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.

Laboratory of Signal Transduction Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

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Peoples RW, Li C, Weight FF. Lipid vs protein theories of alcohol action in the nervous system. Annu Rev Pharmacol Toxicol. 1996; 36: 185–201. 10.1146/annurev.pa.36.040196.001153 PubMed DOI

Meyer HH. Zur theorie der alkoholnarkose. I. Mitt. welche eigenschaft der anästhetika bedingt ihre narkotische wirkung? Arch Exp Pathol Pharmakol. 1899; 42: 109–108.

Meyer KH. Contribution to the theory of narcosis. Trans Faraday Soc. 1937; 33: 1062–1068.

Chin JH, Goldstein DB. Effects of low concentrations of ethanol on the fluidity of spin-labeled erythrocyte and brain membranes. Mol Pharmacol. 1977; 13: 435–441. PubMed

Chen SY, Yang B, Jacobson K, Sulik KK. The membrane disordering effect of ethanol on neural crest cells in vitro and the protective role of GM1 ganglioside. Alcohol. 1996; 13: 589–595. S0741832996000730. PubMed

Rowe ES. Thermodynamic reversibility of phase transitions. Specific effects of alcohols on phosphatidylcholines. Biochim Biophys Acta. 1985; 813: 321–330. PubMed

Gray E, Karslake J, Machta BB, Veatch SL. Liquid general anesthetics lower critical temperatures in plasma membrane vesicles. Biophys J. 2013; 105: 2751–2759. S0006-3495(13)01233-2. 10.1016/j.bpj.2013.11.005 PubMed DOI PMC

Ly HV, Longo ML. The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers. Biophys J. 2004; 87: 1013–1033. 10.1529/biophysj.103.034280 PubMed DOI PMC

Seeman P. The membrane actions of anesthetics and tranquilizers. Pharmacol Rev. 1972; 24: 583–655. PubMed

Franks NP, Lieb WR. Molecular mechanisms of general anaesthesia. Nature. 1982; 300: 487–493. PubMed

Franks NP, Lieb WR. Partitioning of long-chain alcohols into lipid bilayers: implications for mechanisms of general anesthesia. Proc Natl Acad Sci U S A. 1986; 83: 5116–5120. PubMed PMC

Ronald KM, Mirshahi T, Woodward JJ. Ethanol inhibition of N-methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue. J Biol Chem. 2001; 276: 44729–44735. 10.1074/jbc.M102800200 PubMed DOI

Ren H, Zhao Y, Dwyer DS, Peoples RW. Interactions among positions in the third and fourth membrane-associated domains at the intersubunit interface of the N-methyl-D-aspartate receptor forming sites of alcohol action. J Biol Chem. 2012; 287: 27302–27312. 10.1074/jbc.M111.338921 PubMed DOI PMC

Borghese CM, Storustovu S, Ebert B, Herd MB, Belelli D, Lambert JJ, et al. The δ subunit of γ-aminobutyric acid type A receptors does not confer sensitivity to low concentrations of ethanol. J Pharmacol Exp Ther. 2006; 316: 1360–1368. 10.1124/jpet.105.092452 PubMed DOI

Brayton RG, Stokes PE, Schwartz MS, Louria DB. Effect of alcohol and various diseases on leukocyte mobilization, phagocytosis and intracellular bacterial killing. N Engl J Med. 1970; 282: 123–128. 10.1056/NEJM197001152820303 PubMed DOI

Gluckman SJ, MacGregor RR. Effect of acute alcohol intoxication on granulocyte mobilization and kinetics. Blood. 1978; 52: 551–559. PubMed

Szabo G, Dolganiuc A, Dai Q, Pruett SB. TLR4, ethanol, and lipid rafts: a new mechanism of ethanol action with implications for other receptor-mediated effects. J Immunol. 2007; 178: 1243–1249. 178/3/1243. PubMed

Rimland D, Hand WL. The effect of ethanol on adherence and phagocytosis by rabbit alveolar macrophages. J Lab Clin Med. 1980; 95: 918–926. PubMed

Karavitis J, Murdoch EL, Deburghgraeve C, Ramirez L, Kovacs EJ. Ethanol suppresses phagosomal adhesion maturation, Rac activation, and subsequent actin polymerization during FcγR-mediated phagocytosis. Cell Immunol. 2012; 274: 61–71. 10.1016/j.cellimm.2012.02.002 PubMed DOI PMC

Ghare S, Patil M, Hote P, Suttles J, McClain C, Barve S, et al. Ethanol inhibits lipid raft-mediated TCR signaling and IL-2 expression: potential mechanism of alcohol-induced immune suppression. Alcohol Clin Exp Res. 2011; 35: 1435–1444. 10.1111/j.1530-0277.2011.01479.x PubMed DOI PMC

