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 μg/mL). OxyMb (10–300 μg/mL) or oxyHb (30–300 μg/mL) enhanced DCFH oxidation by a concentration‐dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor a (TNF‐α)‐, 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 neighboring 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. J. Leukoc. Biol. 67: 357–368.