Activation Tyrosine Kinases, Btk and Lyn, in Mast Cell Redundant And

Redundant and Opposing Functions of Two Tyrosine Kinases, Btk and Lyn, in Mast Cell Activation

This information is current as Yuko Kawakami, Jiro Kitaura, Anne B. Satterthwaite, of September 25, 2021. Roberta M. Kato, Koichi Asai, Stephen E. Hartman, Mari Maeda-Yamamoto, Clifford A. Lowell, David J. Rawlings, Owen N. Witte and Toshiaki Kawakami J Immunol 2000; 165:1210-1219; ;

doi: 10.4049/jimmunol.165.3.1210 Downloaded from http://www.jimmunol.org/content/165/3/1210

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Redundant and Opposing Functions of Two Tyrosine Kinases, Btk and Lyn, in Mast Cell Activation1

Yuko Kawakami,* Jiro Kitaura,* Anne B. Satterthwaite,† Roberta M. Kato,‡ Koichi Asai,* Stephen E. Hartman,* Mari Maeda-Yamamoto,§ Clifford A. Lowell,¶ David J. Rawlings,‡ Owen N. Witte,†ʈ and Toshiaki Kawakami2*

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (Fc⑀RI). In this study, we have made the following observations on growth properties and Fc⑀RI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, Fc⑀RI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-␥1 and C-␥2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, Fc⑀RI-induced elevation of intracellular Ca2؉ concentrations and Downloaded from activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-␣ and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase C␣ and protein kinase C␤II, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun http://www.jimmunol.org/ N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components. The Journal of Immunology, 2000, 165: 1210–1219.

ross-linking of IgE-bound high-affinity IgE receptors the ␤ subunit in resting cells through interaction of the N-terminal (Fc⑀RI)3 on mast cells by Ag leads to the activation of unique region of Lyn with the C-terminal cytoplasmic domain of C mast cells culminating in the release of a panel of proin- the ␤ subunit (3, 4). Lyn is activated by transphosphorylation upon flammatory mediators. Mast cell activation triggers reactions of Fc⑀RI cross-linking (5). Activated Lyn phosphorylates the tyrosine by guest on September 25, 2021 immediate hypersensitivity (1). Fc⑀RI is composed of an IgE-bind- residues in the immunoreceptor tyrosine-based activation motifs ing ␣ subunit, a four-transmembrane, signal-amplifying ␤ subunit, (ITAM) of the cytoplasmic regions of the ␤ and ␥ subunits (6). and a disulfide-bonded pair of ␥ subunits (reviewed in Ref. 2). The Phosphorylated ITAMs of the ␤ and ␥ subunits recruit Lyn and Src family protein tyrosine kinase (PTK) Lyn is associated with Syk, respectively, through Src homology (SH) 2-phosphotyrosine interactions (7–9). Newly recruited PTKs are activated by transphosphorylation of tyrosine residues in their activation loops *Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA and by conformational changes in the case of Syk (10, 11). Active 92121; †Department of Microbiology and Molecular Genetics, University of Califor- nia, Los Angeles, CA 90095; ‡Department of Pediatrics, University of California, Los Lyn and Syk phosphorylate themselves and other protein sub- Angeles, CA 90095; §National Research Institute of Vegetables, Ornamental Plants strates such as phospholipase C (PLC)-␥1 and -␥2 (12–14). Acti- and Tea, Kanaya, Shizuoka, Japan; ¶Department of Laboratory Medicine, University ʈ vation of PLC leads to the generation of two second messengers, of California, San Francisco, CA 94143; and Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095 inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 mobilizes 2ϩ Received for publication November 11, 1999. Accepted for publication May 9, 2000. Ca from intracellular storage sites, and diacylglycerol together 2ϩ The costs of publication of this article were defrayed in part by the payment of page with Ca activates protein kinase C (PKC) (reviewed in Ref. 15). ϩ charges. This article must therefore be hereby marked advertisement in accordance Both Ca2 and PKC appear to be required for optimal mast cell with 18 U.S.C. Section 1734 solely to indicate this fact. degranulation (16). 1 This work was supported in part by National Institutes of Health Grant AI42244, Bruton’s tyrosine kinase (Btk) is a member of the Tec family of AI33617, and AI38348 (to T.K.). A.B.S. is a Special Fellow of the Leukemia Society of America. D.J.R. is a recipient of a McDonnell Scholar Award and is supported in kinases characterized by N-terminal pleckstrin homology (PH) and part by the facilities of the Jonsson Comprehensive Cancer Center, University of Tec homology domains (17–19). btk mutations affect B cell de- California Los Angeles. O.N.W. is an Investigator of the Howard Hughes Medical velopment and result in X-linked agammaglobulinemia in humans Institute. This article is Publication No. 286 from the La Jolla Institute for Allergy and Immunology. (17, 18) and X-linked immunodeficiency (xid) in mice (20, 21). 2 Address correspondence and reprint requests to Dr. Toshiaki Kawakami, La Jolla Btk is also highly expressed in mast cells and is required for cy- Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA tokine production in response to Fc⑀RI cross-linking (22). Unlike 92121. E-mail address: [email protected] B cells, mast cell development does not require Btk. This suggests 3 ⑀ The abbreviations used are: Fc RI, high-affinity IgE receptor; Btk, Bruton’s tyrosine redundant roles for Btk and other Tec family kinases, such as kinase; DNP, dinitrophenyl; HSA, human serum albumin; IP3, inositol 1,4,5-trisphosphate; MAP, mitogen-activated protein; PKC, protein kinase C; PLC, phospholipase Itk/Emt, in some mast cell signaling pathways. Mechanistically, C; PTK, protein-tyrosine kinase; SH, Src homology; ITAM, immunoreceptor ty- btk mutations result in the loss of extracellular Ca2ϩ influx and the rosine-based activation motif; PH, pleckstrin homology; BCR, B cell receptor; PI3-K, 2ϩ phosphatidylinositol 3-kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular sig- sustained phase of Ca response in B cell receptor (BCR)-stim- nal-related kinase. ulated B cells (23, 24). This defective response may be due in part