Pruett SB, Schwab C, Zheng Q, Fan R. Suppression of innate immunity by acute ethanol administration: a global perspective and a new mechanism beginning with inhibition of signaling through TLR3. J Immunol. 2004; 173: 2715–2724. 173/4/2715 [pii]. PubMed

Mandrekar P, Dolganiuc A, Bellerose G, Kodys K, Romics L, Nizamani R, et al. Acute alcohol inhibits the induction of nuclear regulatory factor κ B activation through CD14/toll-like receptor 4, interleukin-1, and tumor necrosis factor receptors: a common mechanism independent of inhibitory κ B α degradation? Alcohol Clin Exp Res. 2002; 26: 1609–1614. 10.1097/01.ALC.0000036926.46632.57 PubMed DOI

Patel M, Keshavarzian A, Kottapalli V, Badie B, Winship D, Fields JZ. Human neutrophil functions are inhibited in vitro by clinically relevant ethanol concentrations. Alcohol Clin Exp Res. 1996; 20: 275–283. PubMed

Toivari M, Maki T, Suutarla S, Eklund KK. Ethanol inhibits IgE-induced degranulation and cytokine production in cultured mouse and human mast cells. Life Sci. 2000; 67: 2795–2806. S0024320500008638. PubMed

Kennedy RH, Pelletier JH, Tupper EJ, Hutchinson LM, Gosse JA. Estrogen mimetic 4-tert-octylphenol enhances IgE-mediated degranulation of RBL-2H3 mast cells. J Toxicol Environ Health A. 2012; 75: 1451–1455. 10.1080/15287394.2012.722184 PubMed DOI

Rivera J, Kinet J-P, Kim J, Pucillo C, Metzger H. Studies with a monoclonal antibody to the β subunit of the receptor with high affinity for immunoglobulin E. Mol Immunol. 1988; 25: 647–661. PubMed

Rudolph AK, Burrows PD, Wabl MR. Thirteen hybridomas secreting hapten-specific immunoglobulin E from mice with Iga or Igb heavy chain haplotype. Eur J Immunol. 1981; 11: 527–529. PubMed

Tolar P, Tumová M, Dráber P. New monoclonal antibodies recognizing the adaptor protein LAT. Folia Biol (Praha). 2001; 47: 215–217. PubMed

Dráberová L, Amoui M, Dráber P. Thy-1-mediated activation of rat mast cells: the role of Thy-1 membrane microdomains. Immunology. 1996; 87: 141–148. PubMed PMC

Volná P, Lebduška P, Dráberová L, Šímová S, Heneberg P, Boubelík M, et al. Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL. J Exp Med. 2004; 200: 1001–1013. PubMed PMC

Kovárová M, Tolar P, Arudchandran R, Dráberová L, Rivera J, Dráber P. Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcε receptor I aggregation. Mol Cell Biol. 2001; 21: 8318–8328. PubMed PMC

Draberova L, Bugajev V, Potuckova L, Halova I, Bambouskova M, Polakovicova I, et al. Transmembrane adaptor protein PAG/CBP is involved in both positive and negative regulation of mast cell signaling. Mol Cell Biol. 2014; 34: 4285–4300. 10.1128/MCB.00983-14 PubMed DOI PMC

DiVirgilio F, Steinberg TH, Swanson JA, Silverstein SC. Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions. J Immunol. 1988; 140: 915–920. PubMed

Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006; 7: R100 10.1186/gb-2006-7-10-r100 PubMed DOI PMC

Christian AE, Haynes MP, Phillips MC, Rothblat GH. Use od cyclodextrins for manipulating cellular cholesterol content. J Lipid Res. 1997; 38: 2264–2272. PubMed

Li TK, Theorell H. Human liver alcohol dehydrogenase: inhibition by pyrazole and pyrazole analogs. Acta Chem Scand. 1969; 23: 892–902. PubMed

Setiawan I, Blanchard GJ. Ethanol-induced perturbations to planar lipid bilayer structures. J Phys Chem B. 2014; 118: 537–546. 10.1021/jp410305m PubMed DOI

Loftsson T, Magnusdottir A, Masson M, Sigurjonsdottir JF. Self-association and cyclodextrin solubilization of drugs. J Pharm Sci. 2002; 91: 2307–2316. 10.1002/jps.10226 PubMed DOI

Nishijo J, Moriyama S, Shiota S. Interactions of cholesterol with cyclodextrins in aqueous solution. Chem Pharm Bull (Tokyo). 2003; 51: 1253–1257. PubMed

Zidovetzki R, Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim Biophys Acta. 2007; 1768: 1311–1324. 10.1016/j.bbamem.2007.03.026 PubMed DOI PMC

