Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity

Isolated human PMNs served as a model to determine oxyhemoglobin (oxyHb) binding and the effects of oxymyoglobin (oxyMb) or oxyHb on production of both nitric oxide (NO) and superoxide (O-2 .(-)) and the resulting cytotoxicity, Physiologically relevant concentrations of NO. and H2O2 oxidized, to a similar extent, 2,7-dichlorodihydrofluorescein (DCFH) loaded into polymorphonuclear neutrophils (PMNs). Activation of PMNs with phorbol 12-myristate 13-acetate (PMA) markedly increased the internalization of extracellular oxyHb (10-250 mu g/ml). OxyMb (10-300 mu g/mL) or oxyHb (30-300 mu g/mL) enhanced DCFH oxidation by a concentration-dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-, and PMA-stimulated PMNs. This increased DCFH oxidation was eliminated by NO. synthase inhibitors, glutathione and ascorbate, and was reduced by albumin. Nitrite accumulation in PMN filtrates mirrored NO.-iuduced DCF fluorescence, OxyMb-induced increases in NO. levels paralleled alterations in DNA and cell membrane damage and ATP levels in PMNs and co-cultured lymphocytes, and were attenuated by NO. synthase inhibitors. OxyMb eliminated extracellular O-2 .(-) at protein concentrations 100- to 1000-fold above those of superoxide dismutase, These results suggest that heme proteins bind and internalize into PMNs and increase NO.-induced damage in neighboring cells by inhibiting O-2 .(-)-scavenging of NO.. We propose a mechanism whereby heme protein-induced NO. levels may contribute to immunosuppression and increased infection rates associated with transfusions and cellular damage during inflammation.