Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

A truncated splice-variant of the FcεRIß receptor subunit is critical for microtubule formation and degranulation in mast cells.

Human linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIß) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIß (t-FcεRIß) in humans that contains a putative calmodulin-binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FcεRIß is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIß in the presence of Ca2+ could be critical for t-FcεRIß function. In addition, gene targeting of t-FcεRIß attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIß mediates Ca2+ -dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIß has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation.

Exon skipping of FcεRIß eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy.

Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the ß-subunit of the high-affinity IgE receptor (FcεRIß) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIß expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIß is a potential approach for mast cell-specific treatment of allergic diseases.

Mast cells signal their importance in health and disease.

FcεRI is the primary receptor in mast cells that mediates allergic reactions by inducing rapid release of mediators, an adaptive immune response that might have evolved as a host defense against parasites and venoms. Yet it is apparent that mast cells are also activated through non-IgE receptors, the significance of which is just beginning to be understood. This includes the Mas-related G protein-coupled receptor X2, which might contribute to reactions to diverse antimicrobials and polybasic compounds, and the adhesion G protein-coupled receptor E2, variants of which are associated with familial vibratory urticaria and are activated by mechanical vibration. Similarly, mast cells have long been recognized as the main repository for histamine, heparin, and proteases. Recent evidence also points to new functions, modes of delivery, and mechanisms of action of mast cell proteases that add new dimensions to the roles of mast cells in human biology. In addition, exposure of mast cells to environmental cues can quantitatively and qualitatively modulate their responses and thus their effect on allergic inflammation. Illustrating this paradigm, we summarize a number of recent studies implicating the injury/tissue damage cytokine IL-33 as a modulator of allergen-induced mast cell responses. We also discuss the discovery of markers associated with transformed mast cells and new potential directions in suppressing mast cell activity.

Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells.

BACKGROUND: DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown. OBJECTIVE: We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation. METHODS: DJ-1 was silenced in human CD34+-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of ß-hexosaminidase, prostaglandin D2, and GM-CSF and changes in reactive oxygen species levels were measured after FcεRI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies. RESULTS: DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following FcεRI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound to Lyn but not to other Src kinases, and this interaction was specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain-containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling. CONCLUSION: We demonstrate a novel role for DJ-1 in the early activation of Lyn by FcεRI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy.

IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3.

BACKGROUND: IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. OBJECTIVES: We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. METHODS: HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. RESULTS: IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. CONCLUSION: IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.

Activated mast cells synthesize and release soluble ST2-a decoy receptor for IL-33.

IL-33 released from damaged cells plays a central role in allergic inflammation by acting through its membrane-bound receptor, ST2 receptor (ST2L). IL-33 activity can be neutralized by the soluble spliced variant of ST2 (sST2) that has been associated with allergic inflammation but its source is not well defined. We investigated whether mast cells (MCs) are a significant source of sST2 following activation through FcεRI or ST2. We find that antigen and IL-33 induce substantial production and release of sST2 from human and mouse MCs in culture and do so synergistically when added together or in combination with stem cell factor. Moreover, increases in circulating sST2 during anaphylaxis in mice were dependent on the presence of MCs. Human MCs activated via FcεRI failed to generate IL-33 and IL-33 produced by mouse bone marrow-derived MCs was retained within the cells. Therefore, FcεRI-mediated sST2 production is independent of MC-derived IL-33 acting in an autocrine manner. These results are consistent with the conclusion that both mouse and human MCs when activated are a significant inducible source of sST2 but not IL-33 and thus have the ability to modulate the biologic impact of IL-33 produced locally by other cell types during allergic inflammation.

Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.

IL-33 induces a hyporesponsive phenotype in human and mouse mast cells.

IL-33 is elevated in afflicted tissues of patients with mast cell (MC)-dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ(1) and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.

Prevention of F-actin assembly switches the response to SCF from chemotaxis to degranulation in human mast cells.

Following antigen/IgE-mediated aggregation of high affinity IgE-receptors (FcεRI), mast cells (MCs) degranulate and release inflammatory mediators leading to the induction of allergic reactions including anaphylaxis. Migration of MCs to resident tissues and sites of inflammation is regulated by tissue chemotactic factors such as stem cell factor (SCF (KIT ligand)). Despite inducing similar early signaling events to antigen, chemotactic factors, including SCF, produce minimal degranulation in the absence of other stimuli. We therefore investigated whether processes regulating MC chemotaxis are rate limiting for MC mediator release. To investigate this issue, we disrupted actin polymerization, a requirement for MC chemotaxis, with latrunculin B and cytochalasin B, then examined chemotaxis and mediator release in human (hu)MCs induced by antigen or SCF. As expected, such disruption minimally affected early signaling pathways, but attenuated SCF-induced human mast cell chemotaxis. In contrast, SCF, in the absence of other stimuli, induced substantial degranulation in a concentration-dependent manner following actin disassembly. It also moderately enhanced antigen-mediated human mast cell degranulation which was further enhanced in the presence of SCF. These observations suggest that processes regulating cell migration limit MC degranulation as a consequence of cytoskeletal reorganization.

