title: Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity.
abstract: 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-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 microg/mL). OxyMb (10-300 microg/mL) or oxyHb (30-300 microg/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 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.
authors.affiliation: Biochemistry Branch, US Army Institute of Surgical Research, Fort Sam Houston, Texas 78234-6315, USA. RICHARD.CASSIDY@CEN.AMEDD.ARMY.MIL
authors.forename: R A
authors.initials: RA
authors.lastname: Cassidy
authors.forename: D G
authors.initials: DG
authors.lastname: Burleson
authors.forename: A V
authors.initials: AV
authors.lastname: Delgado
authors.forename: D J
authors.initials: DJ
authors.lastname: Kohler
authors.forename: M L
authors.initials: ML
authors.lastname: Salin
authors.forename: B A
authors.initials: BA
authors.lastname: Pruitt
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D005452:Fluoresceins / Q000378:metabolism; D006420:Hemeproteins / Q000633:toxicity*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009211:Myoglobin / Q000494:pharmacology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010108:Oxyhemoglobins / Q000378:metabolism / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D000975:Antioxidants; D005452:Fluoresceins; D006420:Hemeproteins; D009211:Myoglobin; D009573:Nitrites; D016877:Oxidants; D010108:Oxyhemoglobins; D010718:Phosphatidylserines; C017595:oxymyoglobin; C065013:2',7'-dichlorodihydrofluorescein; D013481:Superoxides; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase