Fluvastatin enhances IL-33-mediated mast cell IL-6 and TNF production.

Statins are HMG-CoA reductase inhibitors prescribed for lowering cholesterol. They can also inhibit inflammatory responses by suppressing isoprenylation of small G proteins. Consistent with this, we previously found that fluvastatin suppresses IgE-mediated mast cell function. However, some studies have found that statins induced pro-inflammatory cytokines in macrophages and NK cells. In contrast to IgE signaling, we show that fluvastatin augments IL-33-induced TNF and IL-6 production by mast cells. This effect required the key mast cell growth factor, stem cell factor (SCF). Treatment of IL-33-activated mast cells with mevalonic acid or isoprenoids reduced fluvastatin effects, suggesting fluvastatin acts at least partly by reducing isoprenoid production. Fluvastatin also enhanced IL-33-induced NF-κB transcriptional activity and promoted neutrophilic peritonitis in vivo, a response requiring mast cell activation. Other statins tested did not enhance IL-33 responsiveness. Therefore, this work supports observations of unexpected pro-inflammatory effects of some statins and suggests mechanisms by which this may occur. Because statins are candidates for repurposing in inflammatory disorders, our work emphasizes the importance of understanding the pleiotropic and possible unexpected effects of these drugs.

Human Skin-Derived Mast Cells Spontaneously Secrete Several Angiogenesis-Related Factors.

Mast cells are classically recognized as cells that cause IgE-mediated allergic reactions. However, their ability to store and secrete vascular endothelial growth factor (VEGF) suggests a role in vascular development and tumorigenesis. The current study sought to determine if other angiogenesis-related factors, in addition to VEGF, were also secreted by human tissue-derived mast cells. Using proteome array analysis and ELISA, we found that human skin-derived mast cells spontaneously secrete CXCL16, DPPIV, Endothelin-1, GM-CSF, IL-8, MCP-1, Pentraxin 3, Serpin E1, Serpin F1, TIMP-1, Thrombospondin-1, and uPA. We identified three groups based on their dependency for stem cell factor (SCF), which is required for mast cell survival: Endothelin-1, GM-CSF, IL-8, MCP-1, and VEGF (dependent); Pentraxin 3, Serpin E1, Serpin F1, TIMP-1, and Thrombospondin-1 (partly dependent); and CXCL16, DPPIV, and uPA (independent). Crosslinking of FcεRI with multivalent antigen enhanced the secretion of GM-CSF, Serpin E1, IL-8, and VEGF, and induced Amphiregulin and MMP-8 expression. Interestingly, FcεRI signals inhibited the spontaneous secretion of CXCL16, Endothelin-1, Serpin F1, Thrombospondin-1, MCP-1 and Pentraxin-3. Furthermore, IL-6, which we previously showed could induce VEGF, significantly enhanced MCP-1 secretion. Overall, this study identified several angiogenesis-related proteins that, in addition to VEGF, are spontaneously secreted at high concentrations from human skin-derived mast cells. These findings provide further evidence supporting an intrinsic role for mast cells in blood vessel formation.

Store-Operated Calcium Entry via STIM1 Contributes to MRGPRX2 Induced Mast Cell Functions.

Mast cells are inflammatory immune cells that play an essential role in mediating allergic reactions in humans. It is well-known that mast cell activation is critically regulated by intracellular calcium ion (Ca2+) concentrations. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a G-protein coupled receptor (GPCR) expressed on mast cells that is activated by various ligands, including several FDA approved drugs; consequently, this receptor has been implicated in causing pseudo-allergic reactions in humans. MRGPRX2 activation leads to an increase in intracellular Ca2+ levels; however, the Ca2+ mobilizing mechanisms utilized by this receptor are largely unknown. Previous reports showed that store-operated Ca2+ entry (SOCE) via the calcium sensor, stromal interaction molecule 1 (STIM1), regulates mast cell response induced by the high-affinity IgE receptor (FcεRI). In this study, using complementary pharmacologic and genetic ablation approaches we demonstrate that SOCE through STIM1 promotes MRGPRX2-induced human mast cell response in vitro. Importantly, SOCE also critically modulates MrgprB2 (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Collectively, our data suggests that MRGPRX2/MrgprB2 activation of mast cells is dependent on SOCE via STIM1, and further characterization of the MRGPRX2-SOCE-STIM1 pathway will lead to the identification of novel targets for the treatment of pseudo-allergic reactions in humans.