Grutzkau A, Smorodchenko A, Lippert U, Kirchhof L, Artuc M, Henz BM. LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced secretion of human mast cells. Cytometry A. 2004; 61: 62–68. 10.1002/cyto.a.20068 PubMed DOI

Yeligar SM, Harris FL, Hart CM, Brown LA. Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases. J Immunol. 2012; 188: 3648–3657. 10.4049/jimmunol.1101278 PubMed DOI PMC

Kim MJ, Nepal S, Lee ES, Jeong TC, Kim SH, Park PH. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages. Toxicol Appl Pharmacol. 2013; 273: 77–89. 10.1016/j.taap.2013.08.005 PubMed DOI

Kim MJ, Nagy LE, Park PH. Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway. Mol Pharmacol. 2014; 86: 284–296. 10.1124/mol.114.093039 PubMed DOI PMC

Swindle EJ, Coleman JW, DeLeo FR, Metcalfe DD. FcεRI- and Fcγ receptor-mediated production of reactive oxygen species by mast cells is lipoxygenase- and cyclooxygenase-dependent and NADPH oxidase-independent. J Immunol. 2007; 179: 7059–7071. PubMed

Kuehn HS, Swindle EJ, Kim MS, Beaven MA, Metcalfe DD, Gilfillan AM. The phosphoinositide 3-kinase-dependent activation of Btk is required for optimal eicosanoid production and generation of reactive oxygen species in antigen-stimulated mast cells. J Immunol. 2008; 181: 7706–7712. 181/11/7706. PubMed PMC

Kraft S, Kinet JP. New developments in FcεRI regulation, function and inhibition. Nat Rev Immunol. 2007; 7: 365–378. PubMed

Surviladze Z, Dráberová L, Kovárová M, Boubelík M, Dráber P. Differential sensitivity to acute cholesterol lowering of activation mediated via the high-affinity IgE receptor and Thy-1 glycoprotein. Eur J Immunol. 2001; 31: 1–10. PubMed

Dalton SR, Wiegert RL, Casey CA. Receptor-mediated endocytosis by the asialoglycoprotein receptor: effect of ethanol administration on endosomal distribution of receptor and ligand. Liver Int. 2003; 23: 484–491. PubMed

Methner DN, Mayfield RD. Ethanol alters endosomal recycling of human dopamine transporters. J Biol Chem. 2010; 285: 10310–10317. 10.1074/jbc.M109.029561 PubMed DOI PMC

Pascual-Lucas M, Fernandez-Lizarbe S, Montesinos J, Guerri C. LPS or ethanol triggers clathrin- and rafts/caveolae-dependent endocytosis of TLR4 in cortical astrocytes. J Neurochem. 2014; 129: 448–462. 10.1111/jnc.12639 PubMed DOI

Fattakhova G, Masilamani M, Borrego F, Gilfillan AM, Metcalfe DD, Coligan JE. The high-affinity immunoglobulin-E receptor FcεRI is endocytosed by an AP-2/clathrin-independent, dynamin-dependent mechanism. Traffic. 2006; 7: 673–685. PubMed

Cleyrat C, Darehshouri A, Anderson KL, Page C, Lidke DS, Volkmann N, et al. The architectural relationship of components controlling mast cell endocytosis. J Cell Sci. 2013; 126: 4913–4925. 10.1242/jcs.128876 PubMed DOI PMC

Nourissat P, Travert M, Chevanne M, Tekpli X, Rebillard A, Le Moigne-Muller G, et al. Ethanol induces oxidative stress in primary rat hepatocytes through the early involvement of lipid raft clustering. Hepatology. 2008; 47: 59–70. 10.1002/hep.21958 PubMed DOI

Heneberg P, Draberova L, Bambouskova M, Pompach P, Draber P. Down-regulation of protein tyrosine phosphatases activates an immune receptor in the absence of its translocation into lipid rafts. J Biol Chem. 2010; 285: 12787–12802. 10.1074/jbc.M109.052555 PubMed DOI PMC

Bugajev V, Bambousková M, Dráberová L, Dráber P. What precedes the initial tyrosine phosphorylation of the high affinity IgE receptor in antigen-activated mast cell? FEBS Lett. 2010; 584: 4949–4955. 10.1016/j.febslet.2010.08.045 PubMed DOI

Tobin SJ, Cacao EE, Hong DW, Terenius L, Vukojevic V, Jovanovic-Talisman T. Nanoscale effects of ethanol and naltrexone on protein organization in the plasma membrane studied by photoactivated localization microscopy (PALM). PLoS One. 2014; 9: e87225 10.1371/journal.pone.0087225 PubMed DOI PMC

