Oxidant-induced priming of the macrophage involves activation of p38 mitogen-activated protein kinase through an Src-dependent pathway.
BACKGROUND
Resuscitated hemorrhagic shock predisposes patients to the development of organ dysfunction, particularly to lung injury. Ischemia/reperfusion during shock is believed to prime the immune system for an exaggerated inflammatory response to a second delayed stimulus. We previously reported an in vitro model of oxidant-induced priming of the macrophage to lipopolysaccharide (LPS) involves the Src family of tyrosine kinases. Because the Src family has been shown to activate the p38 mitogen-activated protein kinase (MAPK) pathway, we hypothesize that LPS signaling after oxidant stress involves the p38 pathway and is activated by Src kinases.


METHODS
The murine macrophage cell line, Raw 264.7, was first incubated with H(2)O(2) 100 micromol/L for 1 hour and then with low dose LPS 0.01 microg/mL for 5 to 45 minutes. In a separate experiment, the cells were pretreated with PP2 or SB203580, a specific inhibitor of the Src family and p38 respectively. The phosphorylation of p38, representative of its activation, was assessed in whole cell lysates by use of Western blotting. NF-kappaB translocation was detected by immunofluorescence with anti-p65 antibody.


RESULTS
There is a time dependent earlier activation of p38 by oxidant stress. H(2)O(2) augmented the LPS-induced p38 phosphorylation. The Src inhibitor, PP2, prevented only the LPS-induced earlier phosphorylation after oxidant stress and had no effect on LPS activation of p38 alone. The p38 inhibitor had no effect in preventing NF-kappaB translocation in either the LPS- or H(2)O(2)/LPS-exposed cells.


CONCLUSIONS
Oxidant stress generated during global ischemia/reperfusion activates p38 MAPK in an Src-dependent manner. Oxidants seem to alter the LPS-induced activation of p38. P38 does not seem to have a direct role in leading to oxidant-induced NF-kappaB translocation but may affect other oxidant-induced transcription factors. This altered pathway provides an alternative avenue to target therapy during the oxidant-induced priming of the macrophage induced by trauma resuscitation.
Khadaroo
Rachel G
RG
Department of Surgery, University Health Network, and University of Toronto, Ontario, Canada.
Parodo
Jean
J
Powers
Kinga A
KA
Papia
Giuseppe
G
Marshall
John C
JC
Kapus
Andras
A
Rotstein
Ori D
OD
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C412373:AG 1879; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016877:Oxidants; D011743:Pyrimidines; D006861:Hydrogen Peroxide; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases