Wild-type Bone Marrow-derived Mast Cells Research Articles

IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

Sirtuin 6 is a negative regulator of FcεRI signaling and anaphylactic responses

Binding IgE to a cognate allergen causes aggregation of Fcε receptor I (FcεRI) in mast cells, resulting in activation of receptor-associated Src family tyrosine kinases, including Lyn and Syk. Protein tyrosine phosphatase, receptor type C (PTPRC), also known as CD45, has emerged as a positive regulator of FcεRI signaling by dephosphorylation of the inhibitory tyrosine of Lyn. Sirtuin 6 (Sirt6), a NAD+-dependent deacetylase, exhibits an anti-inflammatory property. It remains to be determined, however, whether Sirt6 attenuates mast cell-associated diseases, including anaphylaxis. FcεRI signaling and mast cell degranulation were measured after IgE cross-linking in murine bone marrow-derived mast cells (BMMCs) and human cord blood-derived mast cells. To investigate the function of Sirt6 in mast cell activation invivo, we used mast cell-dependent animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA). Sirt6-deficient BMMCs augmented IgE-FcεRI-mediated signaling and degranulation compared to wild-type BMMCs. Reconstitution of mast cell-deficient KitW-sh/W-sh mice with BMMCs received from Sirt6 knockout mice developed more severe PSA and PCA compared to mice engrafted with wild-type BMMCs. Similarly, genetic overexpression or pharmacologic activation of Sirt6 suppressed mast cell degranulation and blunted responses to PCA. Mechanistically, Sirt6 deficiency increased PTPRC transcription via acetylating histone H3, leading to enhanced aggregation of FcεRI in BMMCs. Finally, we recapitulated the Sirt6 regulation of PTPRC and FcεRI signaling in human mast cells. Sirt6 acts as a negative regulator of FcεRI signaling cascade in mast cells by suppressing PTPRC transcription. Activation of Sirt6 may therefore represent apromising and novel therapeutic strategy for anaphylaxis.

The Putatively Specific Synthetic REV-ERB Agonist SR9009 Inhibits IgE- and IL-33-Mediated Mast Cell Activation Independently of the Circadian Clock.

The cell-autonomous circadian clock regulates IgE- and IL-33-mediated mast cell activation, both of which are key events in the development of allergic diseases. Accordingly, clock modifiers could be used to treat allergic diseases, as well as many other circadian-related diseases, such as sleep and metabolic disorders. The nuclear receptors REV-ERB-α and -β (REV-ERBs) are crucial components of the circadian clockwork. Efforts to pharmacologically target REV-ERBs using putatively specific synthetic agonists, particularly SR9009, have yielded beneficial effects on sleep and metabolism. Here, we sought to determine whether REV-ERBs are functional in the circadian clockwork in mast cells and, if so, whether SR9009 affects IgE- and IL-33-mediated mast cell activation. Bone marrow-derived mast cells (BMMCs) obtained from wild-type mice expressed REV-ERBs, and SR9009 or other synthetic REV-ERBs agonists affected the mast cell clockwork. SR9009 inhibited IgE- and IL-33-mediated mast cell activation in wild-type BMMCs in association with inhibition of Gab2/PI3K and NF-κB activation. Unexpectedly, these suppressive effects of SR9009 were observed in BMMCs following mutation of the core circadian gene Clock. These findings suggest that SR9009 inhibits IgE- and IL-33-mediated mast cell activation independently of the functional circadian clock activity. Thus, SR9009 or other synthetic REV-ERB agonists may have potential for anti-allergic agents.

Mast cell-deficiency protects mice from streptozotocin-induced diabetic cardiomyopathy