Interleukin-6 potentiates FcεRI-induced PGD2 biosynthesis and induces VEGF from human in situ-matured skin mast cells.

BACKGROUND: Interleukin-6 is a gp130 utilizing cytokine that is consistently associated with allergic diseases like asthma and urticaria in humans where mast cells are known to play a critical role. However, the role of IL-6 in allergic disease in not known. IL-6 was reported to enhance degranulation of in vitro-derived mast cells, but the effect of IL-6 on mediator release from human in situ-matured tissue-isolated mast cells had not been reported. METHODS: Human mature mast cells were isolated and purified from normal skin tissue from different donors. The expression of surface-expressed IL-6 receptors was demonstrated by flow cytometry. The effect of IL-6 on FcεRI-induced degranulation, PGD2 biosynthesis, and cytokine production was determined with ß­hexosaminidase release assay, Western blotting, quantitative real-time PCR, and ELISA. The small molecule inhibitor of STAT-3, C188-9, was used to demonstrate STAT3 dependency. RESULTS: IL-6 significantly potentiated FcεRI-induced PGD2 biosynthesis, but had no effect on degranulation. IL-6 also induced VEGF gene expression and protein secretion, and enhanced FcεRI-induced IL-8 production. Mechanistically, IL-6 enhanced FcεRI-induced COX­2 expression, PGD2 biosynthesis, and VEGF production in a STAT3 dependent manner. CONCLUSION: Here, we demonstrate that IL-6 is a potentiator of FcεRI-induced PGD2 biosynthesis, and can induce or enhance production of pro-angiogenesis factors VEGF and IL-8 from human in situ-matured skin mast cells. GENERAL SIGNIFICANCE: These findings from this study indicate that IL-6 contributes to human allergic disease by enhancing the production of inflammatory PGD2 from tissue-resident mast cells. Moreover, the data suggest a novel role for IL-6 in mast cell-mediated angiogenesis.

Resveratrol preferentially inhibits IgE-dependent PGD2 biosynthesis but enhances TNF production from human skin mast cells.

BACKGROUND: Resveratrol, a natural polyphenol found in the skin of red grapes, is reported to have anti-inflammatory properties including protective effects against aging. Consequently, Resveratrol is a common nutritional supplement and additive in non-prescription lotions and creams marketed as anti-aging products. Studies in mice and with mouse bone marrow-derived mast cells (BMMCs) have indicated anti-allergic effects of Resveratrol. However, the effects of Resveratrol on human primary mast cells have not been reported. METHODS: Human mast cells were isolated and purified from normal skin tissue of different donors. The effect of Resveratrol on IgE-dependent release of allergic inflammatory mediators was determined using various immunoassays, Western blotting, and quantitative real-time PCR. RESULTS: Resveratrol at low concentrations (≤10 µM) inhibited PGD2 biosynthesis but not degranulation. Accordingly, COX-2 expression was inhibited but phosphorylation of Syk, Akt, p38, and p42/44 (ERKs) remained intact. Surprisingly, TNF production was significantly enhanced with Resveratrol. At a high concentration (100 µM), Resveratrol significantly inhibited all parameters analyzed except Syk phosphorylation. CONCLUSIONS: Here, we show that Resveratrol at low concentrations exerts its anti-inflammatory properties by preferentially targeting the arachidonic acid pathway. We also demonstrate a previously unrecognized pro-inflammatory effect of Resveratrol--the enhancement of TNF production from human mature mast cells following IgE-dependent activation. GENERAL SIGNIFICANCE: These findings suggest that Resveratrol as a therapeutic agent could inhibit PGD2-mediated inflammation but would be ineffective against histamine-mediated allergic reactions. However, Resveratrol could potentially exacerbate or promote allergic inflammation by enhancing IgE-dependent TNF production from mast cells in human skin.

Intracellular adenosine inhibits IgE-dependent degranulation of human skin mast cells.

