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Early Divergence of Fc Receptor I Signals for Receptor Up-Regulation and Internalization from Degranulation, Cytokine Production, and Survival¹

Jiro Kitaura^*, Wenbin Xiao^*, Mari Maeda-Yamamoto, Yuko Kawakami^*, Clifford A. Lowell and Toshiaki Kawakami^2,*

^* Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; National Institute of Vegetable and Tea Science, National Agriculture Research Organization, Kakegawa, Shizuoka Prefecture, Japan; and Department of Laboratory Medicine, University of California, San Francisco, CA 94143

	Abstract

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Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.

	Introduction

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Immunoglobulin E-dependent mast cell activation is a major triggering mechanism for acute and chronic allergic reactions and host defense against certain parasites (1). Activated mast cells release preformed proinflammatory chemical mediators (such as histamine and serotonin), proteases, and nucleotides, and release or secrete de novo synthesized lipids (such as leukotrienes and PGs) and polypeptides (such as cytokines and chemokines). These substances contribute to the development of allergy and other forms of inflammation.

The high affinity receptor (FcRI) on murine mast cells consists of four subunits: an IgE-binding subunit; a signal-amplifying, receptor-stabilizing subunit; and two disulfide-bonded subunits that are the main signal transducer (2). The aggregation of FcRI, traditionally induced by stimulation of IgE-sensitized mast cells with multivalent Ag (a stimulation mode referred to as IgE + Ag hereafter) or anti-IgE Ab (referred to as IgE + anti-IgE), leads to the activation of this system: subunit-associated Lyn, a Src family protein tyrosine kinase (PTK), ³ becomes activated, and phosphorylates tyrosine residues in the ITAMs in the cytoplasmic regions of and subunits. Phosphorylated and ITAMs recruit Lyn and Syk (another PTK with two tandem Src homology (SH) 2 domains N-terminal to the catalytic domain), respectively. Another Src family PTK, Fyn, was recently shown to associate with FcRI and to play a complementary role, particularly by activating PI3K (3). These PTKs phosphorylate numerous targets and activate several signaling pathways, including the PI3K, phospholipase C/Ca²⁺, and several MAPK pathways (4, 5). These signaling events lead to degranulation and cytokine production.

Previous studies including our own indicate that binding of monomeric IgE to FcRI enhances mast cell survival (6, 7) and surface expression of FcRI (8, 9, 10, 11). We also found heterogeneity among IgE molecules: highly cytokinergic (HC) IgEs can elicit not only the production and secretion of various cytokines, but also other activation events, including receptor internalization, degranulation, and cytokine production (12, 13, 14), whereas poorly cytokinergic (PC) IgEs can do so less efficiently (12). It is reasonable to assume that IgE stimulation may evoke signaling pathways identical or similar to those recruited by IgE + Ag or IgE + anti-IgE stimulation, as these three modes of mast cell stimulation induce the aggregation of FcRI. However, signaling pathways particularly for receptor up-regulation and internalization have been poorly characterized despite potential importance of these FcRI-mediated phenomena in allergy and other IgE-dependent diseases (5).

In the present study, we investigated how surface expression of FcRI is regulated by receptor-bound IgE in mast cells. Using the three different modes of FcRI stimulation, i.e., IgE, IgE + Ag, and IgE + anti-IgE, we also characterized the relationship among signaling pathways leading to differential receptor expression and other biologic outcomes of FcRI stimulation, namely, degranulation, IL-6 production, and survival. Genetic and pharmacological experiments indicate that signals diverge at the FcRI or membrane-proximal levels, leading to these individual events.

	Materials and Methods

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Abs and other reagents

IgE Abs used were described previously (12). Anti-IgE mAb B1E3 was kindly provided by D. Conrad, Virginia Commonwealth University (Richmond, VA). DNP₃-BSA and DNP₂₁-BSA were purchased from Biosearch Technologies (Novato, CA); DNP-lysine from Sigma-Aldrich (St. Louis, MO); and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), piceatannol, PD98059, SB203580, Gö-6976, protein kinase A inhibitor fragment, KT 5720, and Y-27632 from Calbiochem (San Diego, CA). ER-27319 (15) and Ro31-8425 were a gift from Eisai (Tokyo, Japan). Terreic acid was described previously (16).

