Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes

Sepsis results in hepatic "growth hormone (GH) resistance" with reductions in plasma IGF-I despite a two- to fourfold increase in circulating GH. In this study, we examine the effects of IL-1 on GH receptor (GHR) expression, GH signaling (via the JAK/STAT and MAPK pathways), and the induction of gene expression [IGF-I mRNA and serine protease inhibitor (Spi) 2.1] by GH in CWSV-1 hepatocytes. Incubation of cells with IL-1beta (10 ng/ml, 24 h) had no effect on the relative abundance of GHR or signaling proteins JAK2, STAT5b, and ERK1/2 in cell lysates. Baseline phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 was minimal. After GH stimulation, tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 increased 2- to 10-fold. However, neither the time course nor the magnitude of GHR, JAK2, and ERK1/2 phosphorylation by GH were significantly altered by IL-1. The GH-induced translocation of STAT5b to the nucleus was not prevented by IL-1. Although phosphorylated STAT5 in nuclear extracts from GH + IL-1 cells was decreased by 24% (vs. controls) 15 min after GH stimulation, this did not result in reduced STAT5-DNA binding activity. Pretreatment with IL-1 did not significantly decrease IGF-I mRNA stability. We conclude that IL-1 only minimally affects the time course of JAK2/STAT5 and MAPK signaling by GH. Therefore, an inhibitory effect of IL-1 on IGF-I and Spi 2.1 mRNA synthesis by GH represents the most likely mechanism for IL-1-mediated GH resistance.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.     

A New IRAK-M-Mediated Mechanism Implicated in the Anti-Inflammatory Effect of Nicotine via α7 Nicotinic Receptors in Human Macrophages

Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the ‘endotoxin tolerant’ phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.     

Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A(1), A(2a), A(2b), A(3)) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A(2) receptor agonists and was blocked by selective A(2a) and A(2b) receptor antagonists, indicating that adenosine was mediating its effect through A(2) receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A(2) receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.     

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca²⁺-activated K⁺ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit.     

A Role for Transcription Factor STAT3 Signaling in Oncogene Smoothened-driven Carcinogenesis

Activation of the Hedgehog (Hh) pathway is known to drive development of basal cell carcinoma and medulloblastomas and to associate with many other types of cancer, but the exact molecular mechanisms underlying the carcinogenesis process remain elusive. We discovered that skin tumors derived from epidermal expression of oncogenic Smo, SmoM2, have elevated levels of IL-11, IL-11Rα, and STAT3 phosphorylation at Tyr⁷⁰⁵. The relevance of our data to human conditions was reflected by the fact that all human basal cell carcinomas examined have detectable STAT3 phosphorylation, mostly in keratinocytes. The functional relevance of STAT3 in Smo-mediated carcinogenesis was revealed by epidermal specific knockout of STAT3. We showed that removal of STAT3 from mouse epidermis dramatically reduced SmoM2-mediated cell proliferation, leading to a significant decrease in epidermal thickness and tumor development. We also observed a significant reduction of epidermal stem/progenitor cell population and cyclin D1 expression in mice with epidermis-specific knockout of STAT3. Our evidence indicates that STAT3 signaling activation may be mediated by the IL-11/IL-11Rα signaling axis. We showed that tumor development was reduced after induced expression of SmoM2 in IL-11Rα null mice. Similarly, neutralizing antibodies for IL-11 reduced the tumor size. In two Hh-responsive cell lines, ES14 and C3H10T1/2, we found that addition of Smo agonist purmorphamine is sufficient to induce STAT3 phosphorylation at Tyr⁷⁰⁵, but this effect was abolished after IL-11Rα down-regulation by shRNAs. Taken together, our results support an important role of the IL-11Rα/STAT3 signaling axis for Hh signaling-mediated signaling and carcinogenesis.     

