The Journal of Immunology
Stat5 Expression Is Required for IgE-Mediated Mast Cell Function1 Brian O. Barnstein,* Geqiang Li,† Zhengqi Wang,† Sarah Kennedy,* Charles Chalfant,‡ Hiroshi Nakajima,§ Kevin D. Bunting,† and John J. Ryan2* The mast cell (MC) inflammatory response is now linked not only to atopy, but also to arthritis, multiple sclerosis, heart disease, and resistance to bacterial infection. In the current study, we demonstrate that the signal transducer and activator of transcription 5 (Stat5) is rapidly activated by IgE cross-linkage, and that its expression is critical to the MC response. Stat5-deficient (Stat5KO) MC demonstrated a significant decrease in IgE-mediated degranulation, leukotriene B4 production, cytokine secretion, and survival signals. The defect in cytokine production may be caused by decreased cytokine mRNA stability. Stat5KO MC-induced cytokine mRNAs normally following IgE cross-linkage, but these mRNAs were not sustained over time and were degraded at twice the rate observed in WT cells. Interestingly, the RNA destabilizing protein tristetraprolin was induced following IgE cross-linkage in Stat5KO but not wild-type cells. Moreover, reducing tristetraprolin expression via short hairpin RNA transfection significantly increased IL-13 production in Stat5KO MC. Our work demonstrates that Stat5 is a critical factor in IgE-induced MC activation, acting in part via posttranscriptional control of cytokine mRNA stability. These data have a direct impact on MC-associated inflammatory and autoimmune diseases. The Journal of Immunology, 2006, 177: 3421–3426.
M
ast cells (MC)3 are present in nearly all tissues. When activated, they direct inflammatory reactions through a biphasic response that controls vascular, respiratory, and immune cell function. The immediate phase of MC activation is characterized by the secretion of vasoactive and inflammatory substances such as histamine, heparin, proteases, prostaglandins, and leukotrienes (LT). This is followed by the late phase of MC activation, marked by synthesis and secretion of cytokines that elicit sustained inflammation (1, 2). This response has been best studied in atopic diseases such as asthma (1). However, MC are now implicated in the inflammatory responses associated with rheumatoid arthritis (3), multiple sclerosis (4), heart disease (5), and resistance to bacterial infection (6 –9). We recently showed that the transcription factor Stat5 is essential for MC survival and proliferation in vitro and in vivo (10). Stat5-deficient (Stat5KO) mice, although born with normal MC numbers, are completely MC deficient by 12 wk of age, due to lack of proper survival signals (Ref. 10 and C. Shelburne and J. Ryan, unpublished observations). Stat5KO MC can be derived in vitro,
where they develop and expand normally if maintained in IL-3 plus stem cell factor (SCF), conditions that do not appear to exist in vivo. Thus, Stat5 expression is not required for normal MC development but is necessary to support subsequent survival and proliferation. Given the critical role for Stat5 in survival, we questioned whether this transcription factor also might play a role in MC function. IgE-mediated signaling is a clinically relevant, well-studied model of MC activation. We determined the importance of Stat5 to the immediate and late phases of the IgE-induced MC response. These experiments revealed an essential role for Stat5 in IgE-mediated activation that impacts our understanding of MCmediated inflammatory responses, possibly revealing a new clinical target for MC-associated diseases.
Materials and Methods Cell culture
*Department of Biology, Virginia Commonwealth University, Richmond, VA 23284; † Department of Medicine, Division of Hematology-Oncology, Case Western Reserve University, Cleveland, OH 44106; ‡Department of Biochemistry, Virginia Commonwealth University, Richmond, VA 23298; and §Department of Molecular Genetics, Graduate School of Medicine, Chiba University, Chiba, Japan
Bone marrow (BM) cells were cultured in complete RPMI (cRPMI) 1640 medium (Invitrogen Life Technologies) (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate, and 1 MM HEPES; Biofluids), supplemented with IL-3 (5 ng/ml) and SCF (50 ng/ml). BM cells were harvested from the femurs and tibias of C57BL/6, BALB/c, Stat5A/B-deficient, and IL-3KO (provided by C. Lantz, Harrisonburg, VA) mice as described (10). Bcl-2 transgenic (Bcl-2Tg) ⫻ Stat5KO mice were created by breeding H2K-Bcl-2Tg mice (a gift from J. Domen, Duke University, Durham, NC) to Stat5KO mice.
Received for publication December 9, 2005. Accepted for publication June 9, 2006.
Reagents
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.