Cell culture and FcRI stimulation

Bone marrow cells from wild-type and mutant mice were cultured in IL-3-containing medium for 4–6 wk to generate mast cells (bone marrow-derived mast cells (BMMC)) with >95% purity (c-Kit⁺ FcRI⁺ by flow cytometry). The following mutant mice were used: lyn^–/– (17), fyn^–/– (18), hck^–/– (19), syk^–/– (12, 20), btk^–/– (21), and lyn^–/–/fyn^–/– (22). For IgE + Ag or IgE + anti-IgE stimulation, BMMC were first sensitized by a 24-h incubation with 1 µg/ml H1 DNP--206 (abbreviated thereafter as 206) IgE. BMMC washed twice with buffer were stimulated with indicated concentrations of Ag (DNP₃-BSA or DNP₂₁-BSA) or anti-IgE mAb B1E3. Preliminary experiments showed that Ag stimulation of BMMC, which had been sensitized with 1 µg/ml, but not 0.1 or 10 µg/ml, 206 IgE, exhibited survival enhancement (data not shown).

Flow cytometry

To quantify live cells, BMMC were incubated with 1 µg/ml FITC-labeled annexin V (BD Clontech, Palo Alto, CA) and 2.5 µg/ml propidium iodide (BD Clontech) at room temperature for 20 min in the dark. Flow cytometric analysis of the stained cells was performed with FACSCalibur (BD Biosciences, San Jose, CA) equipped with CellQuest software. For measurement of cell surface expression of FcRI, BMMC were incubated first with 10 µg/ml 2.4G2 mAb (BD Pharmingen, San Diego, CA) at 4°C for 10 min, then with 5 µg/ml 206 IgE for 50 min, and finally with FITC-conjugated anti-mouse IgE (BD Pharmingen) for another 30 min before flow cytometric analysis. In some experiments (for example, those shown in Figs. 1B and 2D), receptor-bound IgE was measured in the same procedures, except that the step of incubation with 206 IgE was omitted. The initial velocity of receptor internalization is defined as the differential in FcRI expression in mean fluorescence intensity (MFI) at 0 and 6 min after FcRI stimulation divided by 6, (MFI^{0 min} – MFI^{6 min})/6.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Regulation of surface expression of FcRI by HC and PC IgEs. A, Naive wild-type BMMC were incubated with various concentrations of 206 or SPE-7 IgE in the presence or absence of 100 µM DNP-lysine for 48 h before flow cytometric measurement of surface FcRI expression. B, MFIs of IgE-bound FcRI are plotted as a function of incubation time.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Receptor internalization induced by HC IgE and IgE + anti-IgE depends on Lyn, but not Syk. A, Naive wild-type BMMC were preincubated with vehicle, 1 µM PP2, 1 µM PP3, 10 µM ER-27319 (ER), or 20 µM terreic acid (TA) for 20 min, and then incubated with PBS, 5 µg/ml 206 IgE, or 5 µg/ml SPE-7 IgE in the presence of vehicle or inhibitor for 48 h before measurement of FcRI. Ratios of MFI (MFI with inhibitor divided by MFI with vehicle) are plotted. B and C, Naive wild-type, Fyn-deficient, Lyn-deficient, and Syk-deficient BMMC were incubated with the indicated concentrations of SPE-7 IgE for 48 h. Wild-type controls were C57BL/6 mice for Fyn- and Lyn-deficient mice, and littermate heterozygous mice for Syk-deficient mice. D, Naive wild-type and Lyn-deficient BMMC were incubated with 10 µg/ml 206 or SPE-7 IgE with or without 100 µM pervanadate for 30 min.

Amounts of histamine in BMMC or in culture supernatants from BMMC that had been stimulated through FcRI were measured, as described (12). Supernatants of FcRI-stimulated BMMC were measured by ELISA for IL-6 and TNF- (BD Pharmingen).