to reduced PLC-␥ activation. Downstream of these early activation Biolabs (Boston, MA). Pansorbin was purchased from Calbiochem (La events in mast cells and B cells, Btk mediates the activation of Jolla, CA). Other chemicals of highest grade were obtained from Sigma JNK1, JNK2, and, to a lesser extent, p38 (25). JNK regulates c-Jun (St. Louis, MO), unless otherwise mentioned. and other transcription factors that induce the transcription of Cells TNF-␣, IL-2, and other cytokine genes (26), accounting for the btkϪ/Ϫ and lynϪ/Ϫ mice, each on a mixed C57BL/6 ϫ 129/Sv genetic reduced cytokine production in Btk-deficient mast cells. ϩ/Ϫ ϩ/Ϫ background, were mated to generate btk lyn F1 progeny. These F1 Activation of Btk in BCR and Fc⑀RI signaling systems requires mice were mated to obtain wild-type, btkϪ/Ϫ, lynϪ/Ϫ, and btkϪ/ϪlynϪ/Ϫ both phosphatidylinositol 3-kinase (PI3-K) and Src family kinases mice (36). Genotyping was done by Southern blotting or PCR analysis of (27, 28). The product of PI3-K, phosphatidylinositol 3,4,5- mouse tail-derived DNAs. Mast cells were cultured as described previously trisphosphate, is believed to recruit Btk to the plasma membrane (44). Briefly, bone marrow cells derived from the femur of the 6- to 10- wk-old mice were cultured in RPMI 1640 medium supplemented with 10% (29), where it is phosphorylated on tyrosine 551 in its activation FCS, 100 ␮M nonessential amino acids, 50 ␮M 2-ME, and 8% conditioned loop by activated Lyn molecules (27). Btk phosphorylated on ty- medium of IL-3 gene-transfected cells (bone marrow-derived mast cell rosine 551 is enzymatically active and autophosphorylates tyrosine medium). More than 95% of the trypan blue-excluding viable cells were 223 in its SH3 domain (30). Although these observations suggest mast cells after 4 wk of culture. No discernible differences in morphology and expression of early signaling proteins, including Fc⑀RI␤,Fc⑀RI␥, Syk, a direct enzyme/substrate relationship for Lyn and Btk, studies in Grb2, PLC-␥2, c-Cbl, and Shc, were detected between these four types of both B and mast cells imply a more complex interaction between mast cells (see Figs. 3–5 and data not shown). Surface expression of Fc⑀RI these two kinases. While btk mutations profoundly impaired cy- was measured by flow cytometry using a FACSCalibur apparatus and tokine production in Fc⑀RI-stimulated mast cells (22), this process CellQuest software (Becton Dickinson, Mountain View, CA). In acute (Ͻ60 min) stimulation experiments, mast cells were sensitized by an over- was not affected by Lyn deficiency as measured by RT-PCR (12). Downloaded from night incubation with 1 ␮g/ml anti-dinitrophenyl (DNP) IgE mAb, washed Similarly, B cell development and activation are differentially af- once in Tyrode buffer (112 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.6 fected by Btk and Lyn deficiencies (31–35). Btk-deficient mice mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.5, 0.05% gelatin, 0.1% lack B-1 cells, while Lyn-deficient mice have normal or increased glucose), resuspended in Tyrode buffer to 2 ϫ 107 cells/ml, and stimulated numbers of this cell type. Serum IgM levels are low in the absence by polyvalent Ag, 100 ng/ml DNP conjugates of human serum albumin (DNP-HSA), for the indicated time intervals. of Btk and high in the absence of Lyn. Aged lynϪ/Ϫ mice develop

autoantibodies, a process that is impaired by btk mutations in sev- Measurements of secreted histamine, leukotrienes, and cytokines http://www.jimmunol.org/ eral models of autoimmunity (36, 37). Lyn clearly plays a role in Histamine released into medium during a 45-min stimulation was mea- the initiation of BCR signals, but its predominant unique role in sured by an automated fluorometric assay (45). Leukotrienes secreted into this pathway is inhibitory as shown by the hypersensitivity of Lyn- medium for 30 min were analyzed by an enzyme immunoassay kit for deficient B cells to anti-IgM stimulation. The inhibitory function of leukotrienes C4/D4/E4 (Amersham Pharmacia Biotech, Piscataway, NJ). ␣ Lyn seems to involve the tyrosine phosphorylation of CD22 and TNF- and IL-2 secreted into the culture medum for 20 h were measured ␥ by ELISA kits (Endogen, Woburn, MA). In this case, mast cells were Fc RIIb and the recruitment of the tyrosine phosphatase SHP-1 stimulated in bone marrow-derived mast cell medium instead of Tyrode and 5Ј-inositol phosphatase SHIP, respectively, to these inhibitory buffer. receptors (38–40). In contrast, Btk-deficient cells fail to respond to BCR cross-linking. Immunoblotting and immunoprecipitation by guest on September 25, 2021 Differences in the in vivo phenotypes of btkϪ/Ϫ (or xid) and Cells were lysed in ice-cold 1% Nonidet P-40-containing lysis buffer (20 lynϪ/Ϫ mice and the in vitro properties of btkϪ/Ϫ and lynϪ/Ϫ mast mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium or- ␮ ␮ ␮ cells suggest that these PTKs may have independent or opposing thovanadate, 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin, 25 M p-nitrophenyl pЈ-guanidinobenzoate, 1 ␮M pepstatin, and 0.1% sodium functions. Indeed, Btk and Lyn are each required for B cell sur- azide) immediately after stimulation. Lysates were centrifuged in an Ep- vival and Ag response but exert opposing functions in generation pendorf microcentrifuge at 4°C for 10 min. Protein concentrations were of autoantibodies and the tuning of BCR-dependent proliferative measured using DC protein assay reagents (Bio-Rad, Richmond, CA). responses (36, 37, 41). To determine whether this complexity is Cleared lysates were either directly analyzed by SDS-PAGE or immunoprecipitated before SDS-PAGE analysis. For immunoprecipitation, lysates observed in alternative cell types and receptor systems, growth and were incubated on ice with an appropriate Ab for 2–4 h, and immune activation properties of mast cells derived from Btk/Lyn-deficient complexes were recovered by brief centrifugation following another 30 mice were compared with wild-type and singly deficient mast min incubation with Pansorbin (Calbiochem) for rabbit polyclonal Abs or cells. Btk and Lyn played both redundant and opposing roles in anti-mouse Ig-conjugated agarose (Sigma) for mouse mAbs. Immune com- Fc⑀RI signaling depending on the signal output measured, empha- plexes were washed in lysis buffer four times before SDS-PAGE analysis. Proteins separated by SDS-PAGE were electrophoretically transferred to sizing that multiple signal outcomes must be evaluated to fully polyvinylidene difluoride membranes (NEN Life Science Products, Bos- understand the functional interactions of individual signaling com- ton, MA). Membranes were blocked, incubated consecutively with primary ponents. Strikingly, all aspects of mast cell activation measured Ab and HRP-conjugated secondary Ab, and immunoreactive proteins were were significantly reduced in Btk/Lyn-deficient cells. This sug- visualized by enhanced chemiluminescence reagents (NEN Life Science Products). To estimate concentrations of PKC isoforms in mast cells, var- gests that simultaneous blockade of Btk and Lyn may be an at- ious amounts of human recombinant PKC proteins (␣, ␤I, and ␤II iso- tractive therapeutic strategy for allergic diseases. forms) expressed in insect cells (Panvera, Madison, WI) were run in par- allel with mast cell lysates and followed by immunoblotting with Materials and Methods respective Abs. Reagents Immune complex kinase assays Culture media and FCS were purchased from Life Technologies (Grand For Syk kinase assays, immune complexes precipitated from 1% Nonidet Island, NY). Anti-Fc⑀RI␤ subunit mAb (42) was kindly donated by Juan P-40 cell lysates were washed five times in lysis buffer and once with Rivera (National Institutes of Health, Bethesda, MD). Sources of commer- kinase buffer without ATP. Washed immune complexes were incubated cial Abs are as follows: anti-Btk (M-138), anti-Lyn (44), anti-Syk (C-20), with kinase buffer (50 mM Tris, pH 7.4, 0.1% Nonidet P-40, 10 mM ␥ ␣ ␤ ␮ anti-PLC- 2 (Q-20), anti-PKC (C20), anti-PKC II (C-18), anti-c-Jun N- MnCl2, 10 mM MgCl2) with 2 g of GST-HS1 (46) containing the se- terminal kinase (JNK) 1 (C-17), anti-extracellular signal-related kinase quence from position 352 to position 486 of the human HS1 protein in the (ERK)1 (C-16), and anti-p38 (C-20) from Santa Cruz Biotechnology (San- presence of [␥-32P]ATP (DuPont NEN, Boston, MA). For PKC assays, ta Cruz, CA); anti-phosphotyrosine mAb 4G10 and anti-PLC-␥1 mAbs immunoprecipitates after similarly washed were incubated with kinase ␮ from Upstate Biotechnology (Lake Success, NY); anti-phospho-mitogen- buffer (20 mM Tris, pH 7.4, 10 mM MgCl2,10 M ATP) in the presence activated protein (MAP) kinase and anti-phospho-p38 from New England of [␥-32P]ATP. For JNK kinase assays, cells were lysed in ice-cold whole- 1212 Btk AND Lyn FUNCTIONS IN MAST CELLS

