to background levels in the Btk-/-/Lyn-/- double knock-out BMMCs, as compared with WT BMMCs. In contrast to degranulation, however, an additive response to the combination of SCF and antigen was still observed in Btk-/- BMMCs, although the net response was still ∼50% that in WT BMMCs. In the Btk-/-/Lyn-/- double knock-out BMMCs, however, production of cytokines was virtually ablated. Similar responses were observed at the message level as determined by RPA (data not shown). Activation of PLCγ1 and PI 3-Kinase—Our previous studies suggested that the ability of SCF to potentiate antigen-mediated degranulation was associated with an enhancement of calcium mobilization (4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar). As both PLCγ1- and PI 3-kinase-dependent pathways control FcϵRI-mediated degranulation in human mast cells via regulation of calcium mobilization (13Tkaczyk C. Beaven M.A. Brachman S.M. Metcalfe D.D. Gilfillan A.M. J. Biol. Chem. 2003; 278: 48474-48484Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), we next examined whether these signaling events were ablated in the Btk-/-, Lyn-/-, and Btk-/-/Lyn-/- BMMCs. PLCγ1 and PI 3-kinase activation was monitored by the phosphorylation of PLCγ1 or AKT, respectively (13Tkaczyk C. Beaven M.A. Brachman S.M. Metcalfe D.D. Gilfillan A.M. J. Biol. Chem. 2003; 278: 48474-48484Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Both SCF and antigen stimulated PLCγ1 phosphorylation in WT BMMCs, and the combination of both stimuli resulted in an additive and more sustained phosphorylation than that induced by either stimulant alone (Fig. 4, a, b, e, and f). We were unable to detect PLCγ2 phosphorylation in response to SCF and antigen by utilizing a commercially available anti-phospho-PLCγ2. However, following immunoprecipitation with an anti-PLCγ2 antibody and then probing with an anti-phosphotyrosine antibody, we observed that although both antigen and SCF induced PLCγ2 phosphorylation, these responses were not additive (data not shown). In contrast to PLCγ1 phosphorylation, the effects of SCF and antigen on AKT phosphorylation (Fig. 4, c, d, g, and h) were not additive. Rather, antigen induced a decrease in the more predominant SCF-mediated AKT phosphorylation (Fig. 4, c and d). This was likely because of Lyn-mediated down-regulation of PI 3-kinase activation, as the inhibitory response was reversed in the Lyn-/- and Btk-/-/Lyn-/- BMMCs but not the Btk-/- BMMCs (Fig. 4, g and h). The lack of synergistic enhancement of AKT phosphorylation in the Lyn-/- and Btk-/-/Lyn-/- BMMCs likely reflects the fact that both a Lyn-dependent inhibitory pathway, potentially via SHIP (25Hernandez-Hansen V. Smith A.J. Surviladze Z. Chigaev A. Mazel T. Kalensnikoff J. Lowell C.A. Krystal G. Sklar L.A. Wilson B.S. Oliver J.M. J. Immunol. 2004; 173: 100-112Crossref PubMed Scopus (104) Google Scholar), and a Lyn-dependent activation pathway, potentially via Syk (26Li H.-L. Davis W.W. Whiteman E.L. Birnbaum M.J. Pure E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6890-6895Crossref PubMed Scopus (57) Google Scholar), for antigen-induced PI 3-kinase activation are blocked in these cells. This conclusion is further supported by the fact that the slight increase in AKT phosphorylation observed in response to antigen in the WT cells is absent in the Lyn-/- and Btk-/-/Lyn-/- BMMCs (Fig. 4, g and h) Btk-deficient BMMCs exhibited a slight reduction in PLCγ1 phosphorylation in response to antigen and SCF (Fig. 4, e and f). However, the ability of SCF to potentiate antigen-mediated PLCγ1 phosphorylation was completely blocked in the Btk-/- BMMCs, and the combination of antigen and SCF appeared to result in phosphorylation levels that were slightly lower than that observed with antigen alone (Fig. 4, e and f). In Lyn-/-, as well as Btk-/-/Lyn-/- BMMCs, the ability of antigen in the absence or presence of SCF to induce PLCγ1 phosphorylation was completely blocked. As a result, the synergistic increase in PLCγ1 phosphorylation in response to SCF and antigen added concurrently was reduced to close to base line in the Btk-/-/Lyn-/- double knock-out cells. Thus, although Lyn was required for phosphorylation of PLCγ1 in response to antigen, Btk was central to the ability of SCF to potentiate this response. Calcium Mobilization—As with human mast cells (4Tkaczyk C. Horejsi V. Iwaki S. Draber P. Samelson L.E. Satterthwaite A.B. Nahm D.-H. Metcalfe D.D. Gilfillan A.M. Blood. 2004; 104: 207-214Crossref PubMed Scopus (103) Google Scholar), SCF and antigen acted in synergy to enhance calcium mobilization in WT BMMCs (Fig. 5a). In the Btk-/- BMMCs, the initial increases in calcium mobilization in response to antigen (Fig. 5b) or SCF (Fig. 5c) when added separately or concurrently (Fig. 5d) were still observed. These responses, however, were substantially lower and less sustained than those observed in the WT BMMCs. In contrast, in the Lyn-/- BMMCs, the increase in calcium levels was delayed but eventually reached levels that were similar to those in WT BMMCs. As was the case with degranulation, the residual calcium flux in the antigen-challenged Btk-/- (Fig. 5e) or Lyn-/- BMMCs (Fig. 5f) could not be further potentiated by SCF. In the Btk-/-/Lyn-/- double knock-out BMMCs, the calcium response to both stimuli was virtually ablated (Fig. 5, b-d). Taken together, the above data support the concept that the ability of SCF to potentiate antigen-mediated calcium mobilization, hence degranulation, was entirely dependent on Btk, and this was at the level of PLCγ1 activation but downstream of PI 3-kinase activation. MAPK and Transcription Factor Phosphorylation—We next examined if the observed deficiencies in cytokine production in the Btk-/-, Lyn-/-, and Btk-/-/Lyn-/- BMMCs correlated to reduced activation of MAPKs and specific transcription factors. In WT BMMCs, the phosphorylation of the ERK1/2, JNK, and p38 MAPKs was augmented by co-stimulation with antigen and SCF compared with the effects of the individual stimulants added alone (Fig. 6). There was no reduction in the synergistic phosphorylation of ERK1/2 in the Btk-/- and Lyn-/- BMMCs (Fig. 6, a and b) and only a slight reduction in the Btk-/-/Lyn-/- BMMCs. In contrast, the synergy between antigen and SCF in the phosphorylation of p38 MAPK and JNK
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