	Results

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IgE-induced receptor up-regulation is not sensitive to monovalent hapten

We first examined effects of three FcRI stimulation modes on the surface expression of FcRI. IgE + Ag and IgE + anti-IgE induced decreases in receptor expression due to internalization (see below), but incubation of mast cells with increasing concentrations of a PC (206) IgE increased surface expression of FcRI (Fig. 1A), as described previously (8, 9). HC IgEs (such as SPE-7) also induced receptor up-regulation at low concentrations (0.05–0.5 µg/ml), whereas they induced receptor internalization at higher concentrations (5–50 µg/ml) (Fig. 1A). Monovalent hapten can disengage IgE + Ag-induced aggregation of FcRI (23). As both PC and HC IgEs appear to induce receptor aggregation (12), we examined whether DNP-lysine (a monovalent hapten) affects anti-DNP IgE-induced FcRI up-regulation and/or internalization. Strikingly, 206 IgE-induced receptor up-regulation was not affected (Fig. 1). By contrast, SPE-7 IgE-induced internalization at high IgE concentrations was prevented by DNP-lysine, and high concentrations of SPE-7 IgE, like PC IgEs, rather increased surface expression of FcRI (Fig. 1). These results suggest that IgE-dependent receptor up-regulation requires no or minimal receptor aggregation, unlike receptor internalization (Fig. 1A), cytokine production, or survival (12).

We next examined whether Src, Syk, and Tec family PTKs are involved in receptor up-regulation. Deficiencies in Lyn, Fyn, Hck, Syk, or Btk did not affect 206 IgE-induced enhancement in FcRI expression (data not shown). The 206 IgE-mediated receptor up-regulation was also not affected by PP2 (Src family PTK inhibitor), ER-27319 (Syk inhibitor), or terreic acid (Tec family PTK inhibitor) (Fig. 2A). These results demonstrate that IgE-dependent receptor up-regulation does not require extensive receptor aggregation or several PTKs. Given that several serine/threonine kinases are activated downstream of the activation of Src, Syk, and Tec family PTKs in IgE + Ag-stimulated mast cells, we tested effects of various inhibitors of serine/threonine kinases on 206 IgE-induced receptor up-regulation (Table I). Consistent with the lack of the role of the PTKs in this phenomenon, inhibitors of MEK, p38, protein kinase C (PKC), protein kinase A (PKA), or Rho kinase did not affect it. Therefore, IgE-dependent receptor up-regulation is a phenomenon very different from other FcRI-induced activation events, such as receptor internalization, degranulation, cytokine production, and survival.

FcRI internalization induced by HC IgE or IgE + anti-IgE is dependent on Lyn, but not Syk

We next examined whether PTKs of Src, Syk, and Tec families are involved in receptor internalization. First, we tested effects of pharmacological inhibitors on HC IgE-induced receptor internalization. Interestingly, the pretreatment with 1 µM PP2 significantly inhibited receptor internalization, while PP3, an inactive relative of PP2, did not (Fig. 2A). By contrast, either ER-27319 or terreic acid did not affect receptor internalization. The same pattern of sensitivity to these inhibitors was observed on IgE + anti-IgE-induced receptor internalization (data not shown).

