[Production of chemoattractant-induced reactive oxygen species in human neutrophils--role of protein kinase C isoforms].

Unlabelled: The aim of our study was to characterize the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced by formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), and platelet activating factor (PAF). Also, we investigated the roles played by different protein kinase C (PKC) isoforms in nucleotide-induced priming. ROS production was determined by an isoluminol-based assay.

Material And Method: Nucleotide-induced priming was concentration- and time-dependent. The concentration of UTP able to cause maximal priming was 10 microM. When UTP (10 microM) was administered prior the agonist, the increase of the amplitude of the response reached the maximum at 1 minute of preincubation with the nucleotide.

Results: Calcium depletion of neutrophil caused significant inhibition of ROS production induced by all agonists tested, but did not affect the priming effect of the nucleotides. We tested the effect of several PKC inhibitors on the nucleotide-induced priming. GF 109203X (5 microM), an inhibitor of all neutrophil's PKC isoforms, or RO 31-8220 (5uM), an inhibitor of classical and novel PKC isoforms, abolished the responses induced by fMLP (10 nM) IL-8 (10 nM), LTB4 (100 nM) or PAF (100 nM). Go 6976 (100 nM), a selective inhibitor of classical PKC isoforms, had no effect on nucleotide-induced priming, suggesting that activation of these PKC isoforms does not play a role in the priming effect. Rottlerin (5 microM), a PKC delta inhibitor, almost abolished the effect of fMLP in the absence or in the presence of UTP, indicating that PKC delta is essential for the fMLP-induced effect; rottlerin also caused inhibition of ROS production induced by IL-8, LTB4 or PAF, however the priming effect of UTP was not affected for these chemoattractants. Our data suggest that classical PKC isoforms do not play a role in chemoattractant-induced ROS production.

Conclusion: Although fMLP induced effect appears to be highly dependent on PKC delta activation, other chemoattractants are able to cause ROS production through PKC delta-independent mechanisms.