Release of TNF-alpha during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers.

Abstract

Our study sought to elucidate the role of oxidative stress for shedding of tumor necrosis factor-alpha (TNF-alpha) and for activating TNF-alpha-converting enzyme (TACE). TNF-alpha, a central inflammatory cytokine, is discussed as one of the mediators of reperfusion injury. Shedding of membrane-bound pro-TNF-alpha is thought to be largely due to TNF-alpha-converting enzyme (TACE). Release of TNF-alpha and TACE dependency were studied in isolated rat hearts and in the human mast cell line HMC-1. In reperfused hearts, interstitial release of TNF-alpha occurred in two phases (2-10 and >45 min). It depended on the presence of oxygen during reperfusion and was attenuated by reduced glutathione. Infusion of the oxidants H(2)O(2) or HOCl elicited release in non-ischemic hearts. TNF-alpha release was inhibited in hearts treated with degranulation inhibitors ketotifen or cromoglycate, suggesting mast cells as major source for myocardial TNF-alpha. This was confirmed by tissue staining. Post-ischemic release of histamine, however, did not parallel that of TNF-alpha. Heart tissue contained mainly mature TACE. HMC-1 expressed abundant pro-TACE and cleaved the pro-TNF-alpha-peptide Ac-SPLAQAVRSSSR-NH(2). However, cleavage was nonspecific and only partly inhibited by TACE inhibitor TAPI-2 (10-100 micromol/l), while it was stimulated by H(2)O(2) and HOCl and fully blocked by the nonspecific metalloprotease inhibitor o-phenanthroline. The mechanism underlying TNF-alpha release from post-ischemic myocardium is oxidation-dependent but largely independent of activation of TACE. Mast cell stabilizers may be useful in preventing TNF-alpha release during reperfusion.