Abstract
BackgroundCytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.ResultsTumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state.ConclusionsThe TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.
Highlights
Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton
We found that cytochalasin B had no effect on the NADPH-oxidase response when added to neutrophils prior to TNF-α treatment
The time course of the induced response was similar to that seen with chemoattractants such as the formylated peptide formylated peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), an agonist that activates cells through the Gprotein coupled formyl peptide receptor, formyl peptide receptors (FPR)
Summary
Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The chemoattractants, including C5a, platelet activating factor (PAF), interleukin-8 (IL8) and formylated peptides, bind to specific receptors [2,3], all of which belong to a family of transmembrane G-protein coupled receptors (GPCRs). Activation of these receptors leads to directed migration, granule mobilization and activation of the neutrophil NADPH-oxidase [2]. Tumor necrosis factor-alpha (TNF-α) is one of the earliest cytokines produced at inflammatory sites by activated monocytes and macrophages This cytokine affects neutrophil function mainly through binding to type I TNF receptor (TNFR1) [5]. Phosphorylation of TNFR1 occurs at a consensus MAPK site on its cytoplasmic domain or through tyrosine phosphorylation [6,7], it is not fully understood how this phosphorylation control receptor signaling or processing
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