Protein Phosphatase 2A and Protein Kinase Cα Are Physically Associated and Are Involved in Pseudomonas aeruginosa-induced Interleukin 6 Production by Mast Cells

Abstract

Pulmonary infection with Pseudomonas aeruginosa is characterized by massive airway inflammation, which comprises significant cytokine production. Although mast cells are abundant in the lung and are potent sources of various cytokines, a role of mast cells in P. aeruginosa infection remains undefined, and P. aeruginosa-induced signaling mechanisms in mast cells have not been studied previously. Here we demonstrate that human cord blood-derived mast cells, mouse bone marrow-derived mast cells, and the mouse mast cell line MC/9 produce significant amounts of interleukin 6 (IL-6) in response to P. aeruginosa. This response was accompanied by a stimulation of protein kinase C␣ (PKC␣) phosphorylation and PKC activity and was significantly blocked by the PKC inhibitors Ro 31-8220 and PKC␣ pseudosubstrate. Interestingly, mast cells treated with P. aeruginosa had reduced protein levels of phosphatase 2A catalytic unit (PP2Ac), which prompted us to determine whether a direct association between PKC␣ and PP2A occurs in mast cells. In mouse bone marrowderived mast cells and MC/9 cells, as well as in the human mast cell line HMC-1, PP2A coimmunoprecipitated with PKC␣ either using PKC␣-or PP2Ac-specific antibodies, suggesting that PKC␣ and PP2Ac are physically associated in mast cells. The PP2A inhibitor okadaic acid induced P. aeruginosa-like responses in mast cells including increased PKC␣ phosphorylation, stimulated PKC activity, and augmented IL-6 production, the last being blocked by the PKC inhibitor Ro 31-8220. Finally, okadaic acid potentiated the P. aeruginosa-induced IL-6 production. Collectively, these data provide, to our knowledge, the first evidence of both a direct physical association of PP2A and PKC␣ in mammalian cells and their coinvolvement in regulating mast cell activation in response to P. aeruginosa.

Pseudomonas aeruginosaActivates Human Mast Cells to Induce Neutrophil Transendothelial Migration Via Mast Cell-Derived IL-1α and β

“…However, current knowledge of MC cytokine production in the context of bacterial infection is extremely limited. In humans, TNF-␣ and IL-6 are the only previously reported cytokines produced by human MC after bacterial infection (17,31,32). We attempted to determine those P. aeruginosa-induced MC mediators responsible for enhanced human PMN transmigration across endothelium.…”

“…Highly purified CBMC were obtained by long-term culture of cord blood progenitor cells, as previously described (16,17). After Ͼ8 wk in culture, mature MC (Ͼ95% purity) were identified by their morphological features and the presence of metachromatic granules (toluidine blue staining).…”

Regulation and Function of the Calcium/Calmodulin-dependent Protein Kinase IV/Protein Serine/Threonine Phosphatase 2A Signaling Complex

“…In the case of protein phosphorylation, macromolecular protein complexes may restrict access of kinases and phosphatases to specific microenvironments and position them to respond rapidly to appropriate signals. In recent years several kinases in addition to CaMKIV, including CK2, p70 S6 kinase, p21-activated kinase, Jak2, I ␤ kinase, and protein kinase C, have also been shown to interact with PP2A (27)(28)(29)(30)(31)(32)(33). However, in most of these cases details describing a precise functional link between the kinase and PP2A are lacking.…”

Participation of mast cells in chronic otitis media

“…Bacterial toxins may activate MCs which, in response to infection, release many mediators causing an increase in vascular permeability and in the expression of adhesion molecules or act chemotactically directly on neutrophils and eosinophils, and also produce anti-microbial peptides like cathelicidins and defensins. Activation of human mast cells has been demonstrated for Staphylococcus aureus and Pseudomonas aeruginosa, which are the most common isolates from chronic otitis media [37][38][39][40]. Chonmaitree et al [19] reported that histamine levels were higher in bacteria-positive than bacteria-negative effusions in acute otitis media.…”