Mast cells (MCs) have been implicated in the pathogenesis of cardiometabolic diseases by releasing pro-inflammatory mediators. Patients and animals with diabetic cardiomyopathy (DCM) also show inflammatory cell accumulation in the heart. Here, we detected MCs in mouse heart after streptozotocin (STZ)-induced DCM. DCM production caused significant systole and diastole interventricular septum and left ventricular (LV) posterior wall thinning, and systolic LV internal dilation in wild-type (WT) mice. DCM production also led to significant reductions of fractional shortening percentage, heart rate, body weight, heart weight, and significant increases of kidney, pancreas, and lung weight to body weight ratios, and blood hemoglobin HbA1c and glucose levels in WT mice. All these changes were improved or disappeared in MC-deficient KitW-sh/W-sh mice. In the myocardium from WT DCM mice, we detected significant decrease of cardiac cell proliferation and increases of cardiac cell death, chemokine expression, macrophage infiltration, inflammatory cytokine expression, and collagen deposition. These changes were also improved or disappeared in KitW-sh/W-sh DCM mice. Adoptive transfer of bone marrow-derived MCs (BMMCs) from WT mice fully or partially reversed these cardiac functional and morphologic changes in KitW-sh/W-sh DCM recipient mice. Yet, adoptive transfer of BMMCs from Il6-/- and Tnf-/- mice failed to make these corrections or at much less extent than the WT BMMCs. Mechanistic studies demonstrated a role of MC and MC-derived IL6 and TNF-α in promoting cardiomyocyte death and cardiac fibroblast TGF-β signaling, and collagen synthesis and deposition. Therefore, MC inhibition may have therapeutic potential in attenuating DCM progression.

MALT1 proteolytic activity is necessary for FcɛRI-mediated cytokine production in mast cells

Abstract IgE mediated mast cell activation triggered by multivalent antigen crosslinking of FcɛRI receptors leads to the release of a wide range of inflammatory mediators. Recent studies revealed that B cell lymphoma 10 (Bcl10) and mucosa-associated lymphoid tissue 1 (MALT1) are required for IgE-induced nuclear factor κB (NF-κB)-induced proinflammatory cytokine production, while dispensible for mast cell degranulation and leukotriene synthesis. MALT1, a cytoplasmic signaling protein, possesses at least two functions: scaffolding activity and proteolytic activity. The contribution of MALT1 proteolytic activity to mast cell function has not yet been evaluated. We hypothesize that MALT1 proteolytic activity is necessary for FcɛRI-dependent proinflammatory cytokine production. In order to test our hypothesis, we compared the activation of bone marrow derived mast cells (BMMC) from wild type mice, MALT1 knockout mice (MALT1−/−), and mice harboring a mutant, proteolytically inactive MALT1 (MALT1PD/PD). We find MALT1 protease is active in wild-type BMMCs as evidenced by RelB cleavage, and is absent, as expected, in MALT1−/− and MALT1PD/PD BMMCs. Cytokine gene expression and production in response to IgE mediated FcɛRI crosslinking is equivalently abrogated in both MALT1−/− and MALT1PD/PD BMMCs as compared to wild type. These findings suggest that the contribution of MALT1 to IgE mediated responses can be largely attributed to its protease activity. The MALT1 protease has been shown to be drugable, and pharmaceutically targeting the MALT1 protease activity may represent a promising approach to abrogating late phase pathologic mast cell activation.

IL-4 Signal Regulates IL-9-producing Mucosal Mast Cell Differentiation And Function During The Development of Experimental Food Allergy

Abstract The prevalence of food allergy is increasing significantly in developed nations. Accumulating evidences suggest a pivotal role of intestinal mast cells (MCs), TH2 cytokines, and IgE in the pathophysiology of food allergy. We have recently identified the novel IL-9 producing mucosal mast cells (MMC9s), which can produce high levels of IL-9, IL-13, and mast cells mediators. Mice deficient of IL-4 signals fail to develop MMC9s and become resistant to IgE-mediated food allergy. However, it remains unclear whether IL-4 can directly regulate the development and function of MMC9s. In a skin sensitization induced food allergy model, Il4RaF709 mice which have gain-of-function mutations in IL-4Ra were found to produce much higher levels of ovalbumin (OVA) specific IgE and mast cell protease-1 in blood and became more susceptible to experimental food allergy. While the occurrence of MMC9s and type-2 innate lymphoid cells (ILC2s) were comparable between wild type (WT) and Il4RaF709 mice, repeated OVA ingestions induced significant increase of CD4+TH2 cell occurrence and MC numbers in the small intestine of Il4RaF709 mice than those in WT mice. Furthermore, IL-4 treatments induced bone marrow derived mast cells (BMMCs) from Il4RaF709 mice to produce much more IL-13 and TNF-α than those by WT BMMCs. These results suggest that IL-4 signal provided by CD4+TH2 cells may directly potentiate TH2 effector function of MMC9s and promote the differentiation of MMC9s into intestinal mature MCs, resulting in the increased intestinal hypersensitivity to ingested food allergens. Detailed mechanistic studies of IL-4 signaling in MMC9 development and function may provide therapeutic insights into the preventions and treatments of food allergy.

Role of CCL7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis.