Stem cell factor programs the mast cell activation phenotype.

Mast cells, activated by Ag via FcεRI, release an array of proinflammatory mediators that contribute to allergic disorders, such as asthma and anaphylaxis. The KIT ligand, stem cell factor (SCF), is critical for mast cell expansion, differentiation, and survival, and under acute conditions, it enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal Ag-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of FcεRI-mediated degranulation and cytokine production. The hyporesponsive phenotype was not a consequence of altered signals regulating calcium flux or protein kinase C, but of ineffective cytoskeletal reorganization with evidence implicating a downregulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders.

DJ-1 regulates mast cell activation and IgE-mediated allergic responses.

BACKGROUND: DJ-1 is an antioxidant protein known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown. OBJECTIVES: We sought to determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo. METHODS: ROS and DJ-1 levels in serum or culture medium were measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches. RESULTS: Patients with atopic dermatitis had increased levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced passive cutaneous anaphylaxis and augmented ROS levels in sera and bone marrow-derived mast cells (BMMCs). Furthermore, antigen-induced degranulation and production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 small interfering RNA-transfected BMMCs compared with that seen in wild-type (WT) BMMCs. Studies with these cells and BMMCs transfected with small interfering RNAs against the phosphatases Src homology domain 2-containing protein tyrosine phosphatase (SHP) 1 and SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity, leading to strengthened signaling through linker for activation of T cells, phospholipase Cγ, and mitogen-activated protein kinases. CONCLUSIONS: A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases, such as asthma and anaphylaxis.

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice.

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

Prostaglandin E2 activates and utilizes mTORC2 as a central signaling locus for the regulation of mast cell chemotaxis and mediator release.

Prostaglandin (PG) E(2), a potent mediator produced in inflamed tissues, can substantially influence mast cell responses including adhesion to basement membrane proteins, chemotaxis, and chemokine production. However, the signaling pathways by which PGE(2) induces mast cell chemotaxis and chemokine production remains undefined. In this study, we identified the downstream target of phosphatidylinositol 3-kinase, mammalian target of rapamycin (mTOR), as a key regulator of these responses. In mouse bone marrow-derived mast cells, PGE(2) was found to induce activation of mTORC1 (mTOR complexed to raptor) as indicated by increased p70S6K and 4E-BP1 phosphorylation, and activation of mTORC2 (mTOR complexed to rictor), as indicated by increased phosphorylation of AKT at position Ser(473). Selective inhibition of the mTORC1 cascade by rapamycin or by the use of raptor-targeted shRNA failed to decrease PGE(2)-mediated chemotaxis or chemokine generation. However, inhibition of the mTORC2 cascade through the dual mTORC1/mTORC2 inhibitor Torin, or through rictor-targeted shRNA, resulted in a significant attenuation in PGE(2)-mediated chemotaxis, which was associated with a comparable decrease in actin polymerization. Furthermore, mTORC2 down-regulation decreased PGE(2)-induced production of the chemokine monocyte chemoattractant protein-1 (CCL2), which was linked to a significant reduction in ROS production. These findings are consistent with the conclusion that activation of mTORC2, downstream of PI3K, represents a critical signaling locus for chemotaxis and chemokine release from PGE(2)-activated mast cells.

Btk-dependent Rac activation and actin rearrangement following FcepsilonRI aggregation promotes enhanced chemotactic responses of mast cells.

Mast cells infiltrate the sites of inflammation associated with chronic atopic disease and during helminth and bacterial infection. This process requires receptor-mediated cell chemotaxis across a concentration gradient of their chemotactic ligands. In vivo, mast cells are likely to be exposed to several such agents, which can cooperate in a synergistic manner to regulate mast cell homing. Here, we report that chemotaxis of mouse bone-marrow-derived mast cells (BMMCs) in response to the chemoattractants stem-cell factor (SCF) and prostaglandin (PG)E(2), is substantially enhanced following antigen-dependent ligation of the high-affinity receptor for IgE (FcεRI). These responses were associated with enhanced activation of phosphoinositide 3-kinase (PI3K), and downstream activation of the tyrosine protein kinase Btk, with subsequent enhanced phospholipase (PL)Cγ-mediated Ca(2+) mobilization, Rac activation and F-actin rearrangement. Antigen-induced chemotaxis, and the ability of antigen to amplify responses mediated by SCF, adenosine and PGE(2) were suppressed following inhibition of PI3K, and were impaired in BMMCs derived from Btk(-/-) mice. There were corresponding decreases in the PLCγ-mediated Ca(2+) signal, Rac activation and F-actin rearrangement, which, as they are essential for BMMC chemotaxis, accounts for the impaired migration of Btk-deficient cells. Taken together, these data demonstrate that, by regulating signaling pathways that control F-actin rearrangement, Btk is crucial for the ability of antigen to amplify mast-cell chemotactic responses.