Murine SCF and IL-3 were purchased from PeproTech and R&D Systems. IgE (clone C38-2), anti-CD16/CD32 (clone 2.4G2), PE-labeled anti-kit, anti-ERK, and phosphotyrosine-specific anti-ERK were purchased from BD Biosciences. PE-labeled rat IgG isotope control was purchased from eBioscience. FITC-labeled rat IgG isotype control and FITC-labeled rat anti-mouse IgE were purchased from Southern Biotechnology Associates. DNP-human serum albumin was purchased from Sigma-Aldrich. Phosphotyrosine-specific anti-Stat5 was purchased from Cell Signaling Technology. Anti-actin was purchased from Sigma-Aldrich.
1 This work was supported in part National Institutes of Health Grants 1R01AI43433 and 1R01CA91839 (to the Ryan laboratory) R01DK059380 (to the Bunting laboratory). 2 Address correspondence and reprint requests to Dr. John J. Ryan, Biology Department, Virginia Commonwealth University, Box 842012, Richmond, VA 23284-2012. E-mail address:
[email protected] 3 Abbreviations used in this paper: MC, mast cell; BMMC, bone marrow-derived MC; SCF, stem cell factor; Stat5, signal transducer and activator of transcription 5; Stat5KO, Stat5 deficient; shRNA, short hairpin RNA; TTP, tristetraprolin; WT, wild type; BFA, brefeldin A; LT, leukotriene; Bcl-2Tg, Bcl-2 transgenic; ActD, actinomycin D; cRPMI, complete RPMI.
Copyright © 2006 by The American Association of Immunologists, Inc.
Western blotting Western blotting was performed essentially as described previously (9) using 50 g of total cell lysate per sample. 0022-1767/06/$02.00
3422 Histamine release assay Histamine release was measured using an ELISA kit (Neogen). BMMC were sensitized to IgE overnight in medium with IL-3 plus SCF, washed, and replated in Tyrode’s buffer with 1 mM MgCl2 and 2.5 mM CaCl2, without cytokines. Cells were then activated for 30 min with Ag at 37°C. Percent histamine release was calculated by dividing histamine reactivity in supernatant by reactivity in cell pellet.
LTB4 assay
Stat5 IS REQUIRED FOR IgE-MEDIATED MC FUNCTION abilized and stained with PE-anti-TNF-␣ or PE-IgG in a solution of PBS containing 0.1% BSA and 0.5% saponin.
Analysis of monomeric IgE effects Monomeric IgE was prepared by centrifuging IgE at 60,000 ⫻ g for 60 min. Monomeric IgE was added to BMMC cultures for 3 days at 0.1 or 1 g/ml, which yielded similar results. FcRI up-regulation was measured by flow cytometry. Cell survival was measured by propidium iodide staining.
Statistics
Cells were incubated with IgE (10 g/ml) for 45 min at 4°C in cRPMI, washed, resuspended in cRPMI, then activated with DNP-human serum albumin (50 ng/ml) for 1 h in cRPMI without cytokines. LTB4 was measured in culture supernatants using an ELISA kit (R&D Systems) as described by the manufacturer.
Results are the mean and SD for experiments with three to four samples. SE measurements are shown for data sets where ⬎4 measurements were made. Values of p ⬍ 0.05, as determined by Student’s t test, were considered to be significant.
ELISA
Results
Cytokine production was measured as described previously, (11). Briefly, BMMC were incubated with IgE (10 g/ml) for 45 min at 4°C in cRPMI, washed, and resuspended in cRPMI supplemented with IL-3 plus SCF, then activated with DNP-human serum albumin (5–50 ng/ml) for 16 –24 h. Cytokines were measured using OptEIA ELISA kits as described by the manufacturer (BD Biosciences).
FcRI signaling activates Stat5
Retroviral transduction
To investigate the possibility that Stat5 may function in IgE-mediated MC activation, we first examined Stat5 activation following cross-linkage with IgE plus Ag. As shown in Fig. 1, Stat5 is rapidly tyrosine phosphorylated by the addition of Ag to IgE-sensitized MC, with maximal activation after 5 min of stimulation. This activity was quite transient, as tyrosine phosphorylated Stat5 was
Stat5A expression was achieved using a murine stem cell virus-based bicistronic retroviral vector expressing Stat5A and coexpressing the GFP. This vector, termed pMSCV/Stat5A-IRES-GFP, has been described previously (10). Because Stat5A expression conveys a growth advantage, nearly 100% of the transfected cells were GFP⫹ at the time of use and expressed Stat5A at levels comparable with that of wild-type (WT) cells.
