title: Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.
abstract: Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs), commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos) would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway), while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD), which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal) or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery); subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2), and an IL-5-dependent increase in interleukin-1β (IL-1β) mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, MβCD+Chol pretreatment attenuated an IL-5-induced increase in cell survival at 48 hours, measured as total cellular metabolism. The reduction in cell survival following cholesterol addition despite unaltered STAT phosphorylation contradicts the current dogma in which JAK/STAT activation is sufficient to promote eosinophil survival, and suggests an additional, unidentified mechanism critically regulates IL-5-mediated human PBEos survival.
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, United States of America.
authors.forename: Mandy E
authors.initials: ME
authors.lastname: Burnham
authors.affiliation: Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, United States of America.
authors.forename: Stephane
authors.initials: S
authors.lastname: Esnault
authors.affiliation: Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, United States of America.
authors.forename: Elon C
authors.initials: EC
authors.lastname: Roti Roti
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, United States of America.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Bates
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, United States of America.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Bertics
authors.affiliation: Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, Madison, WI, United States of America.
authors.forename: Loren C
authors.initials: LC
authors.lastname: Denlinger
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D004804:Eosinophils / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D015848:Interleukin-5 / Q000378:metabolism*; D053612:Janus Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: C508599:IL5 protein, human; D053583:Interleukin-1beta; D015848:Interleukin-5; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D053612:Janus Kinases; D048051:p38 Mitogen-Activated Protein Kinases