Phospholipase C-?3 regulates Fc?RI-mediated mast cell activation by recruiting the protein phosphatase SHP-1.
ABSTRACT: Mast cells are major effectors in high-affinity IgE receptor (Fc?RI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-?3 is crucial for Fc?RI-mediated mast cell activation. Plcb3(-/-) mice showed blunted Fc?RI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, Fc?RI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-?3 constitutively interacts with Fc?RI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-?3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, Fc?RI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-?3- and SHP-1-mediated signaling pathway for Fc?RI-mediated cytokine production.
Project description:Mitochondrial aldehyde dehydrogenase (ALDH2) metabolizes endogenous and exogenous aldehydes and protects cells against oxidative injury. Inactivating genetic polymorphisms in humans are common and associate with alcohol flush reactions. However, whether mast cell Aldh2 activity impacts normal mast cell responses is unknown. Using bone marrow-derived mast cells from Aldh2 knockout mice, we found evidence for a role of mast cell Aldh2 in Kit-mediated responses. Aldh2-deficient mast cells showed enhanced Kit tyrosine kinase phosphorylation and activity after stimulation with its ligand (stem cell factor) and augmentation of downstream signaling pathways, including Stat4, MAPKs, and Akt. The activity of the phosphatase Shp-1, which attenuates Kit activity, was reduced in Aldh2-/- mast cells, along with an increase in reactive oxygen species, known to regulate Shp-1. Reduced Shp-1 activity concomitant with sustained Kit signaling resulted in greater proliferation following Kit engagement, and increased mediator and cytokine release when Aldh2-/- mast cells were co-stimulated via Kit and Fc?RI. However, Fc?RI-mediated signaling and responses were unaffected. Therefore, our findings reveal a functional role for mast cell intrinsic Aldh2 in the control of Kit activation and Kit-mediated responses, which may lead to a better understanding of mast cell reactivity in conditions related to ALDH2 polymorphisms.
Project description:C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (Fc?RI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in Fc?RI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their Fc?RI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of Fc?RI, SYK, and phospholipase C. Interestingly, Fc?RI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in Fc?RI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.
Project description:Atopic dermatitis (AD) is a chronic inflammatory skin disease. Here, we show that phospholipase C-?3 (PLC-?3)-deficient mice spontaneously develop AD-like skin lesions and more severe allergen-induced dermatitis than wild-type mice. Mast cells were required for both AD models and remarkably increased in the skin of Plcb3(-/-) mice because of the increased Stat5 and reduced SHP-1 activities. Mast cell-specific deletion of Stat5 gene ameliorated allergen-induced dermatitis, whereas that of Shp1 gene encoding Stat5-inactivating SHP-1 exacerbated it. PLC-?3 regulates the expression of periostin in fibroblasts and TSLP in keratinocytes, two proteins critically involved in AD pathogenesis. Furthermore, polymorphisms in PLCB3, SHP1, STAT5A, and STAT5B genes were associated with human AD. Mast cell expression of PLC-?3 was inversely correlated with that of phospho-STAT5, and increased mast cells with high levels of phospho-STAT5 were found in lesional skin of some AD patients. Therefore, STAT5 regulatory mechanisms in mast cells are important for AD pathogenesis.
Project description:Antigen-mediated cross-linking of IgE bound to its receptor, Fc?RI, initiates a transmembrane signaling cascade that results in mast cell activation in the allergic response. Using immunogold labeling of intact RBL mast cells and scanning electron microscopy (SEM), we visualize molecular reorganization of IgE-Fc?RI and early signaling proteins on both leaflets of the plasma membrane, without the need for ripped off membrane sheets. As quantified by pair correlation analysis, we observe dramatic changes in the nanoscale distribution of IgE-Fc?RI after binding of multivalent antigen to stimulate transmembrane signaling, and this is accompanied by similar clustering of Lyn and Syk tyrosine kinases, and adaptor protein LAT. We find that Lyn co-redistributes with IgE-Fc?RI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-Fc?RI and Lyn in stimulated clusters. Coupling of these proteins is also decreased when membrane cholesterol is reduced either before or after antigen addition. These results provide evidence for involvement of Fc?RI phosphorylation and cholesterol-dependent membrane structure in the interactions that accompany IgE-mediated activation of RBL mast cells. More generally, this SEM view of intact cell surfaces provides new insights into the nanoscale organization of receptor-mediated signaling complexes in the plasma membrane.
Project description:This study is aimed at determining whether Sesamum indicum Linn. beneficially influences Fc?RI-mediated allergic reactions in RBL-2H3 mast cells; it is also aimed at further investigating Lyn/Fyn and Syk signaling pathways. To examine the antiallergic effect of Sesamum indicum Linn. extract (SIE), we treated antigen/immunoglobulin E- (IgE-) sensitized mast cells with extracts of various concentrations. We examined the degranulation release and concentrations of inflammatory mediators. Additionally, the expressions of genes involved in the Fc?RI and arachidonate signaling pathways were examined. SIE inhibited the degranulation and secretion of inflammatory mediators in antigen/IgE-sensitized mast cells. SIE reduced the expressions of Fc?RI signaling-related genes, such as Syk, Lyn, and Fyn, and the phosphorylation of extracellular signal-regulated kinase in antigen/IgE-sensitized mast cells. Additionally, in late allergic responses, SIE reduced PGD2 release and COX-2 and cPLA2 phosphorylation expression in Fc?RI-mediated mast cell activation. Lastly, 250-500?mg/kg SIE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. The potent effect of SIE on RBL-2H3 mast cell activation indicates that the extract could potentially be used as a novel inhibitor against allergic reactions.