RNase protection assay RNase protection assay was performed using the Riboquant system (BD Biosciences) as directed by the manufacturer. The ratio of gene of interest expression to housekeeping gene (L32 plus GAPDH) expression in the same sample was determined using a Typhoon phosphorimager (Molecular Dynamics). These normalized values were used to calculate the percentage change in gene expression.
Short hairpin RNA (shRNA) expression To inhibit tristetrapolin (TTP) expression, Stat5KO BMMC were transfected with the TTP-A shRNA expression vector, encoding the sequence 5⬘act cgg act cca tcc cgt ct corresponding to nucleotides 160 –179 of the murine TTP mRNA. Control cells were transfected with the pSuppressorNeo vector (Imgenex) alone. Plasmid was introduced into BMMC through use of the Nucleoporator, using Solution V, program T20 (Amaxa). Cells were selected in medium supplemented with 500 g/ml G418 for 10 days before use.
Flow cytometric analysis of IgER expression IgER expression was determined by flow cytometry as described previously (11).
Flow cytometric analysis of TNF production Cells were stimulated with IgE plus anti-IgE for 5 h in the presence of monensin (2 M), fixed in 4% paraformaldehyde for 20 min, then perme-
FIGURE 1. FcRI cross-linkage activates Stat5. BMMC were incubated in medium without cytokines for 3 h, then activated with IgE plus Ag (50 ng/ml) for the indicated times in the presence or absence of BFA (10 g/ml). Total cell lysates were subjected to Western blotting with phosphotyrosine-specific anti-Stat5. The membrane was stripped and reprobed with anti-Stat5 and anti-actin to demonstrate protein loading. Data shown are representative of five experiments.
FIGURE 2. Stat5 deficiency does not alter ERK activation, but inhibits IgE-mediated degranulation and LT secretion. A, BMMC were in medium without cytokines for 3 h, then activated by IgE plus Ag (50 ng/ml) for 15 min. Total cell lysates were subjected to Western blotting with Abs specific for phosphorylated ERK proteins. The membrane was stripped and reprobed with anti-ERK to show protein loading. Data shown are representative of three independent experiments. B, BMMC were cross-linked with IgE plus Ag (50 ng/ml) for 30 min, and histamine release was measured by ELISA as described in Materials and Methods. Data are means and SD of three samples from one of three representative experiments. ⴱ, p ⫽ 0.035, when comparing activated WT with Stat5KO cells using Student’s t test. C, BMMC were cross-linked with IgE plus Ag (50 ng/ml) for 60 min as described in Materials and Methods. LTB4 secretion was measured in culture supernatants by ELISA. Data are means and SD of four samples from one of three representative experiments. ⴱ, p ⬍ 0.001, when comparing activated WT with Stat5KO cells using Student’s t test.
The Journal of Immunology not detectable after 30 min of IgE signaling. Addition of the vesicular transport inhibitor brefeldin A (BFA), which blocks MC cytokine secretion (12), did not affect Stat5 phosphorylation (Fig. 1). We also found that Stat5 was phosphorylated after IgE crosslinkage in IL-3KO BMMC, indicating that IgE-mediated IL-3 secretion is not required for Stat5 activation (data not shown). Hence, Stat5 activation may occur directly through FcRI signaling rather than secondary to cytokine production. Stat5 deficiency does not affect IgE-mediated ERK activation, but reduces degranulation and LT production To determine the importance of Stat5 in IgE-mediated signaling, we compared responses in WT and Stat5KO BMMC. Importantly, we have shown previously that BMMC derived from Stat5KO mice develop normally and express FcRI at levels comparable to that of WT BMMC (10). In keeping with the normal expression of IgER, some early events in FcRI signaling appeared to be intact in Stat5KO MC. For example, the absence of Stat5 did not affect IgE-mediated MC ERK phosphorylation (Fig. 2A). In contrast, degranulation and secretion of the arachidonic acid metabolite LTB4 during the first hour after IgE cross-linkage was reduced ⬃50% in Stat5KO BMMC (Fig. 2, B and C). Thus Stat5 has distinct and important roles in the early phase of the MC response.
