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

R. A. Cassidy, D. G. Burleson, A. V. Delgado, D. J. Kohler, M. L. Salin, Jr Pruitt

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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 α (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.

Original languageEnglish (US)
Pages (from-to)357-368
Number of pages12
JournalJournal of Leukocyte Biology
Issue number3
StatePublished - 2000


  • Apoptosis
  • Cytotoxicity
  • DNA strand breaks
  • Hemoglobin
  • Myoglobin
  • Superoxide

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Cell Biology


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