Dataset Information

Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3.

ABSTRACT: Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marked by increased reactivity to aggregated immunoglobulin-G (IgG). To characterize this phenotype, we quantified protein abundance and phosphorylation by multiplexed isobaric labeling combined with high-resolution mass spectrometry. Purified blood eosinophils of five individuals were treated with IL-3 or no cytokine for 20 h, and comparative data were obtained on abundance of 5385 proteins and phosphorylation at 7330 sites. The 1150 proteins that were significantly up-regulated ( q < 0.05, pairwise t test with Benjamini-Hochberg correction) by IL-3 included the IL3RA and CSF2RB subunits of the IL-3 receptor, the low-affinity receptor for IgG (FCGR2B), 96 proteins involved in protein translation, and 55 proteins involved in cytoskeleton organization. Among the 703 proteins that decreased were 78 mitochondrial proteins. Dynamic regulation of protein phosphorylation was detected at 4218 sites. These included multiple serines in CSF2RB; Y694 of STAT5, a key site of activating phosphorylation downstream of IL3RA/CSF2RB; and multiple sites in RPS6KA1, RPS6, and EIF4B, which are responsible for translational initiation. We conclude that IL-3 up-regulates overall protein synthesis and targets specific proteins for up-regulation, including its own receptor.

SUBMITTER: Esnault S

PROVIDER: S-EPMC5984179 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3.

Esnault Stephane S Hebert Alexander S AS Jarjour Nizar N NN Coon Joshua J JJ Mosher Deane F DF

Journal of proteome research 20180504 6

Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marked by increased reactivity to aggregated immunoglobulin-G (IgG). To characterize this phenotype, we quantified protein abundance and phosphorylation by multiplexed isobaric labeling combined with high-resolution mass spectrometry. Purified blood eosinophils of five individuals were treated with IL-3 or no cytokine for 20 h, and comparative data were obtained on abundance of 5385 proteins and phosphory ...[more]

PMID: 29706072

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Project description: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-?-cyclodextrin (M?CD), which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether M?CD is preloaded with cholesterol. Human PBEos were pretreated with M?CD (cholesterol removal) or M?CD+Cholesterol (M?CD+Chol; cholesterol delivery); subsequent IL-5-stimulated signaling and physiological endpoints were assessed. M?CD 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-1? (IL-1?) mRNA levels. In contrast, M?CD+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, M?CD+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.

Project description:BackgroundIL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the "nucleopod," which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators.ObjectiveDetermine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin.MethodsBlood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed.ResultsAfter 10 minutes in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30-60 minutes, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin.Conclusions and clinical relevanceMigrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target.

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Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3.

Publications

Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3.

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