FIGURE 3. Stat5-deficient MC have decreased cytokine production. A, WT and Stat5KO BMMC were cultured in IL-3 plus SCF and activated with IgE plus Ag for 18 h. Culture supernatants were measured for the presence of the indicated cytokines by ELISA. Data are means and SD of three separate BMMC populations. Data shown are from one of eight experiments that gave similar results. B, Bcl-2Tg and Bcl-2Tg/Stat5 BMMC were cultured in medium lacking growth factors, and stimulated with IgE plus Ag as described in A. C, WT and Stat5KO BMMC cultured in IL-3 plus SCF were activated with IgE plus Ag for 5 h, after which the presence of TNF-␣ was detected by intracellular staining as described in Materials and Methods. Data shown are representative of 6 experiments. D, Stat5KO BMMC transduced to express Stat5A were assessed for GFP expression driven by the vector IRES sequence, and for Stat5 expression by Western blotting. E, WT, Stat5KO, and Stat5KO BMMC transfected with Stat5A expression vector were activated by IgE cross-linkage as in A. Net IL-13 secretion was measured by ELISA. Data shown are the means and SEM of six samples from one experiment. ⴱ, p ⬍ 0.02, compared with WT or Stat5Aexpressing BMMC to Stat5KO cells by Student’s t test.
3423 Stat5 deficiency prevents IgE-mediated cytokine production The late phase of MC activation is denoted by the production of cytokines that promote inflammation (1, 2). We determined the importance of Stat5 expression in IgE-mediated cytokine secretion by activating WT and Stat5KO BMMC with IgE plus Ag. Culture supernatants were tested for the presence of IL-6, IL-13, and TNF-␣ by ELISA. As expected, WT BMMC secreted significant amounts of TNF-␣, IL-6, and IL-13 (Fig. 3A). Stat5KO BMMC were completely defective in secreting these cytokines, producing at most 5–10% of WT levels. To rule out a contribution of SCF or IL-3 in this process, BMMC were activated with IgE plus Ag for 18 h in the absence of growth factors. Because Stat5KO BMMC die rapidly after growth factor withdrawal, we used BMMC prepared from Stat5KO mice crossed to H2K-Bcl-2Tg mice in this experiment. These cells survived normally for 24 h in medium without growth factors (⬎95% viable by trypan blue exclusion; data not shown). As shown in Fig. 3B, Bcl-2Tg BMMC activated in medium without IL-3 and SCF produced TNF-␣, whereas Bcl2/Stat5KO Tg did not. Hence, the cytokine production defect in Stat5KO BMMC is consistent in the absence of IL-3 or SCF signaling. The reduction in cytokine production also was apparent when activated cells were stained for TNF-␣ and analyzed by flow cytometry (Fig. 3C). The less dramatic difference in cytokine
3424
Stat5 IS REQUIRED FOR IgE-MEDIATED MC FUNCTION
expression observed by intracellular staining may indicate that Stat5 exerts its influence by controlling the degree of protein synthesis and secretion rather than completely inhibiting cytokine gene induction. We have shown previously that Stat5A transduction rescues the survival defects of Stat5KO MC, indicating that the loss of Stat5, and not an altered development in vitro, explains the phenotype of these cells (10). To confirm the requirement for Stat5 in cytokine production, Stat5KO BMMC were transduced with retrovirus expressing Stat5A. Nearly 100% of these cells expressed the vector, as judged by expression of GFP from the IRES sequence. Transduction restored Stat5 production to levels comparable to that of WT cells (Fig. 3D). Importantly, Stat5A expression restored IgEinduced IL-13 secretion in Stat5KO MC (Fig. 3E). These results argue strongly that Stat5 expression is a critical aspect of IgEinduced cytokine production. Decreased cytokine mRNA stability in Stat5KO BMMC Cytokines are controlled by both mRNA induction and stability (13–15). To determine the effect of Stat5 deletion on IgE-mediated cytokine mRNAs, we measured the induction and maintenance of IL-13, a cytokine with critical inflammatory functions (16). IL-13 showed significant up-regulation that was matched by protein secretion. As shown in Fig. 4A, IgE cross-linkage greatly up-regulated IL-13 mRNA in both WT and Stat5KO BMMC. These mRNAs were nearly absent within 90 min of signaling in Stat5KO BMMC but were still overtly expressed at this time point in WT cells. A similar pattern of regulation was observed with IL-6 and TNF-␣ (data not shown). To determine whether these differences were related to mRNA stability, BMMC were activated for 30 min