An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells

BackgroundMultidrug resistant cancer cells are hard to eradicate for the inefficacy of conventional anticancer drugs. Besides escaping the cytotoxic effects of chemotherapy, they also bypass the pro-immunogenic effects induced by anticancer drugs: indeed they are not well recognized by host dendritic cells and do not elicit a durable anti-tumor immunity. It has not yet been investigated whether multidrug resistant cells have a different ability to induce immunosuppression than chemosensitive ones. We addressed this issue in human and murine chemosensitive and multidrug resistant cancer cells.ResultsWe found that the activity and expression of indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the conversion of tryptophan into the immunosuppressive metabolite kynurenine, was higher in all the multidrug resistant cells analyzed and that IDO1 inhibition reduced the growth of drug-resistant tumors in immunocompetent animals. In chemoresistant cells the basal activity of JAK1/STAT1 and JAK1/STAT3 signaling was higher, the STAT3 inhibitor PIAS3 was down-regulated, and the autocrine production of STAT3-target and IDO1-inducers cytokines IL-6, IL-4, IL-1β, IL-13, TNF-α and CD40L, was increased. The disruption of the JAK/STAT signaling lowered the IDO1 activity and reversed the kynurenine-induced pro-immunosuppressive effects, as revealed by the restored proliferation of T-lymphocytes in STAT-silenced chemoresistant cells.ConclusionsOur work shows that multidrug resistant cells have a stronger immunosuppressive attitude than chemosensitive cells, due to the constitutive activation of the JAK/STAT/IDO1 axis, thus resulting chemo- and immune-evasive. Disrupting this axis may significantly improve the efficacy of chemo-immunotherapy protocols against resistant tumors.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

Differential regulation of interleukin 5-stimulated signaling pathways by dynamin

Through the yeast two-hybrid screen we have identified dynamin-2 as a molecule that interacts with the alpha subunit of the interleukin (IL) 5 receptor. Dynamin-2 is a GTPase that is critical for endocytosis. We have shown that dynamin-2 interacts with the IL-5 receptor-associated tyrosine kinases, Lyn and JAK2, in eosinophils. Tyrosine phosphorylation of dynamin is markedly enhanced upon IL-5 stimulation. The inhibition of tyrosine kinases results in complete abolition of ligand-induced receptor endocytosis. Inhibition of dynamin by a dominant-negative mutant or by small interfering RNA results in enhancement of IL-5-stimulated ERK1/2 signaling and cell proliferation. In contrast, the absence of a functional dynamin does not affect STAT5 or AKT phosphorylation or cell survival. Thus, we have identified specific functions for dynamin in the IL-5 signaling pathway and demonstrated its role in receptor endocytosis and termination of the ERK1/2 signaling pathway.     

IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCdelta

CD86 expression is up-regulated in activated monocytes and macrophages by a mechanism that is not clearly defined. Here, we report that IL-4-dependent CD86 expression requires activation of ERK1/2 and JAK/STAT6 but is negatively regulated by PKCdelta. PMA differentiated U937 monocytic cells when stimulated with IL-4 increased CD11b and CD86 expression by 52- and 98-fold, respectively. PMA+IL-4 treatment also induced a synergistic enhancement of ERK1/2 activation when compared to the effects of PMA and IL-4 alone. Use of the mitogen or extracellular kinase (MEK) inhibitor, PD98059, completely blocked up-regulation of CD11b and CD86 demonstrating the importance of MEK-activated ERK1/2. JAK inhibition with WHI-P154-abrogated IL-4-dependent CD11b and CD86 up-regulation and inhibited STAT6 tyrosine phosphorylation. Importantly, CD11b and CD86 expression were not reliant on IL-4-dependent activation of phosphatidylinositol 3'-kinase (PI 3-kinase). Blockade of PKCdelta activation with rottlerin prevented CD11b expression but lead to a 75- and 213-fold increase in PMA and PMA+IL-4-dependent CD86 expression, respectively. As anticipated, increasing PKCdelta activity with anti-sense reduction of CD45 increased CD11b expression and reduced CD86 expression. Likewise, rottlerin prevented nuclear localization of activated PKCdelta. We conclude from these data that IL-4-dependent CD11b expression relies predominantly on enhanced activation of ERK1/2, while IL-4-dependent CD86 expression utilizes the JAK/STAT6 pathway.     

