Pentoxifylline modulates p47phox activation and downregulates neutrophil oxidative burst through PKA-dependent and -independent mechanisms.
OBJECTIVE
Pentoxifylline (PTX) has been proven to be an inhibitor of fMLP-induced neutrophil (PMN) oxidative burst and is thought to function by increasing cAMP and Protein kinase A (PKA). We hypothesized that PTX diminishes production of the neutrophil respiratory burst by both PKA-dependent and independent mechanisms.


METHODS
Human neutrophils were isolated and stimulated with fMLP (1microM) alone or in combination with PTX (2mM). PMN activation was determined by the cytochrome C reduction method in the presence and absence of p38 MAPK (SB203580), ERK (PD98059), and PKA inhibitors (H89). Western blot analysis of Ras, Raf, p38 MAPK, ERK, and Akt was performed in PMNs exposed to fMLP and PTX. Cell membranes were fractionated to measure membrane-associated p47 phox. Treated cells were imaged using confocal microscopy to examine changes in localization of Akt and p47phox.


RESULTS
PTX produced a decrease in oxidative burst that was diminished but not abrogated by H89 exposure. The reduction in Ras, Raf, and Akt activation seen with PTX was not effected by the presence of H89. The ability of PTX to attenuate phosphorylation of p38 MAPK and ERK was significantly decreased in the presence of H89, suggesting a PKA-dependent mechanisms. Membrane fractions of neutrophils demonstrate that PTX decreased membrane-associated p47phox, thus diminishing the ability to generate oxidative burst. PTX also decreased membrane localization of Akt and p47phox by confocal microscopy.


CONCLUSIONS
PTX attenuates activation of signaling molecules involved in activation of p47phox and suppress the subsequent assembly of the NADPH machinery through both PKA-dependent and PKA-independent mechanisms.
Costantini
Todd W
TW
Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California-San Diego School of Medicine, San Diego, California.
Deree
Jessica
J
Peterson
Carrie Y
CY
Putnam
James G
JG
Woon
Tiffany
T
Loomis
William H
WH
Bansal
Vishal
V
Coimbra
Raul
R
D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D015536:Down-Regulation; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010431:Pentoxifylline / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects*; D048490:raf Kinases / Q000502:physiology; D018631:ras Proteins / Q000502:physiology
D010726:Phosphodiesterase Inhibitors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051057:Proto-Oncogene Proteins c-akt; D048490:raf Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D018631:ras Proteins; D010431:Pentoxifylline