Molecules that are necessary for ocular hypersensitivity reactions include the receptors CCR1 and CCR3; CCL7 is a ligand for these receptors. Therefore, we explored the role of CCL7 in mast cell activity and motility in vitro and investigated the requirement for CCL7 in a murine model of IgE-mediated allergic conjunctivitis. For mast cells treated with IgE and Ag, the presence of CCL7 synergistically enhanced degranulation and calcium influx. CCL7 also induced chemotaxis in mast cells. CCL7-deficient bone marrow-derived mast cells showed decreased degranulation following IgE and Ag treatment compared with wild-type bone marrow-derived mast cells, but there was no difference in degranulation when cells were activated via an IgE-independent pathway. In vivo, CCL7 was upregulated in conjunctival tissue during an OVA-induced allergic response. Notably, the early-phase clinical symptoms in the conjunctiva after OVA challenge were significantly higher in OVA-sensitized wild-type mice than in control challenged wild-type mice; the increase was suppressed in CCL7-deficient mice. In the OVA-induced allergic response, the numbers of conjunctival mast cells were lower in CCL7-deficient mice than in wild-type mice. Our results demonstrate that CCL7 is required for maximal OVA-induced ocular anaphylaxis, mast cell recruitment in vivo, and maximal FcεRI-mediated mast cell activation in vitro. A better understanding of the role of CCL7 in mediating ocular hypersensitivity reactions will provide insights into mast cell function and novel treatments for allergic ocular diseases.

Critical Role of Mast Cells and Peroxisome Proliferator-Activated Receptor γ in the Induction of Myeloid-Derived Suppressor Cells by Marijuana Cannabidiol In Vivo.

Cannabidiol (CBD) is a natural nonpsychotropic cannabinoid from marijuana (Cannabis sativa) with anti-epileptic and anti-inflammatory properties. Effect of CBD on naive immune system is not precisely understood. In this study, we observed that administering CBD into naive mice triggers robust induction of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the peritoneum, which expressed functional arginase 1, and potently suppressed T cell proliferation ex vivo. Furthermore, CBD-MDSC suppressed LPS-induced acute inflammatory response upon adoptive transfer in vivo. CBD-induced suppressor cells were comprised of CD11b(+)Ly6-G(+)Ly6-C(+) granulocytic and CD11b(+)Ly6-G(-)Ly6-C(+) monocytic subtypes, with monocytic MDSC exhibiting higher T cell-suppressive function. Induction of MDSC by CBD was markedly attenuated in Kit-mutant (Kit(W/W-v)) mast cell-deficient mice. MDSC response was reconstituted upon transfer of wild-type bone marrow-derived mast cells in Kit(W/W-v) mice, suggesting the key role of cKit (CD117) as well as mast cells. Moreover, mast cell activator compound 48/80 induced significant levels of MDSC in vivo. CBD administration in mice induced G-CSF, CXCL1, and M-CSF, but not GM-CSF. G-CSF was found to play a key role in MDSC mobilization inasmuch as neutralizing G-CSF caused a significant decrease in MDSC. Lastly, CBD enhanced the transcriptional activity of peroxisome proliferator-activated receptor γ in luciferase reporter assay, and PPAR-γ selective antagonist completely inhibited MDSC induction in vivo, suggesting its critical role. Together, the results suggest that CBD may induce activation of PPAR-γ in mast cells leading to secretion of G-CSF and consequent MDSC mobilization. CBD being a major component of Cannabis, our study indicates that marijuana may modulate or dysregulate the immune system by mobilizing MDSC.

Intraperitoneal influx of neutrophils in response to IL-33 is mast cell–dependent

AbstractIL-33 is a recently discovered cytokine involved in induction of Th2 responses and functions as an alarmin. Despite numerous recent studies targeting IL-33, its role in vivo is incompletely understood. Here we investigated inflammatory responses to intraperitoneal IL-33 injections in wild-type and mast cell–deficient mice. We found that wild-type mice, but not mast cell–deficient Wsh/Wsh mice, respond to IL-33 treatment with neutrophil infiltration to the peritoneum, whereas other investigated cell types remained unchanged. In Wsh/Wsh mice, the IL-33–induced innate neutrophil response could be rescued by local reconstitution with wild-type but not with T1/ST2−/− mast cells, demonstrating a mast cell–dependent mechanism. Furthermore, we found this mechanism to be partially dependent on mast cell–derived TNF, as we observed reduced neutrophil infiltration in Wsh/Wsh mice reconstituted with TNF−/− bone marrow–derived mast cells compared with those reconstituted with wild-type bone marrow–derived mast cells. In agreement with our in vivo findings, we demonstrate that humanneutrophils migrate toward the supernatant of IL-33–treated human mast cells. Taken together, our findings reveal that IL-33 activates mast cells in vivo to recruit neutrophils, a mechanism dependent on IL-33R expression on peritoneal mast cells. Mast cells activated in vivo by IL-33 probably play an important role in inflammatory reactions.