Amplification of cytokine production through synergistic activation of NFAT and AP-1 following stimulation of mast cells with antigen and IL-33.

IL-33 is associated with atopic and autoimmune diseases and, as reported here, it interacts synergistically with Ag to markedly enhance production of inflammatory cytokines in rodent mast cells even in the absence of degranulation. Investigation of the underlying mechanisms revealed that synergy in signaling occurred at the level of TGF-ß-activated kinase 1, which was then transmitted downstream through JNK, p38 MAP kinase, and AP-1. Stimulation of the Ca(2+) /calcineurin/NFAT pathway by Ag, which IL-33 did not, was critical for the synergy between Ag and IL-33. For example, selective stimulation of the NFAT pathway by thapsigargin also markedly enhanced responses to IL-33 in a calcineurin-dependent manner. As indicated by luciferase-reporter assays, IL-33 failed to stimulate the transcriptional activities of NFAT and AP-1 but augmented the activation of these transcription factors by Ag or thapsigargin. Robust stimulation of NF-κB transcriptional activity by IL-33 was also essential for the synergy. These and pharmacologic data suggested that the enhanced production of cytokines resulted in part from amplification of the activation of AP-1 and NFAT as well as co-operative interactions among transcription factors. IL-33 may retune mast cell responses to Ag toward enhanced cytokine production and thus determine the symptoms and severity of Ag-dependent allergic and autoimmune diseases.

Regulation of mast cell responses in health and disease.

Mast cells are multifunctional cells that initiate not only IgE-dependent allergic diseases but also play a fundamental role in innate and adaptive immune responses to microbial infection. They are also thought to play a role in angiogenesis, tissue remodeling, wound healing, and tumor repression or growth. The broad scope of these physiologic and pathologic roles illustrates the flexible nature of mast cells, which is enabled in part by their phenotypic adaptability to different tissue microenvironments and their ability to generate and release a diverse array of bioactive mediators in response to multiple types of cell-surface and cytosolic receptors. There is increasing evidence from studies in cell cultures that release of these mediators can be selectively modulated depending on the types or groups of receptors activated. The intent of this review is to foster interest in the interplay among mast cell receptors to help understand the underlying mechanisms for each of the immunological and non-immunological functions attributed to mast cells. The second intent of this review is to assess the pathophysiologic roles of mast cells and their products in health and disease. Although mast cells have a sufficient repertoire of bioactive mediators to mount effective innate and adaptive defense mechanisms against invading microorganisms, these same mediators can adversely affect surrounding tissues in the host, resulting in autoimmune disease as well as allergic disorders.

Phospholipase d promotes lipid microdomain-associated signaling events in mast cells.

Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distribution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.

Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14(+) monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)(+) multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14(+) monocytes.

Regulators of Ca(2+) signaling in mast cells: potential targets for treatment of mast cell-related diseases?

A calcium signal is essential for degranulation, generation of eicosanoids and optimal production of cytokines in mast cells in response to antigen and other stimulants. The signal is initiated by phospholipase C-mediated production of inositol1,4,5-trisphosphate resulting in release of stored Ca(2+) from the endoplasmic reticulum (ER) and Golgi. Depletion of these stores activates influx of extracellular Ca(2+), usually referred to as store-operated calcium entry (SOCE), through the interaction of the Ca(2+)-sensor, stromal interacting molecule-1 (STIM1 ), in ER with Orai1(CRACM1) and transient receptor potential canonical (TRPC) channel proteins in the plasma membrane (PM). This interaction is enabled by microtubular-directed reorganization of ER to form ER/PM contact points or "punctae" in which STIM1 and channel proteins colocalize. The ensuing influx of Ca(2+) replenishes Ca(2+) stores and sustains elevated levels of cytosolic Ca(2+) ions-the obligatory signal for mast-cell activation. In addition, the signal can acquire spatial and dynamic characteristics (e.g., calcium puffs, waves, oscillations) that encode signals for specific functional outputs. This is achieved by coordinated regulation of Ca(2+) fluxes through ATP-dependent Ca(2+)-pumps and ion exchangers in mitochondria, ER and PM. As discussed in this chapter, studies in mast cells revealed much about the mechanisms described above but little about allergic and autoimmune diseases although studies in other types of cells have exposed genetic defects that lead to aberrant calcium signaling in immune diseases. Pharmacologic agents that inhibit or activate the regulatory components of calcium signaling in mast cells are also discussed along with the prospects for development of novel SOCE inhibitors that may prove beneficial in the treatment inflammatory mast-cell related diseases.