with IgE plus Ag, after which the transcriptional inhibitor actinomycin D (ActD) was added. RNA was then harvested during the 60 min following ActD addition to determine the rate of mRNA degradation. As shown in Fig. 4, B and C, the rate of IL-13 mRNA degradation in Stat5KO BMMC was twice that observed with WT MC. This was in contrast to c-kit mRNA, which showed a similarly slow rate of degradation in both WT and Stat5KO BMMC. Stat5 deficiency alters TTP expression The observed instability of cytokine mRNAs in Stat5KO BMMC suggests that Stat5 may control the expression of proteins that alter mRNA half-life, rather than directly eliciting cytokine transcription. One candidate is the mRNA destabilizing protein TTP, which is induced by Stat6 (17). We measured TTP protein levels during IgE cross-linkage in WT and Stat5KO BMMC and noted a striking increase in TTP expression that occurred only in Stat5KO cells (Fig. 5A). These data suggest that Stat5 may normally function to inhibit TTP expression in WT cells. To determine whether TTP expression had a causal relationship to cytokine production, Stat5KO BMMC were transfected with pSuppressorNeo plasmid expressing TTP shRNA, which has previously been shown to repress TTP expression (17). As shown in Fig. 5B, TTP shRNA expression significantly repressed TTP expression following IgE cross-linkage, when compared with Stat5KO cells transfected with vector alone. Moreover, TTP shRNA expression led to a significant increase in IL-13 production (Fig. 5C), arguing that TTP has a role in the cytokine defect noted in Stat5KO MC. Stat5 deficiency alters the effects of monomeric IgE Separate from Ag-mediated effects, IgE has been reported to upregulate FcRI surface expression and to induce MC survival (18). Although the increase in IgE receptor expression is thought to occur via protein stabilization, IgE-mediated survival may be
FIGURE 4. Decreased cytokine mRNA stability in Stat5KO MC. A, WT and Stat5KO BMMC were activated with IgE plus Ag for the indicated times, and the presence of IL-13 mRNA was measured by RNase protection assay. Data shown are representative of 12 experiments that showed similar results. In B, cells were activated for 30 min, after which actinomycin D (ActD, 20 g/ml) was added and cells were further incubated for the indicated times. Each set of WT or Stat5KO samples were analyzed on the same gel. Samples shown are representative of four sets of WT and Stat5KO samples. C, The data from B were analyzed by phosphorimaging to determine the percentage decrease in IL-13 mRNA expression after ActD addition, using the 30-min stimulation point as the baseline for comparisons and taking into account RNA loading, as judged by expression of L32 and GAPDH. The graph shows the mean decrease in IL-13 and c-kit mRNA expression (⫾SD) from four independent samples. ⴱ, p ⬍ 0.002, compared with WT and Stat5KO samples, as determined by Student’s t test.
driven by IL-3 secretion (19). We found that monomeric IgE profoundly up-regulated FcRI expression on both WT and Stat5KO BMMC, but only enhanced the survival of WT MC (Fig. 6). Thus, Stat5 is critical to the cytokine-related effects of IgE, both those elicited by Ag, and those that occur independent of IgE–Ag complexes.
Discussion MC-mediated immune responses, once mainly the interest of allergists, are now broadly implicated in many inflammatory diseases. Data from the human system are still largely circumstantial; however, animal models show a clear and critical role for these granulated tissue cells in models of multiple sclerosis, rheumatoid arthritis, coronary artery disease, and host resistance to bacterial infection (3–9). Thus, understanding MC activation has taken on a
The Journal of Immunology
FIGURE 5. Loss of cytokine production in Stat5KO BMMC is associated with TTP induction. A, BMMC were activated by IgE plus Ag (50 ng/ml) for the indicated times, and TTP expression was measured in total cell lysates by Western blotting. Membrane was stripped and reprobed for actin expression. B, Stat5KO BMMC were transfected with pSuppresorNeo vector encoding TTP shRNA or vector alone (control). Neomycinresistant cells were activated with IgE plus Ag for the indicated times and probed for TTP expression as in A. C, Cells from B were activated for 16 h, and IL-13 secretion in culture supernatants was measured by ELISA. Data shown are means and SD from four separate samples harvested in one experiment.