Distinct and opposing roles for Rab27a/Mlph/MyoVa and Rab27b/Munc13‐4 in mast cell secretion

Mediator release from mast cells is a critical step in allergic and inflammatory disease. However, the processes regulating the latter stages of granule release are yet to be fully understood. Rab27 small GTPases regulate release of secretory lysosomes in a variety of cells, including mast cell granules. In the present study, using murine bone marrow-derived mast cells (BMMC) from Rab27-deficient mutant mice, we found that, in contrast to Rab27b, Rab27a primarily plays an inhibitory role in regulating degranulation. Immunofluorescence analysis revealed that resting Rab27a-deficient (ashen) BMMCs display abnormal cortical F-actin distribution. Actin disassembly prior to IgE cross-linking increased wild-type BMMC secretion to ashen levels, suggesting that changes in the integrity of cortical F-actin underlie the ashen phenotype. Comparison of the secretory impairment of Rab27b knockout and Rab27a/b double knockout BMMCs highlighted a secondary positive role for Rab27a in enhancing degranulation. Rab27 is known to interact with actin via its effectors melanophilin (Mlph) and myosinVa (MyoVa) in other cell types. To better understand the differing roles of Rab27 proteins, we analysed the secretory phenotype of BMMCs derived from mice lacking Rab27 effector proteins. These experiments revealed that the phenotype of BMMCs deficient in Mlph (leaden) and BMMCs deficient in MyoVa (dilute) resembles the hyper-secretion of ashen BMMCs, while Munc13-4-deficient (jinx) BMMCs phenocopy the Rab27b knockout and double Rab27a/b knockout secretory impairment. We conclude that Rab27a and Rab27b regulate distinct steps in the BMMC degranulation pathway, with Rab27a/Mlph/MyoVa regulating cortical actin stability upstream of Rab27a/b/Munc13-4-dependent granule exocytosis.

Class I PI3K-mediated Akt and ERK signals play a critical role in FcεRI-induced degranulation in mast cells

Class IA and IB phosphoinositide 3-kinases (PI3Ks) have been shown to regulate mast cell functions such as proliferation, development, survival and degranulation, but the functional redundancy between these two PI3K signaling pathways in mast cells remains unclear. Here, we have generated mice deficient in both class IA regulatory subunit p85α and class IB catalytic subunit p110γ, and show that p85α(-/-)p110γ(-/-) mice exhibit a more severe defect in mast cell development than single-knockout mice. In addition, the in vivo passive cutaneous anaphylaxis reaction of p85α(-/-)p110γ(-/-) mice was nearly completely abrogated, whereas single-knockout mice exhibit just marginal reduction. Pharmacological inactivation of Akt in wild-type bone marrow-derived mast cells (BMMCs) led to partial reduction of degranulation, while over-expression of a constitutively active Akt partially restored the impaired degranulation in p85α(-/-)p110γ(-/-) BMMCs. We also found that the extracellular signal-regulated kinase (ERK) signaling pathway was activated in a PI3K-dependent manner upon FcεRI stimulation and that simultaneous inhibition of Akt and ERK resulted in nearly complete blockade of FcεRI-induced degranulation. Our data provide evidence that Akt and ERK pathways play redundant roles in FcεRI-induced degranulation.

The Receptor LMIR3 Negatively Regulates Mast Cell Activation and Allergic Responses by Binding to Extracellular Ceramide

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

Mast cell activation and degranulation promotes renal fibrosis in experimental unilateral ureteric obstruction