To further investigate roles of individual PTKs, we used BMMC derived from Lyn-, Hck-, Fyn-, Syk-, and Btk-deficient mice. As shown for wild-type cells incubated with SPE-7 IgE (Fig. 1A), a similar mixed pattern of receptor up-regulation predominant at low IgE concentrations and receptor internalization predominant at high concentrations was seen with Fyn-, Hck-, Syk-, and Btk-deficient cells (Fig. 2, B and C, and data not shown). By contrast, Lyn-deficient cells behaved very differently: receptor expression at low concentrations (0.05–0.5 µg/ml) of SPE-7 IgE was similar to, or slightly higher than, that in wild-type cells, but it was remarkably higher at high concentrations (5–50 µg/ml) than in wild-type cells, indicating that HC IgE-induced receptor internalization is defective in Lyn-deficient cells (Fig. 2B). Receptor internalization induced by IgE + anti-IgE was also dependent on Lyn, but not the other PTKs tested above (Fig. 3A, and data not shown). However, IgE + Ag-induced internalization was less dependent on Lyn (Fig. 3, B and C). Higher concentrations of DNP₃-BSA or DNP₂₁-BSA (low and high valency Ag, respectively) were required to reach the same levels of receptor internalization in Lyn-deficient cells than in wild-type cells, and the lowest levels of receptor expression attained by the highest tested concentrations of Ag were substantially higher in Lyn-deficient cells compared with wild-type cells (Fig. 3, B and C). Fyn-deficient cells exhibited a less severe defect in IgE + Ag-induced receptor internalization (Fig. 3, B and C). Fyn/Lyn doubly-deficient cells exhibited greater defects than Lyn-deficient cells (data not shown). By contrast, Hck or Btk deficiency did not affect IgE + Ag-induced receptor internalization (data not shown). In contrast, Syk-deficient cells exhibited slightly higher sensitivity to Ag in IgE + Ag-induced receptor internalization (Fig. 4).

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Receptor internalization induced by IgE + anti-IgE or IgE + Ag in wild-type and mutant mast cells. Wild-type, Fyn- and Lyn-deficient BMMC sensitized with 206 IgE were incubated with various concentrations of anti-IgE mAb B1E3 (A), DNP3-BSA (B), or DNP21-BSA (C) for the indicated periods before flow cytometric measurement of surface expression of FcRI.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Receptor internalization induced by IgE + Ag in Syk-deficient mast cells. Wild-type and Syk-deficient BMMC sensitized with 206 IgE were incubated with various concentrations of Ag, DNP21-BSA, for 48 h before flow cytometric measurement of surface expression of FcRI.

These pharmacological and genetic data collectively indicate that Lyn and Fyn to a lesser extent, but not Hck, Syk, or Btk, are required for HC IgE- and IgE + anti-IgE-induced receptor internalization, and IgE + Ag-induced receptor internalization was weakly affected by Lyn and Fyn deficiencies.

Relation between signal strength and kinetics of receptor internalization

Fig. 3 shows the time courses of receptor internalization induced by various concentrations of anti-IgE mAb B1E3, DNP₃-BSA, or DNP₂₁-BSA in 206 IgE-sensitized mast cells. Higher concentrations of Ag or anti-IgE induced more rapid internalization (Fig. 3), and there was a nice proportional relationship between Ag concentrations (up to 100 ng/ml) in a logarithmic scale and initial velocities of internalization (Fig. 5A). These results suggest that the initial velocity of receptor internalization might serve as a surrogate indicator of signal strength when signal strengths are compared between IgE + Ag and IgE + anti-IgE (see below).

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Initial velocities of receptor internalization can serve as a surrogate indicator of stimulus strength of IgE + Ag and IgE + anti-IgE. A, A proportional relation between Ag concentrations in a logarithmic scale and initial velocities of receptor internalization induced by IgE + Ag. BMMC sensitized with 206 IgE were incubated with various concentrations of Ag, DNP3-BSA, or DNP21-BSA, for 48 h before flow cytometric measurement of surface expression of FcRI. MFIs are plotted as a function of Ag concentration. B, Survival effects of IgE + Ag and IgE + anti-IgE (see Figs. 7 and 8) are plotted as a function of initial velocities of receptor internalization.