cell extraction buffer (25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5 mM ␮ MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 2 M DTT, 0.5 mM PMSF, 20 mM ␤-glycerophosphate, and 0.5 mM sodium orthovanadate). Cleared lysates in this buffer were diluted with three volumes of dilution buffer (20

mM HEPES, [pH 7.5, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05% Triton X-100, 0.5 mM sodium orthovanadate, 0.5 mM PMSF, and 20 mM ␤-glycerophosphate). Cleared lysates were immunoprecipitated with anti-JNK1 (C-17, Santa Cruz Biotechnology). Immune complexes were washed ﬁve times with lysis buffer and once with kinase buffer without ATP and substrate. Then, immunoprecipitates were incubated with 3 ␮g GST-c-Jun (1–79) in 15-min reactions at 30°C in 20 mM HEPES, pH 7.4, 10 mM ␤ ␮ ␮ MgCl2, 22 mM DTT, 20 mM -glycerophosphate, 50 MNa3VO4,20 M ATP, and 10 ␮Ci [␥-32P]ATP. Reaction products were analyzed by SDS- PAGE followed by electroblotting onto polyvinylidene diﬂuoride membranes and autoradiography.

IP3 measurement

A commercial kit (Amersham Pharmacia Biotech) was used. Cells were ⑀ extracted with chloroform/methanol (1/2 v/v) on ice for 10 min. Methanol FIGURE 1. Expression of Fc RI on the surface of wild-type and mutant fractions containing phosphorylated inositols were lyophilized and mixed mast cells. Mast cells derived from mouse bone marrow cells were incubated with anti-DNP mouse IgE mAb and then with ﬂuosceinated anti- with bovine adrenal IP3-binding proteins in the presence of a limiting 3 amount of tracer D-myo-[ H]IP . The mixtures were centrifuged at 2000 ϫ mouse Ig. Flow cytometric analysis of these cells was performed. 3 Downloaded from g for 10 min and radioactivity bound to IP3-binding protein was measured in a ␤-scintillation counter. Intracellular calcium analysis (0.05–5 ␮g/ml) in a concentration-dependent manner. Comparable ⑀ Mast cells were sensitized with 0.5 ␮g/ml anti-DNP IgE in bone marrow- IgE-mediated enhancement in Fc RI expression was observed in derived mast cell medium for2hat37°C and loaded with 10 ␮M INDO-1 mast cells derived from the other genotypes as well (data not Ϫ Ϫ acetoxymethyl (Molecular Probes, Eugene, OR) in RPMI 1640 and 2% /

shown). As described previously (25), btk mice yielded more http://www.jimmunol.org/ FCS for 40 min at 30°C with constant agitation. Cells were washed once, mast cells than wild-type counterparts under these culture condi- 2ϩ resuspended in HBSS with 1.7 mM Ca (Sigma), and maintained on ice Ϫ/Ϫ ϫ 7 tions, while lyn bone marrow generated normal numbers of at a concentration of 1 10 cells/ml until analysis. Cells were resus- Ϫ/Ϫ Ϫ/Ϫ pended at a concentration of 5 ϫ 105 cells/ml in the same buffer and mast cells (Fig. 2A). btk lyn bone marrow also generated warmed for 2 min at 37°C with rapid stirring before analysis. Bulk intra- mast cell numbers that were intermediate between those present in cellular calcium levels were monitored by excitation at 350 nm with de- wild-type and btkϪ/Ϫ mice. The large number of mast cells pro- tection of the 405 nm bound and 440 nm unbound emissions of INDO-1 duced in the absence of Btk could have resulted from increased using a DeltaRam spectrofluorometer (Photon Technology Instruments, Princeton, NJ) at a rate of five measurements per second. Analysis at base- number of precursors in the bone marrow, faster cell cycle time, or line was acquired for 20 s before Fc⑀RI cross-linking with 100 ng/ml reduced cell death. The latter hypothesis is supported by the ob- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ DNP-HSA. Calcium analysis was continued for 280 s, followed by addi- servation that both btk and btk lyn cells were more re- by guest on September 25, 2021 tion of 10 ␮M ionomycin to determine the peak population response using sistant to growth factor (IL-3) deprivation-induced apoptosis (Fig. an additional 60-s data acquisition. 2B). Btk has been suggested to play a role in both proapoptotic Transcriptional activity assay with luciferase reporter constructs (Fig. 2B, Refs. 25 and 49) and anti-apoptotic pathways (50, 51), implying cell type- or receptor-specific outcomes of Btk signaling. Luciferase reporter constructs, the mouse IL-2 (-321)-Luc and the human ␣ ϫ 7 TNF- (-200)-Luc, were described previously (22). A total of 1–1.5 10 Btk and Lyn are redundant for mast cell degranulation and mast cells were transfected with 5–10 ␮g reporter plasmid by electropo- ration at 400V, 950 ␮F using a Gene Pulser II apparatus (Bio-Rad). Trans- leukotriene release fected cells were sensitized overnight with anti-DNP IgE and left unstimu- Fc⑀RI-mediated activation of mast cells results in degranulation lated or stimulated with 30 ng/ml DNP-HSA for 8 h before cell harvest. Cells were lysed in 0.2% Triton-X-100 in 100 mM potassium phosphate (measured by histamine release) and secretion of leukotriene. We buffer (pH 7.8)/1 mM DTT. Luminescence of cleared lysates was measured examined the interaction of Btk and Lyn in regulating these pro- after addition of luciferin solution using a model Monolight 2010 lumi- cesses. Histamine release was normal or near normal and leuko- nometer (Analytical Luminescence Laboratory, San Diego, CA). triene release was normal in cells lacking either Btk or Lyn alone (Fig. 3). Surprisingly, the secretion of both histamine and leuko- Results triene was nearly completely abrogated in btkϪ/ϪlynϪ/Ϫ mast cells Generation of mast cells from bone marrow cells derived from (Fig. 3), indicating that Btk and Lyn together are essential for Btk-, Lyn-, and Btk/Lyn-deficient mice Fc⑀RI-induced mast cell degranulation and leukotriene release. Ϫ Ϫ Culturing murine bone marrow cells in IL-3-containing medium Normal degranulation in lyn / mast cells may be due to a func- for 4 wk leads to the generation of a Ͼ95% pure population of tion fulfilled by another Src family PTK(s) expressed in mast cells. immature mast cells. This process is independent of both Btk and Fyn/Lyn doubly deficient mast cells degranulated significantly less Lyn, as bone marrow cells derived from wild-type, btkϪ/Ϫ, lynϪ/Ϫ, efficiently than wild-type or singly deficient mast cells (data not and btkϪ/ϪlynϪ/Ϫ littermates gave rise to similarly pure popula- shown), indicating the redundant function exhibited by two Src tions of mast cells. The genotypes of these cells determined by family PTKs in mast cell degranulation. Full description of our Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Southern blot or PCR analysis were confirmed by immunoblotting study on fyn / and fyn / lyn / mast cells will be provided cell lysates with anti-Lyn or anti-Btk Abs (data not shown). These elsewhere. mast cells express similar levels of Fc⑀RI on their surfaces (Fig. 1). Ϫ/Ϫ Ϫ/Ϫ Btk/Lyn-deficient mast cells exhibit impaired substrate tyrosine Although the btk lyn cells exhibited a broader distribution 2ϩ of Fc⑀RI expression in this cell preparation, the distribution in phosphorylation and defective IP3 /Ca signaling another preparation was similar to that of wild-type cells. As pre- We examined Fc⑀RI-induced tyrosine phosphorylation of several viously shown (47, 48), Fc⑀RI expression on the surface of wild- key substrate molecules to begin to understand the role(s) for Btk type mast cells increased 2–4 days after incubation with IgE vs Lyn in mast cell degranulation. The overall pattern of tyrosine The Journal of Immunology 1213

