Class IA Phosphatidylinositide 3-Kinases, rather than p110γ, Regulate Formyl-Methionyl-Leucyl-Phenylalanine-Stimulated Chemotaxis and Superoxide Production in Differentiated Neutrophil-Like PLB-985 Cells

Abstract

Class I PI3Ks, through the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), are thought of as essential elements of the neutrophil response to chemotactic factors. Moreover, the recent development of PI3K-deficient mice and isoform-specific inhibitors enabled examinations of the contribution of the distinct PI3K isoforms in neutrophil activation. However, the results of these various studies are conflicting, and the exact role of the different PI3K isoforms is not yet clearly established, particularly in human cells. In the present study, we used a different approach to assess the role of the distinct PI3K isoforms in response to the chemotactic agent fMLP. We inhibited PI3K activities by the transient expression following nucleofection of dominant negative mutants of either p85α or p110γ in the human myeloid cell line PLB-985, which can be induced to express a neutrophil-like phenotype. The data obtained with this approach showed that the production of PI(3,4,5)P3 triggered by fMLP is biphasic, with a peak of production observed in a short time period that entirely depends on p110γ activity, and a delayed phase that is mediated by class IA PI3K. We also provide evidence that the PI3K-dependent functional responses (i.e., superoxide production and chemotaxis) induced by the chemotactic factor mainly involve PI3K IA and, by implication, the delayed phase of PI(3,4,5)P3 production, whereas p110γ and the early peak of PI(3,4,5)P3 do not play major roles in the initiation or the control of these responses.

Blockade of Class IA Phosphoinositide 3-Kinase in Neutrophils Prevents NADPH Oxidase Activation- and Adhesion-dependent Inflammation

“…Prior studies in PI3K␥-deficient mice showed impaired fMLP-stimulated PMN respiratory burst activity (17,41) indicating the important role of GPCR-coupled PI3K␥ in the activation of NADPH oxidase. Using dominant negative protein inhibitors of class IA and IB PI3K, a recent study has demonstrated the dominant role of class IA PI3K in fMLPstimulated superoxide generation (36). In the present study, we addressed the role of class IA PI3K in mediating TNF-␣-induced activation of PMN NADPH oxidase and its consequences in the mechanism of lung inflammation and injury.…”

PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP

“…One example of these conflicting data is neutrophil chemotaxis towards the bacterial peptide formyl-Met-Leu-Phe (fMLP). Some groups have proposed that PI3K activity is an absolute requirement for migration towards fMLP (Sadhu et al, 2003), whereas other groups have identified a partial requirement for PI3K in migration towards fMLP (Boulven et al, 2006;Ferguson et al, 2007), and still other groups have demonstrated that chemotaxis towards fMLP is independent of PI3K (Heit et al, 2002). In addition, recent studies using Dictyostelium have demonstrated that chemotaxis, at least under some circumstances, can occur in the absence of both PI3K and its negative regulator PTEN (Andrew and Insall, 2007;Loovers et al, 2006).…”

Pharmacological profile of a bifunctional ligand of the formyl peptide receptor1 fused to the myc epitope

“…An undifferentiated myeloid cell line was used in most experiments, namely, the PLB 985 cell line [8], originally obtained from the German Collection of Microorganisms and Cell Cultures. These lines were cultured in RPMI 1640 medium containing 10% FBS, pyruvate and antibiotics at 37°C in a 5% CO 2 humidified atmosphere and chemical induction of differentiation was based on adding dimethylsulfoxide (DMSO, 1.3% v/v) for 5 to 7 days in the culture medium, a step known to upregulate the expression of the human FPR 1 in these cells [9].…”

“…Differentiated PLB 985 cells support f-Met-Leu-Phe-induced the O 2 -release assay based on cytochrome c reduction that was applied as described [9].…”