Insights from knock-out models concerning postischemic release of TNF alpha from isolated mouse hearts

The inflammatory cytokine tumor necrosis factor alpha (TNF alpha) is controversially discussed in ischemia/reperfusion damage of the heart. Purpose of this study was to elucidate cellular sources of TNF alpha and parameters which possibly influence its release in the heart following ischemia. Isolated hearts of mice were subjected to 15 min of global ischemia and 90 min of reperfusion. We employed hearts of various mice knock-out strains (interteukin-6(-/-), matrix metalloprotease-7(-/-), mast-cell deficient WBB6F1-Kit(W)/Kit(W-v), TNF-R1(-/-)) and wildtype mice, the latter perfused without and with infusion of cycloheximide or TNF alpha-cleaving-enzyme inhibitor (TAPI-2). Normoxic control hearts showed basal release of TNF alpha during the whole experiment. Immunohistology identified cardiac mast cells, macrophages and endothelial cells as main sources. TNF alpha release was stimulated during postischemic reperfusion, occurring in a two-peak pattern: directly after ischemia (0-10 min) and again after 60-90 min. The first peak mainly reflects tissue washout of TNF alpha accumulated during ischemia. The second, protracted peak arose continuously from the basal level and was abolished by protein synthesis inhibitor cycloheximide. Both properties are characteristic for de novo synthesis of TNF alpha, e.g., in cardiac muscle cells. However, immunohistological staining for TNF alpha failed in cardiomyocytes after 90 min of reperfusion. In contrast to hearts of TNF-R1(-/-) and Kit(W/W-v)-mice, those of IL-6(-/-) and MMP-7(-/-) mice lacked the late TNF alpha peak. TAPI did not suppress release of TNF alpha. While autostimulation via TNF-R1 also does not seem obligatory and mast cell can be ignored as source of the second peak, IL-6 may support de novo synthesis of TNF alpha. Additionally, TNF alpha release may essentially involve cleavage of membrane bound TNF alpha by MMP-7. (c) 2006 Elsevier Inc. All rights reserved.