PURPOSE: Adenosine (ADO) can enhance and inhibit mast cell degranulation. Potentiation of degranulation occurs at relatively low concentrations of ADO (10−6­10−5 M) through triggering of A3AR, whereas, inhibition occurs at higher concentrations of ADO reportedly through triggering of A2aAR. However, the discrepancy in the concentration of ADO that inhibits degranulation and that required to trigger ADORs suggests a different mechanism. The purpose of this study is to determine the mechanism by which ADO inhibits human mast cell degranulation. METHODS: We compare the effectiveness of A2aAR specific antagonist ZM241385 and equilibrative nucleoside transporter inhibitors Dipyridamole and NBMPR in preventing ADO-mediated inhibition of FcεRI-induced degranulation of human skin mast cells (hSMCs). Western blotting is done to analyze the effect of ADO on FcεRI-induced Syk phosphorylation. RESULTS: Dipyridamole and NBMPR completely and dose-dependently prevented ADO from inhibiting FcεRI-induced degranulation in all hSMC preparations. In contrast, ZM241385 at 10−5 M was effective in only 3 of 10 hSMC preparations. Moreover, NBMPR was effective even in those hSMC preparations not responsive to ZM241385. ADO inhibited degranulation induced by FcεRI crosslinking, but not that induced by complement component 5a (C5a), Substance P or calcium ionophore. Accordingly, ADO significantly attenuated FcεRI-induced phosphorylation of Syk at the critical activating tyrosine (Y525). CONCLUSION: Blocking the influx of ADO, but not A2aAR signals, is necessary and sufficient to prevent ADO from inhibiting FcεRI-induced mast cell degranulation. Thus, ADO specifically inhibits FcεRI-induced degranulation of hSMCs primarily by an intracellular mechanism that requires its influx via equilibrative nucleoside transporter 1 (ENT1).

In vitro desensitization of human skin mast cells.

Desensitization is a clinical procedure whereby incremental doses of a drug are administered over several hours to a sensitive patient until a therapeutic dose and clinical tolerance are achieved. Clinical tolerance may occur in part by attenuating the mast cell response. In the present study, primary human skin mast cells were used to establish and characterize an in vitro model of desensitization. Mast cells in culture were armed with allergen-specific (4-hydroxy-3-nitrophenylacety and Der p2) and non-specific IgE antibodies, and then desensitized by incremental exposures to 4-hydroxy-3-nitrophenylacety-BSA. This desensitization procedure abrogated the subsequent degranulation response to the desensitizing allergen, to an unrelated allergen, and to IgG anti-FcεRI, but not to C5a, substance P, compound 48/80, and calcium ionophore. Desensitized cells regained their FcεRI-dependent degranulation capability by 24-48 h after free allergen had been removed. Therefore, sensitized human skin mast cells are reversibly desensitized in vitro by exposure to incremental doses of that allergen, which also cross-desensitizes them to an unrelated allergen.

Processing of human protryptase in mast cells involves cathepsins L, B, and C.

Human ß-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. ß-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human ß-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature ß-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.

Disparity in FcεRI-induced degranulation of primary human lung and skin mast cells exposed to adenosine.

Inhaled and intravenously administered adenosine induces mast cell-mediated (histamine-dependent) bronchospasm in asthmatics without causing urticaria. A differential response to adenosine by human lung and skin mast cells is shown: low concentrations potentiate FcεRI-induced degranulation of human lung mast cells but not that of skin mast cells. Human lung mast cells were found to express ∼ 3-fold more A3AR messenger RNA (mRNA) than skin mast cells, suggesting the involvement of the G(i)-linked A3AR. Indeed, the adenosine-induced potentiation was sensitive to inhibition by pertussis toxin and, furthermore, could be induced with an A3AR-specific agonist. This study reveals a previously unrecognized disparity in the response to adenosine by primary human mast cells from lung and skin that might explain why adenosine induces a pulmonary but not dermatologic allergy-like response in vivo. In addition, we identify the A3AR as a potentiating receptor of FcεRI-induced degranulation, thereby implicating it in the in vivo bronchoconstrictive response to adenosine in asthmatics.

Differential susceptibility to chemically induced thymic lymphomas in SENCARB and SSIN inbred mice.