FIGURE 2. In vitro growth curves of bone marrow cells derived from wild-type and mutant mice and survival curves of mast cells upon growth factor deprivation. A, numbers of trypan blue-excluding cells in bone marrow cell cultures in IL-3-containing medium were counted on the second day of culture and on the ﬁrst medium change (the sixth day). Weekly cell counting was done at the time of weekly medium changes. A representative result of three Downloaded from independent experiments is shown. B, A total of 5 ϫ 105 mast cells in duplicate were cultured without IL-3. Live cell numbers were counted over the time course of 5 days. Essentially the same results were obtained in another set of experiments.

phosphorylation was very similar between btkϪ/Ϫ and wild-type although its kinetics were similar to those in wild-type cells (Fig. Ϫ/Ϫ cells (Fig. 4A). This response was partially blunted in lyn cells 5C). Consistent with this data and previous studies demonstrating http://www.jimmunol.org/ and severely affected in Btk/Lyn-deficient cells. Tyrosine phos- an important role for Btk in the generation of BCR-dependent phorylation of the Fc⑀RI ␤ subunit (Fig. 4B) and Syk (Fig. 4, C and calcium signaling (23, 24), btkϪ/Ϫ mast cells exhibited a signifi- D) was reduced in both lynϪ/Ϫ and btkϪ/ϪlynϪ/Ϫ cells but not in cantly reduced total calcium flux relative to wild-type mast cells in btkϪ/Ϫ cells. This is consistent with the current model (7, 52) response to receptor cross-linking (Fig. 5D). Following BCR whereby Lyn phosphorylates the ITAMs of both the ␤ and ␥ sub- cross-linking, phosphatidylinositol 3,4,5-trisphosphate generated units of Fc⑀RI and that ␥ITAM phosphorylation leads to recruit- by PI3-K initiates Btk activation in concert with Src kinases by ment and activation of Syk. As previously described for lynϪ/Ϫ targeting the Btk PH domain to the plasma membrane (Refs. 24 mouse B cells (31), the kinetics of Fc⑀RI-dependent tyrosine phos- and 29, and reviewed in Ref. 53). This model is consistent with phorylation of several proteins including Fc⑀RI␤ (Fig. 4B), Syk data obtained from wild-type and Btk-deficient mast cells. Wort- by guest on September 25, 2021 (Fig. 4C), and PLC-␥1 (Fig. 5A) were delayed in lynϪ/Ϫ mast cells mannin pretreatment resulted in a marked reduction in calcium and the degree of phosphorylation was lower than in wild-type or signaling in wild-type mast cells. In contrast, only a minimal re- btkϪ/Ϫ cells. (Tyrosine phosphorylation of PLC-␥1inbtkϪ/Ϫ cells duction in sustained calcium signaling was observed in btkϪ/Ϫ was variable among several experiments, although it was higher mast cells under identical conditions (data not shown). Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ than in wild-type cells in the experiment shown in Fig. 5A.) Ty- IP3 synthesis in lyn and btk lyn cells was much de- rosine phosphorylation of PLC␥-1 was significantly reduced in layed but strikingly augmented at later time points (Fig. 5C). The btkϪ/ϪlynϪ/Ϫ cells relative to cells lacking either Btk or Lyn alone difference in this response between these cell types indicates that ␥ Ϫ/Ϫ (Fig. 5A). Tyrosine phosphorylation of PLC- 2 was lower in the augmented IP3 response in lyn cells are at least partly de- btkϪ/Ϫ, lynϪ/Ϫ, and btkϪ/ϪlynϪ/Ϫ cells than in wild-type cells pendent on Btk. The acute phase of Ca2ϩ increase (within 50 s (Fig. 5B). after Fc⑀RI stimulation) was completely blocked in both lynϪ/Ϫ ␥ Ϫ/Ϫ Ϫ/Ϫ Consistent with the reduced phosphorylation of PLC- 2, IP3 and btk lyn mast cells (Fig. 5D) despite normal peak re- synthesis in btkϪ/Ϫ cells was lower than that in wild-type cells, sponses to ionomycin (data not shown), indicating an essential role

FIGURE 3. Release of histamine and leukotriene from wild-type and mutant mast cells upon Fc⑀RI cross-linking. Mast cells (4–5 wk old in IL- 3-containing cultures) were sensitized overnight with anti-DNP IgE and stimulated with Ag, DNP- HSA for 45 min (A)or30min(B). Histamine released into medium was measured as described (44). Leukotriene released into medium was measured by an enzyme immunoassay. 1214 Btk AND Lyn FUNCTIONS IN MAST CELLS