Progressive renal fibrosis is the final common pathway leading to renal failure irrespective of the initiating cause. Clinical studies of renal fibrosis found that prominent mast cell accumulation correlated with worse outcomes. Mast cells are pluripotent innate immune cells that synthesize and secrete profibrotic mediators. Here we use mast cell-deficient (Kit(W-sh/W-sh)) mice to define a functional pathogenic role for these cells in the development of renal fibrosis. Intrarenal collagen deposition was significantly decreased in mast cell-deficient compared to wild-type mice 7 and 14 days after unilateral ureteric obstruction. The intrarenal expression of mRNAs for transforming growth factor-β, α-smooth muscle actin, chemokines, and renal macrophages and CD4(+) T cells were also decreased in mast cell-deficient mice. Reconstitution of the mast cell population in mast cell-deficient mice with wild-type bone marrow-derived mast cells restored the pattern and intensity of renal fibrosis to levels seen in wild-type mice following ureteric ligation. Interestingly, the mast cells were recruited, activated, and degranulated within 6 h of ureteric ligation. A mast cell stabilizer that impairs degranulation, disodium chromoglycate, significantly attenuated renal fibrosis following ureteric ligation in wild-type mice. Thus, mast cells promote renal fibrosis and their targeting may offer therapeutic potential in the treatment of renal fibrosis.

Mast Cells in Tumor Microenvironment Promotes the In Vivo Growth of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death. No effective therapy is currently available for PDAC because of the lack of understanding of the mechanisms leading to its growth and development. Inflammatory cells, particularly mast cells, have been shown to play key roles in some cancers. We carried out this study to test the hypothesis that mast cells in the tumor microenvironment are essential for PDAC tumorigenesis. The presence of inflammatory cells at various stages of PDAC development was determined in a spontaneous mouse model of PDAC (K-ras(G12V)). The importance of mast cells was determined using orthotopically implanted PDAC cells in mast cell-deficient Kit(w-sh/w-sh) mice and further confirmed by reconstitution of wild-type bone marrow-derived mast cells. Clinical relevance was assessed by correlating the presence of mast cells with clinical outcome in patients with PDAC. In the spontaneous mouse model of PDAC (K-ras(G12V)), there was an early influx of mast cells to the tumor microenvironment. PDAC tumor growth was suppressed in mast cell-deficient Kit(w-sh/w-sh) mice, but aggressive PDAC growth was restored when PDAC cells were injected into mast cell-deficient mice reconstituted with wild-type bone marrow-derived mast cells. Mast cell infiltration into the tumor microenvironment was predictive of poor prognosis in patients with PDAC. Mast cells play an important role in PDAC growth and development in mouse models and are indicative of poor prognosis in humans, which makes them a potential novel therapeutic target.

PEST‐domain‐enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice

PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP(-/-) and PEP(+/+) mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP(+/+) and PEP(-/-) mice. Bone marrow-derived mast cells from PEP(-/-) mice showed impaired PLCγ1 phosphorylation and Ca(2+) mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)-phosphine complex that selectively inhibits PEP activity produced defects in Ca(2+) signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.

Analysis of Two Major Intracellular Phospholipases A2 (PLA2) in Mast Cells Reveals Crucial Contribution of Cytosolic PLA2α, Not Ca2+-independent PLA2β, to Lipid Mobilization in Proximal Mast Cells and Distal Fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.

BLT2 Is Upregulated in Allergen-Stimulated Mast Cells and Mediates the Synthesis of Th2 Cytokines

Mast cells are effector cells that mediate the allergic response through Ag stimulation of IgE bound to FcεRI. In allergic reactions, cross-linking of the surface receptors for IgE on mast cells results in the synthesis of Th2 cytokines such as IL-4 and IL-13, which are critical for the initiation and progression of the allergic response. Despite the important roles of these cytokines, the signaling mechanism by which Ag stimulation mediates the production of IL-4 and IL-13 in mast cells is not clearly understood. In the present study, we found that Ag-stimulated bone marrow-derived mast cells (BMMCs) highly upregulated the expression of BLT2, a leukotriene B(4) receptor, and that blockade of BLT2 with the specific antagonist LY255283 or small interfering RNA knockdown completely abolished the production of Th2 cytokines. Furthermore, BMMCs overexpressing BLT2 showed significantly enhanced production of Th2 cytokines compared with wild-type BMMCs. Additionally, we found that the generation of Nox1-derived reactive oxygen species occurs downstream of BLT2, thus mediating the synthesis of Th2 cytokines. Taken together, our results suggest that the BLT2-Nox1-reactive oxygen species cascade is a previously unsuspected mediatory signaling mechanism to Th2 cytokine production in Ag-stimulated BMMCs, thus contributing to allergic response.

