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 (O2-) and the resulting cytotoxicity. Physiologically relevant concentrations of NO and H2O2 oxidized, to a similar extent, 2,7-dichlorodihydro-fluorescein (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 micro(g)/mL). OxyMb (10 300 g/mL) or oxyHb (30 300 micro(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 -induced 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 O2- 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 neigh- boring cells by inhibiting O2- -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. Published in the Journal of Leukocyte Biology, v67 p357-368, 1 March 2000.