Priming of Eosinophils by GM-CSF Is Mediated by Protein Kinase CβII-Phosphorylated L-Plastin

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 which we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. In addition, co-immunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with Protein Kinase C βII (PKCβII), GM-CSF receptor α chain, and two actin associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis (4-fluoroanilino)phtalimide or PKCβII specific siRNA blocked GM-CSF induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of αMβ2 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 ECP and EPX in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2–19) phosphorylated on Ser5 upregulated αMβ2 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.

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

“…PKC inhibitors have previously been demonstrated to inhibit fMLP-mediated phosphorylation of L-plastin in neutrophils (48), whereas PKC b II and PKC d have recently been implicated in L-plastin phosphorylation in eosinophils (49) and breast cancer cells (17), respectively. Furthermore, several PKC isoforms such as PKC b I , PKC d, PKC u, and PKC z colocalized with L-plastin at the leading edge of T lymphocytes after SDF-1a stimulation.…”

GM-CSF: An immune modulatory cytokine that can suppress autoimmunity

“…It can augment eosinophil survival and phagocytosis (73, 74). Furthermore, GMCSF can also direct eosinophil priming by which they become receptive to chemotaxins and degranulating factors (75). In one study using a murine model of autoimmune gastritis, it was show that hypertrophy of gastric mucosa was associated with infiltrating eosinophils and correlated with higher GM-CSF expression in the spleen (76).…”

Airway exposure to staphylococcal enterotoxin A potentiates allergen-induced bone marrow eosinophilia and trafficking to peripheral blood and airways

“…MF, mean fluorescence. (34,39). CCR3 behaves as a functional receptor responsible for transducing the chemotactic responses after binding to eotaxin (12).…”

“…In BM, immunopathological correlations between GM-CSF and eosinophil activation have been reported (34,35). In addition to its critical role in eosinophil survival, GM-CSF initiates a priming state whereby eosinophils become responsive to chemotactic, which may be accompanied by upregulation of adhesion molecules in cell surface (35).…”