Mast cells influence disease severity in a murine model of relapsing-remitting multiple sclerosis. (135.15)

Abstract Although mast cells (MCs) are critical mediators of allergic and innate immune responses, they also modulate adaptive immune responses. Utilizing WBB6c-kitW/Wv mast cell-deficient mice, MCs have been implicated in the promotion of disease severity in several murine models of human autoimmune disease. WBB6c-kitW/Wv mice have two independent mutations that profoundly inhibit MC development. We previously demonstrated that MCs influence disease severity in a murine model of chronic-progressive multiple sclerosis (CP-MS), specifically by influencing cellular trafficking into the CNS and blood brain barrier permeability. However, this model does not mimic the most common form of the human disease, relapsing-remitting MS (RR-MS). Therefore, to test the effect of a MC deficiency on a RR form of disease, we transferred the c-kitW/Wv mutations onto the SJL background, a strain that is susceptible to RR disease. These animals are MC-deficient in all tissues examined, carry mild hematopoietic deficits, but have relatively normal thymic T cell compartments. SJLc-kitW/Wv mice display significantly decreased cumulative EAE disease scores and a delay in the day of peak acute disease compared to their wild type littermates. These differences in disease phenotypes are abrogated upon reconstitution of wild type bone marrow-derived MCs. Thus, MCs also influence disease severity in a RR murine model of MS, a finding with potential clinical relevance for the heterogeneous MS patient population.

Transcriptional Regulation of Mouse Mast Cell Protease-2 by Interleukin-15

Mast cells (MCs) play a critical role in innate and adaptive immunity through the release of cytokines, chemokines, lipid mediators, biogenic amines, and proteases. We recently showed that the activities of MC proteases are transcriptionally regulated by intracellularly retained interleukin-15 (IL-15), and we provided evidence that this cytokine acts as a specific regulator of mouse mast cell protease-2 (mMCP-2). Here, we show that in wild-type bone marrow-derived mast cells (BMMCs) IL-15 inhibits mMCP-2 transcription indirectly by inducing differential expression and mMCP-2 promoter binding of the bifunctional transcription factors C/EBPbeta and YY1. In wild-type BMMCs, C/EBPbeta expression predominates over YY1 expression, and thus C/EBPbeta preferentially binds to the mMCP-2 promoter. In IL-15-deficient BMMCs, the opposite is found: YY1 expression predominates and binds to the mMCP-2 promoter at the expense of C/EBPbeta. Hypertranscription of the mMCP-2 gene in IL-15-deficient BMMCs is associated with histone acetylation and, intriguingly, with methylation of non-CpG dinucleotides within the MCP-2 promoter. This suggests a novel model of cytokine-controlled protease transcription: non-CpG methylation maintains a chromosomal domain in an "open" configuration that is permissive for gene expression.

SH2 Domain-Containing Phosphatase-2 Protein-Tyrosine Phosphatase Promotes FcεRI-Induced Activation of Fyn and Erk Pathways Leading to TNFα Release from Bone Marrow-Derived Mast Cells

Clustering of the high affinity IgE receptor (Fc(epsilon)RI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Initiation of FFc(epsilon)RI signaling involves rapid tyrosine phosphorylation of Fc(epsilon)RI and membrane-localized adaptor proteins that recruit additional SH2 domain-containing proteins that dynamically regulate downstream signaling. SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in Fc(epsilon)RI signaling, but whose function is not well defined. In this study, using a mouse model allowing temporal shp2 inactivation in bone marrow-derived mast cells (BMMCs), we provide insights into SHP2 functions in the Fc(epsilon)RI pathway. Although no overt defects in Fc(epsilon)RI-induced tyrosine phosphorylation were observed in SHP2 knock-out (KO) BMMCs, several proteins including Lyn and Syk kinases displayed extended phosphorylation kinetics compared with wild-type BMMCs. SHP2 was dispensable for Fc(epsilon)RI-induced degranulation of BMMCs, but was required for maximal activation of Erk and Jnk mitogen-activated protein kinases. SHP2 KO BMMCs displayed several phenotypes associated with reduced Fyn activity, including elevated phosphorylation of the inhibitory pY531 site in Fyn, impaired signaling to Grb2-associated binder 2, Akt/PKB, and IkappaB kinase, and decreased TNF-alpha release compared with control cells. This is likely due to elevated Lyn activity in SHP2 KO BMMCs, and the ability of Lyn to antagonize Fyn activity. Overall, our study identifies SHP2 as a positive effector of Fc(epsilon)RI-induced activation of Fyn/Grb2-associated binder 2/Akt and Ras/Erk pathways leading to TNF-alpha release from mast cells.