Project description:Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following Fc?RI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in Fc?RI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to Fc?RI activation. These responses were accompanied by a significant reduction in PLC?2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell Fc?RI-induced cytokine secretion.
Project description:Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-?3-dependent manner. Thus, PLC-?3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-?3 doubly deficient lyn(-/-);PLC-?3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-?3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-?3-mediated regulatory mechanism of SHP-1 and Stat5 activities.
Project description:The allergic response is initiated on the plasma membrane of mast cells by phosphorylation of the receptor for immunoglobulin E (IgE), Fc?RI, by Lyn kinase after IgE-Fc?RI complexes are cross-linked by multivalent antigen. Signal transduction requires reorganization of receptors and membrane signaling proteins, but this spatial regulation is not well defined. We used fluorescence localization microscopy (FLM) and pair-correlation analysis to measure the codistribution of IgE-Fc?RI and Lyn on the plasma membrane of fixed cells with 20- to 25-nm resolution. We directly visualized Lyn recruitment to IgE-Fc?RI within 1 min of antigen stimulation. Parallel FLM experiments captured stimulation-induced Fc?RI phosphorylation and colocalization of a saturated lipid-anchor probe derived from Lyn's membrane anchorage. We used cytochalasin and latrunculin to investigate participation of the actin cytoskeleton in regulating functional interactions of Fc?RI. Inhibition of actin polymerization by these agents enhanced colocalization of IgE-Fc?RI with Lyn and its saturated lipid anchor at early stimulation times, accompanied by augmented phosphorylation within Fc?RI clusters. Ising model simulations provide a simplified model consistent with our results. These findings extend previous evidence that IgE-Fc?RI signaling is initiated by colocalization with Lyn in ordered lipid regions and that the actin cytoskeleton regulates this functional interaction by influencing the organization of membrane lipids.
Project description:Background: As the number of allergic disease increases, studies to identify new treatments take on new urgency. Epigallocatechin gallate (EGCG), a major component of green tea, has been shown to possess a wide range of pharmacological properties, including anti-inflammation and anti-viral infection. In previous study, gallic acid (GA), a part of EGCG, has shown anti-allergic inflammatory effect. To improve on preliminary evidence that GA has allergy mitigating effect, we designed SG-SP1 based on GA, and aimed to assess the effects of SG-SP1 on mast cell-mediated allergic inflammation using various animal and in vitro models. Methods: For in vitro experiments, various types of IgE-stimulated mast cells (RBL-2H3: mast cell-like basophilic leukemia cells, and primary cultured peritoneal and bone marrow-derived mast cells) were used to determine the role of SG-SP1 (0.1-1 nM). Immunoglobulin (Ig) E-induced passive cutaneous anaphylaxis and ovalbumin-induced systemic anaphylaxis, standard animal models for immediate-type hypersensitivity were also used. Results: For in vitro, SG-SP1 reduced degranulation of mast cells by down-regulating intracellular calcium levels in a concentration-dependent manner. SG-SP1 decreased expression and secretion of inflammatory cytokines in activated mast cells. This suppressive effect was associated with inhibition of the phosphorylation of Lyn, Syk and Akt, and the nuclear translocation of nuclear factor-?B. Due to the strong inhibitory effect of SG-SP1 on Lyn, the known upstream signaling to Fc?RI-dependent pathway, we confirmed the direct binding of SG-SP1 to Fc?RI, a high affinity IgE receptor by surface plasmon resonance experiment. Oral administration of SG-SP1 hindered allergic symptoms of both anaphylaxis models evidenced by reduction of hypothermia, serum IgE, ear thickness, and tissue pigmentation. This inhibition was mediated by the reductions in serum histamine and interleukin-4. Conclusions: We determined that SG-SP1 directly interacts with Fc?RI and propose SG-SP1 as a therapeutic candidate for mast cell-mediated allergic inflammatory disorders via inhibition of Fc?RI signaling.
Project description:Activation of the high-affinity receptor for IgE (Fc?RI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5'-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen (Ag) stimulation. The low-affinity IgG receptor, Fc?RIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, Ag/IgE-dependent and progressive co-localization of Fc?RI and Fc?RIIB, which is not dependent on IgG. Upon supra-optimal Fc?RI cross-linking, Fc?RIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of Fc?RIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon Fc?RI cross-linking. Absence of Fc?RIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca2+ mobilization. Though, degranulation and IL-6 production were only weakly altered. Altogether our data establish the LYN/Fc?RIIB/SHIP1 signalosome in the context of Fc?RI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on Fc?RIIB, highlights the necessity for its tight backup control.