FIGURE 4. Tyrosine phosphorylation including those of the Fc⑀RI␤ subunit and Syk in wild-type and mutant mast cells. A, Bone marrow-derived mast Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ cells from wild-type (wt), btk (btk-), lyn (lyn-), and btk lyn (btk-/lyn-) mice were sensitized overnight with anti-DNP-IgE and stimulated with Downloaded from DNP-HSA for the indicated intervals. Cell lysates containing equal protein amounts were analyzed by SDS-PAGE and blotting followed by probing the blots with anti-phosphotyrosine mAb 4G10. The position of the 30-kDa protein, which was prominently tyrosine-phosphorylated upon Fc⑀RI cross-linking in wild-type and btkϪ/Ϫ cells, but not in lynϪ/Ϫ and btkϪ/ϪlynϪ/Ϫ cells, is indicated by an asterisk. This protein was identiﬁed as Fc⑀RI␤ subunit in B.In B, mast cell lysates were immunoprecipitated with anti-Fc⑀RI␤ mAb JRK (20 ␮l of culture supernatants of the hybridoma). Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. The blot was consecutively probed with anti-phosphotyrosine mAb and anti-Fc⑀RI␤ mAb. C, Mast cell lysates were immunoprecipitated with anti-Syk Ab (2 ␮g). Immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine mAb (upper panel). The portion of the Syk bands is shown. The same blot was reprobed with anti-Syk (lower panel). D, Mast cell lysates were immunoprecipitated http://www.jimmunol.org/ with anti-Syk Ab, and immunoprecipitates were subjected to in vitro kinase assays using GST-HS1 as substrate. The portion of the autoradiogram covering the phosphorylated GST-HS1 region is shown.

for Lyn in this event. In most experiments, these cells exhibited a and a less abundant (1.25 ng per 106 cells) PKC␤I isoform, as slow, but sustained, increase in intracellular Ca2ϩ concentration, measured by immunoblotting cell lysates with respective Abs us- which correlates with the delayed response of IP3 synthesis. These ing recombinant PKC isoforms as reference (data not shown). Ϫ Ϫ ϩ data are similar to those previously observed in lyn / chicken B PKC␤ is the major isoform of PKC, which together with Ca2 is by guest on September 25, 2021 cells (54) and lynϪ/Ϫ mast cells (12). Effects of Btk and Lyn de- sufficient to elicit degranulation in permeabilized RBL-2H3 mast 2ϩ ␣ ficiencies on IP3 production and Ca response suggest that ty- cells (16). Therefore, we compared the activities of PKC and rosine phosphorylation (therefore enzymatic activation) of PLC-␥2 PKC␤II in mast cells of the four genotypes. Autophosphorylating 2ϩ ␣ ␤ Ͻ is more relevant to IP3 production and Ca mobilization than that activities of PKC and PKC II were slightly ( 3-fold) increased of PLC-␥1 in mast cells. at their peak (15 min) after Fc⑀RI cross-linking in wild-type and btkϪ/Ϫ mast cells (Fig. 6B). Interestingly, basal activities of PKC␣ Regulation of PKC activity by Lyn and Btk and PKC␤II in unstimulated lynϪ/Ϫ cells were significantly lower Ϫ Ϫ Robust degranulation in spite of delayed (or blunted in some than wild-type and btk / cells, but became higher at their peaks Ϫ Ϫ Ϫ Ϫ lynϪ/Ϫ cell preparations as shown in one of the tracings in Fig. 5D) upon Fc⑀RI stimulation. In contrast, btk / lyn / cells exhibited Ca2ϩ response in lynϪ/Ϫ mast cells and blunted degranulation with little or no activation of PKC␣ or PKC␤II (a longer exposure of similar Ca2ϩ response in btkϪ/ϪlynϪ/Ϫ cells prompted us to eval- the autoradiograms shown in Fig. 6B and in three other experi- uate the involvement of Ca2ϩ and PKC in degranulation from mast ments). Therefore, the robust degranulation correlates with the vig- cells. Chelation of extracellular calcium by EGTA inhibited his- orous activity of PKC, more specifically that of PKC␤II, in lynϪ/Ϫ tamine release from Fc⑀RI-stimulated lynϪ/Ϫ mast cells as well as cells while the drastically reduced degranulation correlates with from wild-type and btkϪ/Ϫ cells, indicating that a Ca2ϩ flux was the poor PKC activation in btkϪ/ϪlynϪ/Ϫ cells. Immunoblotting of important for this response and that the delayed-phase calcium total cell lysates showed that the amounts of PKC␣ and PKC␤II in response in lynϪ/Ϫ cells was sufficient (Fig. 6A). Inhibition of PKC unstimulated and Fc⑀RI-stimulated btkϪ/ϪlynϪ/Ϫ cells were re- by Ro 31-8425 also blocked histamine release from Fc⑀RI-stimu- duced apparently by degradation, because the amount of degrada- lated wild-type, btkϪ/Ϫ or lynϪ/Ϫ mast cells (Fig. 6A). PMA treat- tion products reactive with the C-terminal peptide-specific Abs ment of wild-type cells in the presence of EGTA induced a modest was dramatically increased (data not shown). but significant degranulation (data not shown). These results indi- Kinetics of histamine release were determined to examine the cate that PKC activation is required and sufficient for degranula- relationship between degranulation, Ca2ϩ response, and PKC␤II tion in mouse bone marrow-derived mast cells and that a minimal activity in lynϪ/Ϫ cells vs other cell types. As shown in Fig. 6C, threshold of Ca2ϩ flux is also required for efficient degranulation. histamine release from Fc⑀RI-stimulated lynϪ/Ϫ cells exhibited a In the same line of study, human basophils are known to degranu- lag time (Ͼ3 min) before a significant amount of histamine was late upon Fc⑀RI cross-linking without the requirement for a phar- detected in the medium, whereas it was detected within 1 min in macologic Ca2ϩ release signal (55) and that PKC is essential for wild-type cells. This delayed histamine release in lynϪ/Ϫ cells cor- degranulation in these cells. relates with the delayed Ca2ϩ response and the low basal and Mouse mast cells express two predominant Ca2ϩ-dependent induced activity of PKC␤II, suggesting the importance of these PKC isoforms, 40 ng of PKC␣ and 50 ng of PKC␤II per 106 cells, signals for degranulation. The Journal of Immunology 1215 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Involvement of Ca2ϩ and PKC in mast cell degranulation. A, IgE-sensitized wild-type and mutant mast cells were pretreated with solvent, 5 mM EGTA (for 1 min), or 2 ␮M Ro 31-8425 (for 10 min) before stimulation with 30 ␥ ␥ FIGURE 5. Tyrosine phosphorylation of PLC- 1 and PLC- 2 and IP3/ ng/ml DNP-HSA for 45 min. Histamine released into media (Tyrode buffer con- calcium response in wild-type and mutant mast cells. Mast cell lysates were taining 1.6 mM CaCl2) was measured. Representative results of two experiments immunoprecipitated with 2 ␮g each of anti-PLC-␥1(A) or anti-PLC-␥2 giving essentially the same results are shown. Controls (45 min treatment of cells (B). Immunoprecipitates were subjected to immunoblotting with anti- with solvent alone, EGTA alone, and Ro 31-8425 alone, i.e., without Ag stimu- phosphotyrosine mAb (upper panel) and after stripping the blots with the lation) induced Ͻ3% of the cellular histamine content and were omitted from the respective Abs (lower panel). C, IgE-sensitized mast cells were stimulated ﬁgure. B, IgE-sensitized wild-type and mutant mast cells were stimulated with with multivalent DNP-HSA (30 ng/ml) for the indicated periods. IP3 pro- DNP-HSA for the indicated periods. Cell lysis, immunoprecipitation with 2 ␮g duction was measured as described in Materials and Methods. Early (0–1 each of anti-PKC␣ or anti-PKC␤II, and autophosphorylating reactions were done min, left scale) and later (5–15 min, right scale) time points from the same as described in Materials and Methods. Blots of kinase assays were probed with experiments (performed in duplicate with Ͻ5% differences in values of respective Abs to measure the amounts of PKC␣ and PKC␤II. Results represen- duplicate samples) are plotted with different scales. D, IgE-sensitized mast tative of three similar experiments are shown. C, IgE-sensitized mast cells were ϩ cells were stimulated with DNP-HSA (arrow), and Ca2 mobilization of washed and incubated at 37°C with 30 ng/ml DNP-HSA for the indicated periods INDO-1-loaded cells was monitored by spectroﬂuorometry in the presence of time before sampling media for histamine measurements. Note the different ϩ of extracellular Ca2 . Representative data from more than three similar scales for the time points before (left) and after (right) 15 min incubation. One of experiments are shown. two experiments is shown. Less than 2% variance at each time point was observed between duplicate samples.

