Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells

J Immunol. 2004 Dec 15;173(12):7462-70. doi: 10.4049/jimmunol.173.12.7462.

Abstract

A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox), which respond to phorbol ester and arachidonic acid with O()(2) production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCdelta, but not PKCalpha, -betaII, and -zeta, is necessary for the COS-phox cells to respond to fMLP. A role of PKCdelta in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCdelta translocation and the sensitivity of fMLP-induced O()(2) production to rottlerin, a PKCdelta-selective inhibitor. Optimal reconstitution also requires phospholipase C-beta2 and PI3K-gamma. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40(phox) potentiated fMLP-induced O()(2) production and raised the level of O()(2) in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O()(2) production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Enzyme Activation / genetics
  • Enzyme Activation / immunology
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism
  • GTP-Binding Protein alpha Subunits, Gi-Go / physiology
  • Humans
  • Isoenzymes / genetics
  • Isoenzymes / physiology
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology*
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / genetics*
  • NADPH Oxidases / metabolism*
  • Neutrophils / enzymology*
  • Neutrophils / immunology*
  • Neutrophils / metabolism
  • Oxidation-Reduction
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / physiology
  • Phospholipase C beta
  • Phosphoproteins / biosynthesis
  • Phosphoproteins / genetics*
  • Phosphoproteins / physiology
  • Protein Kinase C / genetics
  • Protein Kinase C / physiology
  • Protein Kinase C-delta
  • RAC2 GTP-Binding Protein
  • Receptors, Formyl Peptide / biosynthesis
  • Receptors, Formyl Peptide / genetics
  • Receptors, Formyl Peptide / physiology*
  • Signal Transduction / genetics
  • Signal Transduction / immunology
  • Superoxides / metabolism
  • Transfection / methods*
  • Type C Phospholipases / genetics
  • Type C Phospholipases / physiology
  • Up-Regulation / genetics
  • Up-Regulation / immunology
  • rac GTP-Binding Proteins / genetics
  • rac GTP-Binding Proteins / physiology
  • rac1 GTP-Binding Protein / genetics
  • rac1 GTP-Binding Protein / physiology

Substances

  • Isoenzymes
  • Phosphoproteins
  • Receptors, Formyl Peptide
  • neutrophil cytosol factor 40K
  • Superoxides
  • N-Formylmethionine Leucyl-Phenylalanine
  • NADPH Oxidases
  • PRKCD protein, human
  • Protein Kinase C
  • Protein Kinase C-delta
  • Type C Phospholipases
  • PLCB2 protein, human
  • Phospholipase C beta
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • rac GTP-Binding Proteins
  • rac1 GTP-Binding Protein