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MAP Kinase Inhibition Ameliorates Lung Ischemia-Reperfusion Injury
P. S. Wolf, H. E. Merry, A. S. Farivar, A. S. McCourtie, M. S. Mulligan*. University of Washington, Seattle, WA,
BACKGROUND: Lung ischemia-reperfusion injury (LIRI) is a significant clinical problem, affecting up to 25% of lung transplant recipients and increasing the risk for both early graft dysfunction and late graft failure. LIRI is driven by transcriptional activation in the alveolar macrophage (AM) that leads to the release of inflammatory mediators which amplify inflammatory signaling in pulmonary endothelial cells (PAEC) and type 2 pneumocytes (T2P). Inhibition of individual cytokines offers only modest protection from tissue injury in animal models. More effective strategies would aim to selectively inhibit the transcriptional activation that occurs in response to oxidative stress. Mitogen-activated protein kinases (MAPK) are a group of intracellular signaling proteins thought to regulate transcriptional activation of several inflammatory cytokines. Three specific MAPK, p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK 1/2) have been shown to be activated after oxidative stress and in animal models of acute inflammatory injury. Therefore, we hypothesized that inhibition of MAPKs, would block upstream transcriptional activation, thereby inhibiting signaling amplification, resulting in more robust protection from tissue injury in response to lung ischemia and reperfusion.
METHODS: Male Long-Evans rats weighing 280-320 grams were subjected to left lung hilar occlusion for a period of 90 minutes followed by up to four hours of reperfusion. Experimental animals were pre-treated 45 minutes prior to ischemia with specific inhibitors of p38 (FR167653), JNK (SP600125), and ERK 1/2 (U0126). Immunohistochemistry was performed to localize cell specific activation of MAPKs. Injury was assessed by measuring lung vascular permeability, myeloperoxidase content, and BAL cell count analysis. BAL effluent was analyzed for soluble cytokine and chemokine content via sandwich ELISA. Western blotting was performed to assess for MAPK phosphorylation while electron mobility shift assay assessed nuclear translocation of transcription factors.
RESULTS: In positive control animals, immunohistochemistry demonstrated p38 and JNK activation in AM but not PAEC or T2P. Conversely, ERK 1/2 was activated only in PAEC and T2P. Animals treated with MAPK inhibitors demonstrated significant reductions in transcription factor activation, vascular permeability, BAL cytokine content and tissue inflammatory cell infiltration and myeloperoxidase content. p38 and JNK inhibition was more protective than ERK 1/2 inhibition. Western blotting confirmed highly specific inhibition for each inhibitor, therefore the relative importance of p38 and JNK versus ERK1/2 could be assessed.
CONCLUSIONS: Inhibition of MAPKs provided significant protection from injury. This correlated with a decreased production of inflammatory cytokines, providing evidence that MAPK activation is an early upstream signaling event in lung ischemia reperfusion injury. The alveolar macrophage appears to be the key coordinator of injury in response to oxidative stress, as targeted inhibition of JNK or p38 yielded greater protection than ERK 1/2 inhibition (which localized to PAEC and T2P). Therapeutically targeting specific cell populations (like the AM) has the potential benefit of reducing LIRI severity while leaving host immune responses intact.
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