Opposing effects of btk and lyn mutations on cytokine secretion Late-phase reactions of immediate hypersensitivity appear to be at on cytokine production by activated mast cells (Fig. 7). As shown least partly dependent on TNF-␣ secreted from Fc⑀RI-stimulated previously (22), btkϪ/Ϫ mast cells produced and secreted less mast cells (56). We determined the effects of btk and lyn mutations TNF-␣ and IL-2 than wild-type counterparts. Intriguingly, lynϪ/Ϫ 1216 Btk AND Lyn FUNCTIONS IN MAST CELLS

FIGURE 7. Secretion of TNF-␣ and IL-2 from Fc⑀RI-stimulated wild-type and mutant mast cells. Mast cells were sensitized overnight with anti-DNP IgE and stimulated with Ag in the complete culture medium for 20 h. Cytokines secreted into culture supernatants were measured by ELISAs.

mast cells secreted 3-fold more of these cytokines than wild-type MAP kinase activities in mutant mast cells Downloaded from cells. This enhanced response was abrogated in the absence of Btk, Because ERK and JNK pathways can induce transcription of IL-2 indicating that Btk and Lyn play opposing roles in the production and TNF-␣ genes (57), we evaluated the activity of these kinases ⑀ of specific cytokines in response to Fc RI activation. in Fc⑀RI-stimulated mast cells of all four genotypes (Fig. 9). Ϫ/Ϫ Transcriptional activation of cytokine genes in bone marrow- lyn mast cells exhibited prolonged activation of both ERK derived mast cells (Fig. 9A) and JNK1 (Fig. 9B), potentially accounting for the enhanced cytokine secretion from these cells. As shown previously http://www.jimmunol.org/ To begin to understand the mechanism(s) by which Btk and Lyn (25), JNK1 activity was significantly reduced in Btk-deficient cells exerted opposing effects on cytokine secretion, we evaluated the (Fig. 9A). Strikingly, JNK1 activity was completely abrogated in ␣ Ϫ Ϫ Ϫ Ϫ signaling pathways potentially regulating IL-2 and TNF- expres- btk / lyn / cells. In contrast, ERK1 and ERK2 phosphorylation sion in activated mast cells. Because transcriptional regulation of was unaltered in either btkϪ/Ϫ or btkϪ/ϪlynϪ/Ϫ mast cells. Nota- cytokine genes is a critical step in cytokine production in activated bly, Fc⑀RI-induced activation of the p38 MAP kinase pathway was mast cells, we analyzed transcriptional activity of the IL-2 gene promoter by transfecting the IL-2/Luc reporter plasmid into mast cells of the four genotypes. As described previously (22, 26), Fc⑀RI cross-linking induced a robust transactivation of IL-2 pro- by guest on September 25, 2021 moter in wild-type mast cells while btkϪ/Ϫ cells exhibited much lower activity (Fig. 8). Consistent with the data on cytokine secretion, transcriptional activation in lynϪ/Ϫ cells was about twice more than that in wild-type cells. In contrast, btkϪ/ϪlynϪ/Ϫ cells gave little activation of the IL-2 promoter. Similar to TNF-␣ secretion, TNF-␣/Luc transcriptional activity was less remarkably affected by Lyn and Btk deficiencies (data not shown). Therefore, these data confirm that transcriptional regulation of cytokine genes is an important regulatory step in mast cell activation.

FIGURE 9. Activities of MAP kinases in wild-type and mutant mast cells. A, mast cell lysates were analyzed by immunoblotting with an Ab specific for the phosphorylated, activated form of ERK1 and ERK2 (upper panel) and with anti-ERK Ab that recognizes both ERK1 and ERK2 (lower panel). B, JNK1 was immunoprecipitated with 1 ␮g anti-JNK1(C-17) from mast cell lysates. Immunoprecipitates were subjected to immune complex kinase assays using GST-c-Jun(1–79) as substrate. Phosphorylated GST- FIGURE 8. Activity of IL-2 promoter upon Fc⑀RI stimulation in wild- c-Jun(1–79) bands detected by autoradiography are shown (upper panel). type and mutant mast cells. Wild-type and mutant mast cells were elec- Expression of JNK1 was checked by immunoblotting of cell lysates with troporated with 8 ␮g of IL-2/Luc plasmid. Cell stimulation, lysis, and anti-JNK1 (lower panel). C, p38 activity in mast cell lysates was measured luciferase assays were performed as described (26). Fold luciferase activ- by immunoblotting with an Ab specific for the phosphorylated, activated ities relative that of unstimulated lynϪ/Ϫ cells are shown. A representative form of p38 (upper panel). The amounts of p38 in mast cells were mea- result of three experiments is shown. sured by reprobing the same blot with anti-p38 Ab (lower panel). The Journal of Immunology 1217 unchanged in singly deficient cells but was significantly impaired requires both modestly increased intracellular Ca2ϩ concentrations in doubly deficient mast cells (Fig. 9C). p38 has been shown to and activation of PKC isoforms (16). The impaired activation of ␥ ␥ regulate c-jun expression via the phosphorylation of another tran- both PLC- 1 and - 2, the consequent defects in IP3/calcium sig- scription factor MEF2C (58). This combined impairment of JNK1 naling, and little or no activation of Ca2ϩ-dependent PKC iso- and p38 activation may be responsible for the significantly reduced forms, especially PKC␤II, may explain the severe reduction in production of TNF-␣ and IL-2 in Btk/Lyn-deficient cells. BCR- degranulation in Btk/Lyn-deficient mast cells. Release of arachi- dependent p38 activation in chicken B cells required both Lyn and donic acid, the precursor of leukotrienes, involves Syk, the ERK Syk activity but was unaltered in Btk, Syk, or Lyn singly deficient pathway, and Ca2ϩ (66). However, it is unlikely that the abroga- Ϫ Ϫ Ϫ Ϫ cells (59). Our data suggest that doubly deficient (btk / lyn / )B tion of leukotriene release in Btk/Lyn-deficient cells resulted sim- cells are also likely to exhibit suboptimal BCR-dependent p38 ply from decreased Syk activation because lynϪ/Ϫ mast cells also activation. exhibited reduced Syk activity yet released normal levels of leukotrienes. The reduction in ERK phosphorylation in btkϪ/ϪlynϪ/Ϫ Discussion cells is likely to be related to the decrease in leukotriene Regulation of mast cell growth and survival by Btk and Lyn production. Most lynϪ/Ϫ cell preparations exhibited delayed, but aug- Signaling pathways used by the two major mast cell growth fac- mented, IP production and delayed Ca2ϩ responses, similar to tors, IL-3 and stem cell factor (c-Kit ligand), activate both Src and 3 lynϪ/Ϫ chicken B cells (54). This result is distinct from the en- Tec family PTKs as proximal events (reviewed in Ref. 60). The hanced peak and sustained Ca2ϩ following BCR stimulation ob-