In the past 20 yr, several inbred strains have been derived from SENCAR outbred mice. These strains display different susceptibility to the induction of papillomas and progression to squamous cell carcinomas (SCC) in the skin after chemical carcinogenesis. In the present study, we showed that one of these strains SENCARB/Pt was highly susceptible to the development of N-methyl-N-nitrosourea (MNU)- and 7,12-dimethylbenz[a]anthracene (DMBA)-induced lymphomas. In contrast, the SSIN/Sprd inbred strain is completely resistant to T-cell lymphomagenesis by both carcinogens. Within 175 d after a single injection of 75 mg/kilogram body weight (kbw) of MNU, SENCARB/Pt mice exhibited a 91.6% incidence of lymphoma. In addition, during an independent tumorigenesis study with repeated doses of intragastric DMBA, SENCARB/Pt mice showed an incidence of 75% lymphoma development 300 d after the last treatment. Histopathological and flow cytometric parameters indicated that the lymphomas were of the T-cell lineage. In order to study the genetics of MNU-induced tumorigenesis, we generated F1 hybrid mice between SSIN/Sprd and SENCARB/Pt mice. Tumor incidence in MNU-injected F1 mice suggested that the high tumor incidence is a dominant trait. Loss of heterozygosity (LOH) analysis in these tumor samples revealed allelic imbalances on chromosomes 15 and 19. Given that these inbred strains are closely related, it is likely that a relatively small number of loci are responsible for the observed differences in susceptibility. Therefore, these SENCAR inbred strains constitute important new tools to study the genetic basis of resistance and susceptibility to chemically induced thymic lymphoma formation.

Impaired FcepsilonRI-dependent gene expression and defective eicosanoid and cytokine production as a consequence of Fyn deficiency in mast cells.

Fyn kinase is a key contributor in coupling FcepsilonRI to mast cell degranulation. A limited macroarray analysis of FcepsilonRI-induced gene expression suggested potential defects in lipid metabolism, eicosanoid and glutathione metabolism, and cytokine production. Biochemical analysis of these responses revealed that Fyn-deficient mast cells failed to secrete the inflammatory eicosanoid products leukotrienes B4 and C4, the cytokines IL-6 and TNF, and chemokines CCL2 (MCP-1) and CCL4 (MIP-1beta). FcepsilonRI-induced generation of arachidonic acid and normal induction of cytokine mRNA were defective. Defects in JNK and p38 MAPK activation were observed, whereas ERK1/2 and cytosolic phospholipase A2 (S505) phosphorylation was normal. Pharmacological studies revealed that JNK activity was associated with generation of arachidonic acid. FcepsilonRI-mediated activation of IkappaB kinase beta and IkappaBalpha phosphorylation and degradation was defective resulting in a marked decrease of the nuclear NF-kappaB DNA binding activity that drives IL-6 and TNF production in mast cells. However, not all cytokine were affected, as IL-13 production and secretion was enhanced. These studies reveal a major positive role for Fyn kinase in multiple mast cell inflammatory responses and demonstrate a selective negative regulatory role for certain cytokines.

TGF-beta1 inhibits late-stage mast cell maturation.

OBJECTIVE: The objective of this study was to determine the effects of transforming growth factor (TGF)-beta1 on mast cell development. MATERIALS AND METHODS: Mast cells were cultured from mouse bone marrow in interleukin (IL)-3 + stem cell factor, in the presence or absence of TGF-beta1. We assessed mast cell development by measuring the expression of kit, T1/ST2, FcvarepsilonRI, and Fcgamma receptors. Cell morphology was determined by histochemical staining. Alterations in FcvarepsilonRI subunit expression were measured by Western blot analysis. Adoptive transfer of cultured mast cells into mast cell-deficient W/W(v) mice was used to determine if the in vivo environment could reverse the inhibitory effects of TGF-beta1. RESULTS: TGF-beta1 decreased FcvarepsilonRI, c-kit, T1/ST2, and FcgammaR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3 deficiency did not alter the response of bone marrow cells to TGF-beta1. TGF-beta1 inhibited expression of the FcvarepsilonRI alpha subunit protein, without decreasing beta or gamma proteins. Mast cells derived in the presence of TGF-beta1 were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to W/W(v) mice. CONCLUSIONS: TGF-beta1 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGF-beta1 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGF-beta1 can serve as a potent and multifunctional regulator of mast cell maturation.

TGF-beta 1 inhibits mast cell Fc epsilon RI expression.