Furlow M, Diamond SL. Interplay between membrane cholesterol and ethanol differentially regulates neutrophil tether mechanics and rolling dynamics. Biorheology. 2011; 48: 49–64. 10.3233/BIR-2011-0583 PubMed DOI

Daragan VA, Voloshin AM, Chochina SV, Khazanovich TN, Wood WG, Avdulov NA, et al. Specific binding of ethanol to cholesterol in organic solvents. Biophys J. 2000; 79: 406–415. 10.1016/S0006-3495(00)76302-8 PubMed DOI PMC

Salous AK, Ren H, Lamb KA, Hu XQ, Lipsky RH, Peoples RW. Differential actions of ethanol and trichloroethanol at sites in the M3 and M4 domains of the NMDA receptor GluN2A (NR2A) subunit. Br J Pharmacol. 2009; 158: 1395–1404. 10.1111/j.1476-5381.2009.00397.x PubMed DOI PMC

Das J, Pany S, Rahman GM, Slater SJ. PKC ε has an alcohol-binding site in its second cysteine-rich regulatory domain. Biochem J. 2009; 421: 405–413. 10.1042/BJ20082271 PubMed DOI

Olsen RW, Li GD, Wallner M, Trudell JR, Bertaccini EJ, Lindahl E, et al. Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol. Alcohol Clin Exp Res. 2014; 38: 595–603. 10.1111/acer.12283 PubMed DOI PMC

Polley A, Vemparala S. Partitioning of ethanol in multi-component membranes: effects on membrane structure. Chem Phys Lipids. 2013; 166: 1–11. 10.1016/j.chemphyslip.2012.11.005 PubMed DOI

Lisboa FA, Peng Z, Combs CA, Beaven MA. Phospholipase d promotes lipid microdomain-associated signaling events in mast cells. J Immunol. 2009; 183: 5104–5112. 10.4049/jimmunol.0802728 PubMed DOI PMC

Jenkins GM, Frohman MA. Phospholipase D: a lipid centric review. Cell Mol Life Sci. 2005; 62: 2305–2316. 10.1007/s00018-005-5195-z PubMed DOI PMC

Zhu M, Zou J, Li T, O'Brien SA, Zhang Y, Ogden S, et al. Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function. J Immunol. 2015. 10.4049/jimmunol.1500665 PubMed DOI PMC

Zhou JS, Xing W, Friend DS, Austen KF, Katz HR. Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J Exp Med. 2007; 204: 2797–2802. 10.1084/jem.20071391 PubMed DOI PMC

Happel KI, Nelson S. Alcohol, immunosuppression, and the lung. Proc Am Thorac Soc. 2005; 2: 428–432. 10.1513/pats.200507-065JS PubMed DOI

Gluckman SJ, Dvorak VC, MacGregor RR. Host defenses during prolonged alcohol consumption in a controlled environment. Arch Intern Med. 1977; 137: 1539–1543. PubMed

Szabo G, Chavan S, Mandrekar P, Catalano D. Acute alcohol consumption attenuates interleukin-8 (IL-8) and monocyte chemoattractant peptide-1 (MCP-1) induction in response to ex vivo stimulation. J Clin Immunol. 1999; 19: 67–76. PubMed

Boe DM, Nelson S, Zhang P, Bagby GJ. Acute ethanol intoxication suppresses lung chemokine production following infection with Streptococcus pneumoniae. J Infect Dis. 2001; 184: 1134–1142. 10.1086/323661 PubMed DOI

Dolganiuc A, Bakis G, Kodys K, Mandrekar P, Szabo G. Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts. Alcohol Clin Exp Res. 2006; 30: 76–85. 10.1111/j.1530-0277.2006.00003.x PubMed DOI

Dai Q, Pruett SB. Ethanol suppresses LPS-induced Toll-like receptor 4 clustering, reorganization of the actin cytoskeleton, and associated TNF-α production. Alcohol Clin Exp Res. 2006; 30: 1436–1444. 10.1111/j.1530-0277.2006.00172.x PubMed DOI

Mandrekar P, Catalano D, Szabo G. Inhibition of lipopolysaccharide-mediated NFκB activation by ethanol in human monocytes. Int Immunol. 1999; 11: 1781–1790. PubMed

Bagasra O, Howeedy A, Dorio R, Kajdacsy-Balla A. Functional analysis of T-cell subsets in chronic experimental alcoholism. Immunology. 1987; 61: 63–69. PubMed PMC

Glassman AB, Bennett CE, Randall CL. Effects of ethyl alcohol on human peripheral lymphocytes. Arch Pathol Lab Med. 1985; 109: 540–542. PubMed

Brodie C, Domenico J, Gelfand EW. Ethanol inhibits early events in T-lymphocyte activation. Clin Immunol Immunopathol. 1994; 70: 129–136. S0090122984710208 [pii]. PubMed

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