Strengths of FcRI stimulation required for receptor internalization, degranulation, cytokine production, and survival

Initial velocity of HC IgE-induced internalization, as estimated from FcRI expression in the presence or absence of DNP-lysine (Fig. 1B), was very low (3 MFI/min) at 10 µg/ml SPE-7 IgE (Fig. 6). PC IgEs did not induce appreciable receptor internalization. Therefore, weak signals that induce no or slow internalization seem capable of inducing survival effects because both PC and HC IgEs induce potent survival effects (12). We next examined whether survival effects can be exerted by IgE + Ag or IgE + anti-IgE. Incubation of 206 IgE-sensitized mast cells with low concentrations (0.01–1 ng/ml) of DNP₃-BSA failed to affect mast cell survival in the absence of growth factors. But, higher concentrations (10–1000 ng/ml) of DNP₃-BSA significantly enhanced survival (Fig. 7A). A similar pattern of survival enhancement was induced by DNP₂₁-BSA, except that survival effects were seen at 1–10 ng/ml (Fig. 7B). In contrast, stimulation with anti-IgE mAb B1E3 enhanced survival at all tested concentrations of 0.02–20 µg/ml (Fig. 7C). When the survival data were plotted as a function of initial velocity of receptor internalization (Fig. 5B), the lowest initial velocity for IgE + Ag- and IgE + anti-IgE-induced survival enhancement was determined to be 19 MFI/min. The highest initial velocities for survival effects were estimated to be 152–180 MFI/min. These results indicate that survival effects can be seen only with weak (only slightly above the thresholds for receptor internalization) to moderate signal strengths of IgE + Ag or IgE + anti-IgE (Fig. 5).

View larger version (28K): [in this window] [in a new window]   FIGURE 6. HC IgE-induced receptor internalization as estimated from FcRI levels in the presence or absence of monovalent hapten. Naive wild-type BMMC were incubated with various concentrations of 206 or SPE-7 IgE in the presence or absence of 100 µM DNP-lysine for 48 h before flow cytometric measurement of surface FcRI expression (Fig. 1B). Internalization was estimated by subtracting MFIs of FcRI expression in the absence of DNP-lysine from those in the presence of DNP-lysine and plotted as a function of incubation time.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. IgE + Ag and IgE + anti-IgE induce survival enhancement and histamine release with unique dose-response relationships. BMMC were sensitized by incubation with 1 µg/ml anti-DNP IgE 206 for 24 h. After washed, IgE-sensitized BMMC were incubated with the indicated concentrations of DNP3-BSA (A), DNP21-BSA (B), or anti-IgE mAb B1E3 (C) in the absence of growth factors for 60 h (for survival assays) or 45 min (for histamine measurements). Cell survival is plotted with (see the left for scale), and histamine amounts detected in culture supernatants are plotted with (see the right for scale).

View larger version (29K): [in this window] [in a new window]   FIGURE 8. IgE + Ag and IgE + anti-IgE induce survival enhancement and IL-6 production and secretion with unique dose-response relationships. The 206 IgE-sensitized cells were incubated with the indicated concentrations of DNP3-BSA (A), DNP21-BSA (B), or anti-IgE mAb B1E3 (C) in the absence of growth factors for 60 h (for survival assays) or 20 h (for IL-6 measurements). Cell survival is plotted with (see the left for scale), and IL-6 amounts detected in culture supernatants were plotted with (see the right for scale).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

FcRI levels modulate the sensitivity and intensity of effector responses. Mast cells that have undergone IgE-induced enhancement of FcRI surface expression exhibit increased sensitivity to Ag and increased degranulation and cytokine production in response to Ag challenge (9, 26, 27). Therefore, it is important to better understand how receptor levels are regulated. IgE-induced receptor up-regulation is a unique phenomenon: unlike other activation events, such as receptor internalization, degranulation, cytokine production, and survival enhancement, receptor up-regulation cannot be inhibited by monovalent hapten and does not require FcRI-associated PTKs such as Lyn and Fyn, other PTKs such as Syk and Btk, or several serine/threonine kinases. Previous studies demonstrated that IgE-induced receptor up-regulation can be reconstituted in FcRI expressed in heterologous cells, and that hemopoietic-specific factors, including the ITAM of FcRI subunit, are not required for receptor up-regulation, but the subunit is required (28, 29, 30). Our results are consistent with these studies and the proposed mechanism for IgE-induced receptor up-regulation mainly by stabilization of the receptor at the cell surface, which prevents receptor internalization and degradation (11, 28, 29).

There is no universal indicator of the strength of FcRI stimulation to compare those induced by different stimulation modes, i.e., monomeric IgE, IgE + Ag, and IgE + anti-IgE. However, initial velocity of receptor internalization could serve such a function when signal strengths are compared between IgE + Ag and IgE + anti-IgE. Weak to moderate receptor-internalizing stimuli could induce survival enhancement, and degranulation and IL-6 production were induced by stronger stimuli. However, the initial velocity of receptor internalization cannot be used for monomeric IgE stimulation, because both PC and HC IgEs, which can respectively induce no or very slow receptor internalization, can induce survival enhancement. These results together with differential signaling requirements for receptor internalization among the stimulation modes suggest that different stimulation modes trigger not only quantitatively, but also qualitatively different signals.

FcRI internalization induced by HC IgE or IgE + anti-IgE is dependent on Lyn, but not Syk (Figs. 2 and 3), similar to Ag receptor internalization in B cells (31). Our previous study showed that Syk is absolutely required for mast cell survival induced by both HC and PC IgEs, while Lyn is required for survival induced by PC IgEs or weak HC IgEs, but it is dispensable for strong HC IgE (SPE-7)-induced survival (12). Syk is also absolutely required for IgE + Ag-induced degranulation and cytokine production (24, 25). In a widely supported model of IgE + Ag-induced mast cell activation, Syk is activated by its recruitment to the tyrosine-phosphorylated ITAM of the FcRI subunit and by phosphorylation by Lyn or already activated Syk, and activated Syk phosphorylates numerous substrates along multiple signaling pathways, leading to degranulation, cytokine production, and other activation outcomes (4). Assuming that signaling events induced by IgE + Ag are identical or similar to those by HC IgE and IgE + anti-IgE, our results that HC IgE- and IgE + anti-IgE-induced FcRI internalization requires Lyn, but not Syk, would imply that Lyn functions in other pathways beyond the linear view of ITAM phosphorylation-mediated signaling. Therefore, signal requirements for HC IgE- and IgE + anti-IgE-induced receptor internalization are very different from similarly induced degranulation, cytokine production, and survival.

A minor role for Fyn in receptor internalization may be worthy of a note. Unlike IgE + anti-IgE stimulation, IgE + Ag-induced receptor internalization is less dependent on Lyn (Fig. 3), suggesting a complementary function by Fyn or other Src family PTK(s). In addition, fyn ^–/– cells exhibited minor defects in IgE + Ag-induced receptor internalization, particularly at low Ag concentrations. These results are consistent with recent reports by Rivera and colleagues (3, 32) that Fyn is preferentially activated by weak stimulation and plays a role complementary to Lyn.

Our observations on differential requirements for Lyn and Syk in receptor internalization vs degranulation, cytokine production, and survival enhancement are important in considering signaling molecules as therapeutic targets for the treatment of allergic diseases; such targeting might not inhibit an entire array of activation events, leaving some events intact, even if a target is a molecule located close to receptor-proximal PTKs in signal transduction. In contrast, our data support the notion that targeting IgE or FcRI is advantageous, as evidenced by a recent success of anti-IgE mAb in the treatment of asthma and food allergy (33, 34). This study also corroborates the need to further define signaling pathways for individual biologic outcomes.

	Acknowledgments

 
We are grateful to Dr. Daniel H. Conrad for his kind gift of mAb E1B3, and members of the Kawakami laboratory for providing BMMC used in this study. This is Publication 635 from the La Jolla Institute for Allergy and Immunology.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by National Institutes of Health Grants AI50209 and AI/GM38348 (to T.K.).

² Address correspondence and reprint requests to Dr. Toshiaki Kawakami, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: toshi{at}liai.org

³ Abbreviations used in this paper: PTK, protein tyrosine kinase; BMMC, bone marrow-derived mast cell; HC, highly cytokinergic; MFI, mean fluorescence intensity; PC, poorly cytokinergic; PKA, protein kinase A; PKC, protein kinase C; SH, Src homology; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.

Received for publication April 22, 2004. Accepted for publication July 26, 2004.

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