Mast cell activation through the high affinity IgE receptor (FcepsilonRI) is a critical component of atopic inflammation. The cytokine TGF-beta1 has been shown to inhibit IgE-dependent mast cell activation, possibly serving to dampen mast cell-mediated inflammatory responses. We present proof that TGF-beta1 inhibits mast cell FcepsilonRI expression through a reversible pathway that diminishes protein, but not mRNA, expression of the FcepsilonRI subunit proteins alpha, beta, and gamma. The stability of the expressed proteins and the assembled cell surface complex was unaltered by TGF-beta1 treatment. However, TGF-beta1 decreased the rate of FcepsilonRI beta-chain synthesis, arguing that this inhibitory cytokine exerts its effects at the level of mRNA translation. TGF-beta1 consistently diminished FcepsilonRI expression on cultured human or mouse mast cells as well as freshly isolated peritoneal mast cells. The related cytokines, TGF-beta2 and TGF-beta3, had similar effects. We propose that TGF-beta1 acts as a negative regulator of mast cell function, in part by decreasing FcepsilonRI expression.

The role of Src family kinases in mast cell effector function.

Src family protein tyrosine kinases (SrcPTK) play a central role in immunoglobulin E (IgE)-mediated activation of mast cells. Functional coupling of the high-affinity IgE receptor (FcepsilonRI) is initiated by the SrcPTK family member, Lyn, through an antigen aggregation-dependent transphosphorylation. Because Lyn is the 'initiating' kinase, an essential role in mast cell effector function was conferred. Recent studies challenge this view. Evidence demonstrating that Lyn kinase is dispensable for mast cell degranulation is now available. In contrast, another SrcPTK family member, Fyn, is required for degranulation and cytokine production. New studies, on mast cells expressing FcepsilonRIbeta ITAM mutants, show that the loss of Lyn interaction with FcepsilonRI has only a modest inhibitory effect on mast cell degranulation and an enhancing effect on lymphokine production, although many of the biochemical signals (including FcepsilonRI phosphorylation) were significantly impaired. In vivo studies on Lyn-null mice also demonstrated that this kinase is a negative regulator of IgE production and anaphylaxis, whereas Fyn kinase is required for anaphylaxis but not for IgE production. Collectively, these studies argue that sustained Lyn kinase activity negatively regulates mast cell responses. This suggests the possible existence of Lyn polymorphisms that may contribute in allergic disease.

Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase.

A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn-/- bone marrow-derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn-/- mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn-/- mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcepsilonRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcepsilonRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn-/- mice because both lyn-/- and lyn-/- fyn-/- mice showed high IgE levels. Thus, lyn-/- mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.

Differential requirement for A2a and A3 adenosine receptors for the protective effect of inosine in vivo.

Inosine is an endogenous nucleoside with immunosuppressive properties that is known to inhibit the accumulation of proinflammatory cytokines and protect mice from endotoxin-induced inflammation and lung tissue damage. There are no known receptors specific for inosine, but A3 adenosine receptors (A3Rs) have been shown to bind inosine, resulting in mast cell degranulation and increased vascular permeability. The present study specifically addresses the requirement for A2aR and/or A3R for the protective effect of inosine in 2 experimental in vivo models of inflammatory disease. The data show that A3R is essential for protection against ConA-induced fulminant hepatitis since only A3R-expressing mice were protected by inosine whereas wild-type and A2aR-deficient mice exhibited severe liver damage even after administration of inosine. In addition, we show in a model of LPS-induced endotoxemia that inosine protected both A2aR-/- and A3R-/- mice from inflammation, but not A2aA3R double-null mice, indicating that in this model both A2aR and A3R were used by inosine. Thus, we demonstrate that A2a and A3 adenosine receptors are differentially utilized by inosine for the down-regulation of tissue damage under different inflammatory conditions in vivo.

Targeting G protein-coupled A2a adenosine receptors to engineer inflammation in vivo.

G protein-coupled adenosine receptors are the subject of intense study as immunomodulators of inflammation especially since the recent demonstration that the A2a receptor acts to down-regulate inflammation and inhibit tissue damage in vivo [Nature 414 (6866) (2001) 916]. The adverse effects of overactive inflammation are evident in diseases e.g. sepsis, rheumatoid arthritis, and multiple sclerosis underscoring the importance of inhibiting inflammation or selectively enhancing inflammatory processes. It has been shown recently that the A2a adenosine receptor is a critical component of an endogenous "immunosuppressive loop" in which extracellular adenosine that accumulates due to local hypoxia caused by inflammatory insult signals through cAMP-elevating A2a receptors in a delayed negative feedback manner. Understanding how tissues regulate inflammation will provide the information necessary to allow for the engineering, or selective targeting, of endogenous inflammatory pathways. Recognition of A2a receptors as "natural" or endogenous brakes of inflammation provides the intellectual scaffolding needed to pursue these goals.