data presented here demonstrate that neither Btk nor Lyn are es- Downloaded from served using lynϪ/Ϫ B cells from the same mouse strain (38), fur- sential for IL-3-dependent expansion of bone marrow-derived ther emphasizing the importance of cellular context in determining mast cells. In fact, loss of Btk function leads to the production of functions of Lyn. Despite the loss of the initial calcium response, a significantly greater number of mast cells (25). Btk-deficient Ϫ/Ϫ mast cells exhibit similar proliferative response to IL-3 as wild- lyn mast cells degranulate almost normally, suggesting that a 2ϩ ⑀ type cells but survive better than their wild-type counterparts upon low level or delayed Ca signal remains sufficient for Fc RI- growth factor withdrawal (25), supporting a negative role for Btk induced degranulation. This situation may be similar to human http://www.jimmunol.org/ in regulating cell survival in this system. In contrast to these re- basophils, in which PMA, a potent PKC activator, can induce his- 2ϩ sults, Btk has also been shown to play an anti-apoptotic role in B tamine release without pharmacologic Ca release (55). The re- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ cells (50). Btk-deficient B cells survive poorly in vitro and in vivo duced initial peak calcium flux in lyn and btk lyn mast and fail to up-regulate Bcl-xL in response to Ag receptor cross- cells likely results from the combined effects of reduced Lyn-de- linking. JNK, which is regulated by Btk, has been implicated in pendent transphosphorylation of both Syk and Btk and a reduced both positive and negative regulation of apoptosis. Stress-induced activation of PI3-K isoforms. In contrast, the dramatic late-phase JNK activation is crucial for induction of apoptosis (61–63), while increase in IP3 production and gradual increase in intracellular 2ϩ SEK-1 (a direct activator of JNK)-deficient T cells exhibit en- Ca concentration may represent a loss of Lyn-dependent inhibitory response. This may be mediated at least in part via reduced hanced activation-induced cell death (64, 65). These observations by guest on September 25, 2021 suggest that cell type, signal context, and/or additional factors may activation of the 5Ј inositol phosphatase SHIP leading to Btk be essential for determining the outcome of Btk-dependent signals. membrane targeting and activation. This mechanism would Ϫ/Ϫ Ϫ/Ϫ btk lyn mast cells exhibited an intermediate phenotype (in be consistent with the relative reduction in peak IP3 response in Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ comparison with wild-type cells and btkϪ/Ϫ cells) in production btk lyn vs lyn mast cells. and the rate of growth factor deprivation-induced cell death. This Interestingly, our present study showed that the activity of suggests that Btk and Lyn may have opposing functions in mast PKC␣ and PKC␤II, low before stimulation, is strongly increased Ϫ Ϫ cell survival. This suggestion of a weak positive role for Lyn in upon Fc⑀RI stimulation in lyn / mast cells, consistent with data Ϫ Ϫ mast cell survival was observed only in the context of Btk defi- on lyn / B cells (67). This data and the following observations ciency, as lynϪ/Ϫ cells were indistinguishable from wild-type cells suggest that PKC␤II is involved in mast cell degranulation. First, with respect to both overall production and cell death. The mech- Ozawa et al. (16) showed that PKC␤ (␤Ior␤II is not clear from ϩ anism for this effect is currently unclear, but may be related to the the paper) plus Ca2 can reconstitute degranulation in permeabil- differential regulation of specific MAP kinases and/or PKC iso- ized RBL-2H3 cells. Second, although the autophosphorylating forms by Btk and Lyn. In contrast to these observations, Btk and activity of PKC␤II is not strongly increased by Fc⑀RI stimulation, Lyn appear to have redundant rather than opposing roles in medi- this PKC isoform is translocated from the cytosol to the membrane ating B cell survival (36), again stressing the importance of cellular compartment as vigorously as PKC␣ and PKC␤I (data not shown). context in signal outcome. Third, the translocation of PKC␤II is not severely affected but that of PKC␤I is greatly reduced in btkϪ/Ϫ cells (data not shown). Redundant roles for Btk and Lyn in mast cell degranulation and These differences correlate with the mild effect of Btk deficiency leukotriene release on degranulation (Ref. 22 and this study). Finally, PKC␤Ϫ/Ϫ mast Activated mast cells contribute to allergic responses primarily cells exhibited drastically reduced degranulation (68). However, ␤ ⑀ through the secretion of proinflammatory mediators. We examined relative contribution of PKC II vs other PKC isoforms to Fc RI- the requirement for Btk and Lyn in the secretion of histamine (as induced degranulation remains to be studied. a marker for degranulation) and leukotrienes. Btk and Lyn exhibited redundant or independent roles for maximal degranulation and Opposing functions of Btk vs Lyn in mast cell cytokine secretion leukotriene release. Singly deficient mast cells produced normal or near normal levels of each of these chemical mediators. In con- Cytokines and chemokines comprise an important subset of proin- trast, Btk/Lyn doubly deficient cells had severely blunted activity flammatory mediators that participate in the induction of the late- in all of these assays. phase allergic responses following mast cell activation (1, 56). Our The signaling mechanisms controlling degranulation and leuko- results demonstrate that Btk and Lyn have opposing roles in cy- triene release remain poorly understood. Optimal degranulation tokine production/secretion in mast cells. Strikingly, Lyn-deficient 1218 Btk AND Lyn FUNCTIONS IN MAST CELLS cells secreted 3-fold more cytokines than wild-type cells. This en- References hanced response was dependent on Btk, as Btk/Lyn doubly defi- 1. Galli, S. J., and C. S. Lantz. 1998. Allergy. In Fundamental Immunology, 4th Ed. cient cells exhibited markedly reduced (TNF-␣) or absent (IL-2) W. Paul, ed. Lippincott-Raven Publishers, Philadelphia, PA, p. 1127. ⑀ 2. Ravetch, J. V., and J.-P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457. secretion upon Fc RI cross-linking. A similar Btk-dependent, in- 3. Eiseman, E., and J. B. Bolen. 1992. Engagement of the high-affinity IgE receptor hibitory role for Lyn is observed in proliferative response to BCR activates src protein-related tyrosine kinases. Nature 355:78. cross-linking in B lymphocytes (36, 37). In B cells, Lyn mediates 4. Vonakis, B. M., H. Chen, H. Haleem-Smith, and H. Metzger. 1997. The unique domain as the site on Lyn kinase for its constituttive association with the high down-regulation of BCR signals by phosphorylating CD22 (38– affinity receptor for IgE. J. Biol. Chem. 272:24072. 41) and paired Ig-like receptor B (69) and is also involved in 5. Pribluda, V., C. Pribluda, and H. Metzger. 1994. Transphosphorylation as the Fc␥RIIb-mediated inhibition (31, 40). Analogous Lyn-dependent mechanism by which the high-affinity receptor for IgE is phosphorylated upon ⑀ aggregation. Proc. Natl. Acad. Sci. USA 91:11246. signals may be involved in the negative regulation of Fc RI- 6. Cambier, J. C. 1995. New nomenclature for the Reth motif (or ARH1/TAM/ dependent responses in mast cells, as Fc␥RIIb was shown to be ARAM/YXXL). Immunol. Today 16:110. tyrosine-phosphorylated by Fc⑀RI-associated Lyn upon coliga- 7. Jouvin, M.-H., M. Adamczewski, R. Numerof, O. Letourneur, A. Valle, and J.-P. Kinet. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two tion (70). signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. MAP kinase activation in response to either Fc⑀RI cross-linking 269:5918. in mast cells (Fig. 9) or BCR cross-linking in B cells is enhanced 8. Kihara, H., and R. P. Siraganian. 1994. Src homlogy 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J. Biol. in the absence of Lyn (31). This may explain the increased pro- Chem. 269:22427. duction of cytokines by activated lynϪ/Ϫ mast cells, because the 9. Rowley, R. B., A. L. Burkhardt, H.-G. Chao, G. R. Matsueda, and J. B. Bolen. 1995. Syk protein-tyrosine kinase is regulated by tyrosine-phosphorylated Ig␣/ duration of MAP kinase activation determines the outcome of re- Ig␤ immunoreceptor tyrosine activation motif binding and autophosphorylation. ceptor stimulation (e.g., proliferation vs differentiation (reviewed J. Biol. Chem. 270:11590. Downloaded from in Ref. 71)), which probably involves the differential expression of 10. Kimura, T., H. Sakamoto, E. Appella, and R. P. Siraganian. 1996. Conforma- tional changes induced in the protein tyrosine kinase p72syk by tyrosine phos- transcription factors such as Fos and Jun families (72). Transcrip- phorylation or by binding of phosphorylated immunoreceptor tyrosine-based ac- tion of TNF-␣ and IL-2 involves several transcription factors. For tivation motif peptides. Mol. Cell. Biol. 16:1471. example, TNF-␣ gene activation induced by the TCR/CD3 com- 11. El-Hilal, O., T. Kurosaki, H. Yamamura, J.-P. Kinet, and A. M. Scharenberg. 1997. Syk kinase activation by a src kinase-initiated activation loop phosphory- plex requires NF-AT, c-Jun, and ATF-2 (73–75). c-Jun and ATF-2 lation chain reaction. Proc. Natl. Acad. Sci. USA 94:191.

