Priming of eosinophils by GM-CSF is mediated by protein kinase CbII-phosphorylated L-plastin

Konrad Pazdrak, Travis W. Young, Christof Straub, Susan Stafford, Alexander Kurosky

    Research output: Contribution to journalArticle

    28 Citations (Scopus)

    Abstract

    The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCbII with 4,5-bis (4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of αMβ 2 integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser5 upregulated αMβ 2 integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.

    Original languageEnglish (US)
    Pages (from-to)6485-6496
    Number of pages12
    JournalJournal of Immunology
    Volume186
    Issue number11
    DOIs
    StatePublished - Jun 1 2011

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    Granulocyte-Macrophage Colony-Stimulating Factor
    Eosinophils
    Protein Kinases
    Integrins
    Chemotaxis
    Actins
    Up-Regulation
    Granulocyte-Macrophage Colony-Stimulating Factor Receptors
    Eosinophil Peroxidase
    Phosphorylation
    plastin
    Actin Depolymerizing Factors
    Eosinophil Cationic Protein
    Phosphoproteins
    Chemotactic Factors
    Proteome
    Protein Kinase Inhibitors
    Small Interfering RNA
    Asthma
    Cytokines

    ASJC Scopus subject areas

    • Immunology

    Cite this

    Priming of eosinophils by GM-CSF is mediated by protein kinase CbII-phosphorylated L-plastin. / Pazdrak, Konrad; Young, Travis W.; Straub, Christof; Stafford, Susan; Kurosky, Alexander.

    In: Journal of Immunology, Vol. 186, No. 11, 01.06.2011, p. 6485-6496.

    Research output: Contribution to journalArticle

    Pazdrak, K, Young, TW, Straub, C, Stafford, S & Kurosky, A 2011, 'Priming of eosinophils by GM-CSF is mediated by protein kinase CbII-phosphorylated L-plastin', Journal of Immunology, vol. 186, no. 11, pp. 6485-6496. https://doi.org/10.4049/jimmunol.1001868
    Pazdrak, Konrad ; Young, Travis W. ; Straub, Christof ; Stafford, Susan ; Kurosky, Alexander. / Priming of eosinophils by GM-CSF is mediated by protein kinase CbII-phosphorylated L-plastin. In: Journal of Immunology. 2011 ; Vol. 186, No. 11. pp. 6485-6496.
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    abstract = "The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCbII with 4,5-bis (4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of αMβ 2 integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser5 upregulated αMβ 2 integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.",
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