The Macrophage Cholesterol Exporter ABCA1 Functions as an Anti-inflammatory Receptor

ATP-binding cassette transporter A1 (ABCA1) is a cell membrane protein that exports excess cholesterol from cells to apolipoprotein (apo) A-I, the major protein in high density lipoproteins. Genetic studies have shown that ABCA1 protects against cardiovascular disease. The interaction of apoA-I with ABCA1 promotes cholesterol removal and activates signaling molecules, such as Janus kinase 2 (JAK2), that optimize the lipid export activity of ABCA1. Here we show that the ABCA1-mediated activation of JAK2 also activates STAT3, which is independent of the lipid transport function of ABCA1. ABCA1 contains two candidate STAT3 docking sites that are required for the apoA-I/ABCA1/JAK2 activation of STAT3. The interaction of apoA-I with ABCA1-expressing macrophages suppressed the ability of lysopolysaccaride to induce the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α, which was reversed by silencing STAT3 or ABCA1. Thus, the apoA-I/ABCA1 pathway in macrophages functions as an anti-inflammatory receptor through activation of JAK2/STAT3. These findings implicate ABCA1 as a direct molecular link between the cardioprotective effects of cholesterol export from arterial macrophages and suppressed inflammation.     

Intracellular Interaction of Interleukin (IL)-32α with Protein Kinase C? (PKC?) and STAT3 Protein Augments IL-6 Production in THP-1 Promonocytic Cells

IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCϵ inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCϵ, and this interaction was totally inhibited by the PKCϵ inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCϵ and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCϵ and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.     

Delta-Tocotrienol Suppresses Radiation-Induced MicroRNA-30 and Protects Mice and Human CD34+ Cells from Radiation Injury

We reported that microRNA-30c (miR-30c) plays a key role in radiation-induced human cell damage through an apoptotic pathway. Herein we further evaluated radiation-induced miR-30 expression and mechanisms of delta-tocotrienol (DT3), a radiation countermeasure candidate, for regulating miR-30 in a mouse model and human hematopoietic CD34+ cells. CD2F1 mice were exposed to 0 (control) or 7–12.5 Gy total-body gamma-radiation, and CD34+ cells were irradiated with 0, 2 or 4 Gy of radiation. Single doses of DT3 (75 mg/kg, subcutaneous injection for mice or 2 μM for CD34+ cell culture) were administrated 24 h before irradiation and animal survival was monitored for 30 days. Mouse bone marrow (BM), jejunum, kidney, liver and serum as well as CD34+ cells were collected at 1, 4, 8, 24, 48 or 72 h after irradiation to determine apoptotic markers, pro-inflammatory cytokines interleukin (IL)-1β and IL-6, miR-30, and stress response protein expression. Our results showed that radiation-induced IL-1β release and cell damage are pathological states that lead to an early expression and secretion of miR-30b and miR-30c in mouse tissues and serum and in human CD34+ cells. DT3 suppressed IL-1β and miR-30 expression, protected against radiation-induced apoptosis in mouse and human cells, and increased survival of irradiated mice. Furthermore, an anti-IL-1β antibody downregulated radiation-induced NFκBp65 phosphorylation, inhibited miR-30 expression and protected CD34+ cells from radiation exposure. Knockdown of NFκBp65 by small interfering RNA (siRNA) significantly suppressed radiation-induced miR-30 expression in CD34+ cells. Our data suggest that DT3 protects human and mouse cells from radiation damage may through suppression of IL-1β-induced NFκB/miR-30 signaling.     

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.