are phosphorylated and regulated by JNK (reviewed in Ref. 57). 12. Nishizumi, H., and T. Yamamoto. 1997. Impaired tyrosine phosphorylation and http://www.jimmunol.org/ 2ϩ We have evidence that activators of ERK and JNK can also stim- Ca mobilization, but not degranulation, in Lyn-deficient bone marrow-derived ␣ mast cells. J. Immunol. 158:2350. ulate transcription from both the IL-2 and TNF- promoters in 13. Zhang, J., E. H. Berenstein, R. L. Evans, and R. P. Siraganian. 1996. Transfection mast cells (data not shown). As previously described (25), Btk- of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated deficient cells exhibited reduced JNK activation, accounting for degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells. J. Exp. Med. 184:71. the lower cytokine production in these cells (26). Both JNK acti- 14. Costello, P. S., M. Turner, A. E. Walters, C. N. Cunningham, P. H. Bauer, vation and IL-2 secretion were completely abrogated in Btk/Lyn- J. Downward, and V. L. J. Tybulewicz. 1996. Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells. Oncogene deficient cells. IL-2 production in mast cells is more strictly de- 13:2595. pendent on Btk than TNF-␣ both in the presence and absence of 15. Nishizuka, Y. 1984. The role of protein kinase C in cell surface signal transduc- Lyn. This phenomenon is consistent with the recent finding that tion and tumour promotion. Nature 308:693. by guest on September 25, 2021 16. Ozawa, K., Z. Szallasi, M. G. Kazanietz, P. M. Blumberg, H. Mischak, JNK not only regulates the transcriptional activation of the IL-2 J. F. Mushinski, and M. A. Beaven. 1993. Ca2ϩ-dependent and Ca2ϩ-indepen- gene but also controls the stability of IL-2 mRNA in T cells (76). dent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells: reconstituttion of secretory responses with Ca2ϩ and purified isozymes in washed permeabilized cells. J. Biol. Chem. 268:1749. 17. Tsukada, S., D. Saffran, D. J. Rawlings, O. Parolini, R. C. Allen, I. Klisak, Implications R. S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, et al. 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agamma- We demonstrate that Btk and Lyn have both redundant and op- globulinemia. Cell 72:279. posing functions in mast cell growth and Fc⑀RI-dependent signal- 18. Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, ing. Similar observations have been made in B and T lymphocytes L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow, C. I. E. Smith, and D. R. Bentley. 1993. The gene involved in X-linked agammaglobulinemia is a (36, 37, 41, 43). The present study extends these previous obser- member of the src family of protein-tyrosine kinases. Nature 361:226. vations in two important ways. First, the loss of function of Btk 19. Yamada, N., Y. Kawakami, H. Kimura, H. Fukamachi, G. Baier, A. Altman, and/or Lyn in B cells significantly alters the generation of B cell T. Kato, Y. Inagaki, and T. Kawakami. 1993. Structure and expression of novel protein tyrosine kinases, Emb and Emt, in hematopoietic cells. Biochem. Biophys. developmental subpopulations, making it difficult to directly com- Res. Commun. 192:231. pare BCR-dependent signals in cells derived from these animals. 20. Thomas, J. D., P. Sideras, C. I. E. Smith, I. Vorechovsky, V. Chapman, and In the current study, the functional interaction between Btk and W. E. Paul. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261:355. 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Med. 187:1235. sion of alternative Src and Tec family PTKs. Together, these find- 23. Takata, M., and T. Kurosaki. 1996. A role for Bruton’s tyrosine kinase in B cell ing strongly suggest that Btk and Lyn represent a critical combined antigen receptor-mediated activation of phospholipase C-␥2. J. Exp. Med. 184:31. target for pharmacological intervention in allergic diseases and 24. Fluckiger, A.-C., Z. Li, R. M. Kato, M. I. Wahl, H. D. Ochs, R. Longnecker, J.-P. related disorders involving mast cells. Kinet, O. N. Witte, A. M. Scharenberg, and D. J. Rawlings. 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2ϩ following B-cell receptor activation. EMBO J. 17:1973. 25. Kawakami, Y., T. Miura, R. Bissonnette, D. Hata, W. N. Khan, T. Kitamura, Acknowledgments M. Maeda-Yamamoto, S. E. Hartman, L. Yao, F. W. Alt, and T. Kawakami. 1997. 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