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Oxidant-induced priming of the macrophage involves activation of p38 mitogen-activated protein kinase through an
Rachel G Khadaroo1, Jean Parodo, Kinga A Powers
1Department of Surgery, University Health Network, and University of Toronto, Ontario, Canada.
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.
Insights
Oxidant stress primes macrophages for an exaggerated inflammatory response. Src kinases activate the p38 MAPK pathway, influencing lipopolysaccharide (LPS) signaling after oxidant stress.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Medicine
Background:
- Resuscitated hemorrhagic shock can lead to organ dysfunction, especially lung injury.
- Ischemia/reperfusion during shock may prime the immune system for heightened inflammatory responses.
- Previous research indicated Src kinases are involved in oxidant-induced macrophage priming.
Purpose of the Study:
- To investigate the role of the p38 MAPK pathway in LPS signaling following oxidant stress.
- To determine if Src kinases activate the p38 pathway in this context.
Main Methods:
- Murine macrophage cell line (Raw 264.7) exposed to hydrogen peroxide (H2O2) and lipopolysaccharide (LPS).
- Inhibition of Src family kinases (PP2) and p38 MAPK (SB203580).
- Western blotting to assess p38 phosphorylation; immunofluorescence for NF-kappaB translocation.
Main Results:
- Oxidant stress led to earlier, time-dependent activation of p38.
- H2O2 augmented LPS-induced p38 phosphorylation.
- Src inhibition blocked earlier p38 phosphorylation after oxidant stress but not LPS alone; p38 inhibition did not affect NF-kappaB translocation.
Conclusions:
- Oxidant stress activates p38 MAPK via Src kinases during ischemia/reperfusion.
- Oxidants alter LPS-induced p38 activation.
- p38 MAPK may not directly mediate oxidant-induced NF-kappaB translocation but influences other transcription factors, offering therapeutic targets.