new degree of importance, especially given the range of MC-targeted therapies already in use for atopic diseases that could be expanded to other settings. It is in this arena that we find Stat5 to be a central factor and potential target for clinical intervention. The MC response can be simplistically separated into early and late events, largely demarcated by degranulation and lipid mediator release in the first minutes after activation and cytokine secretion over the following 3– 6 h. These phases collectively work to alter vascular flow and recruit leukocytes (1, 2). We find that Stat5 deletion inhibits histamine release and LT production, markers of the early phase, and essentially ablates late-phase cytokine secretion. Given this regulation of the inflammatory response and its obligatory role in MC survival (10), the utility of targeting Stat5 in MC diseases warrants exploration. Because our data showed a complete loss of cytokine production in Stat5KO MC, we chose to pursue this issue in more detail. However, the 50% decline in histamine release and LTB4 secretion are clinically important, because these are key mediators of bronchoconstriction and vasodilation. More work is needed to understand how Stat5 contributes to the immediate phase of MC activation. For example, it is striking that MAPK, a pathway that appears to be intact in Stat5KO BMMC, has been implicated in activating the arachidonic acid cascade (20 –24), yet LTB4 production remains low in Stat5KO MC. Our current study is aimed at understanding how Stat5 controls this response, focusing specifically on expression and function of phospholipase A2.
3425
FIGURE 6. Stat5 deficiency alters the effects of monomeric IgE. A, WT and Stat5KO BMMC were cultured in IL-3 plus SCF with or without monomeric IgE for 3 days, and FcRI expression was determined as described in Materials and Methods. B, Cells were cultured for 72 h as in A, with the exception that IL-3 plus SCF was added only where indicated. Cell viability was determined by propidium iodide exclusion and flow cytometry analysis. Data are means and SD of three samples from one of two independent experiments. ⴱ, p ⬍ 0.001, when comparing medium plus IgE sample with medium-alone sample by Student’s t test.
Loss of IgE-induced cytokine production was the most overt defect in Stat5KO MC. This is a clinically important finding, because MC-derived cytokines may be particularly important to disease pathology, perhaps because of the sentinel role these cells play in immunity. For example, IL-13 alone elicits many of the symptoms of asthma (16). Further, MC-derived TNF-␣ appears to be the critical determining factor in bacterial immunity and lymphadenopathy (6 –9). The ability of cytokines to modulate the immune response makes Stat5 control of these factors particularly important. Our data suggest that Stat5 may mediate repression of the mRNA-destabilizing protein TTP because of the mechanism controlling cytokine expression in MC. TTP expression was up-regulated following IgE cross-linkage only in Stat5KO BMMC, arguing that Stat5 may actively suppress TTP in WT MC. In support of this theory, TTP suppression by shRNA partially restored IL-13 secretion to 30% of WT levels. It is possible that TTP induction is only part of the cytokine defect in Stat5KO MC. Stat5-mediated repression of TTP is an interesting contrast to Stat6-mediated TTP induction (17). This is particularly striking because Stat6, as well as Stat3, is activated by FcRI (25, 26). A unifying hypothesis to these opposing activities is that Stat transcription factors regulate MC cytokine production by recruiting transcriptional repressors or
3426 activators to the TTP promoter. By this theory, Stats would effectively integrate extrinsic signals, providing homeostasis to a potentially pathological inflammatory response. At present, we do not know the mechanism by which IgE crosslinkage activates Stat5. Given its rapid phosphorylation and the inability of BFA to hamper this process, it does not appear that autocrine signaling by cytokine receptors is the explanation. Further, the induction of IL-3 during IgE sensitization also does not conform to our findings. BMMC were sensitized in the cold, conditions that preclude IL-3 secretion (confirmed by ELISA; data not shown). Moreover, IL-3KO BMMC demonstrated IgE-mediated Stat5 phosphorylation (data not shown). We hypothesize that Stat5 is directly activated by kinases associated with the FcRI complex, an area of our current investigations. It is important to note that most of our cytokine production assays were performed in medium containing IL-3 and SCF. The inclusion of SCF in these assays is particularly noteworthy, because this cytokine activates Stat5 (27, 28) and has been shown to contribute to MC cytokine production (29 –33). Because we have found that Stat5 is necessary for IL-3 and SCF function (10), the possibility that Stat5 is needed for SCF-mediated augmentation of IgE signaling is a real possibility. However, Bcl-2Tg/Stat5KO BMMC activated in the absence of IL-3 and SCF confirmed the cytokine production defect. These results argue that our data reflect an inherent deficiency in FcRI signaling, separate from the enhancing ability of SCF. These data demonstrate the critical contributions made by Stat5 to MC biology. In addition to supporting MC survival and proliferation, Stat5 regulates IgE-induced degranulation, LT secretion, and cytokine production. This central role in the MC response points to Stat5 as a potential clinical target in MC-associated diseases, a group of disorders that has grown to include both allergic inflammation and autoimmunity.
Acknowledgments The authors gratefully acknowledge the assistance of Chris Lantz and Nicole Urtz.
Disclosures The authors have no financial conflict of interest.
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