title: Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages.
abstract: The objectives of this investigation were (1) to determine the effects of hemoglobin on the production of reactive oxygen species by activated rat alveolar macrophages, (2) to determine a possible mechanism for these effects, and (3) to determine which part of the hemoglobin molecule is responsible for these effects. Production of reactive oxygen species by phorbol myristate acetate (PMA)-stimulated cells was assessed by measuring luminol-enhanced chemiluminescence (CL). Hemoglobin enhances PMA-stimulated CL in a dose-dependent manner. The effect is maximal at 0.5-1.0 microM hemoglobin where PMA-induced CL is increased by approximately 20-fold. Superoxide anion release from PMA-stimulated cells is not affected by hemoglobin. However, the hemoglobin-induced enhancement of PMA-stimulated CL is inhibited by superoxide dismutase, catalase, dimethylthiourea, or deferoxamine. These results suggest that hydroxyl radical may be formed from hydrogen peroxide which is derived from superoxide anion. Measurements of electron spin resonance spectra following spin trapping of radicals verify that hydroxyl radicals are produced by the cells in the presence of PMA and hemoglobin. The hemoglobin effects appear to require iron in a protoporphyrin complex, because hemin stimulates PMA-induced CL, whereas neither ferrous nor ferric iron has any effect. These findings taken together suggest that hemoglobin can act as a biological Fenton reagent to enhance the production of reactive oxygen species from alveolar macrophages and potentially contribute to lung damage during leakage of blood into the alveolar spaces.
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA. ljh3@cdc.gov
authors.forename: L J
authors.initials: LJ
authors.lastname: Huffman
authors.forename: P R
authors.initials: PR
authors.lastname: Miles
authors.forename: X
authors.initials: X
authors.lastname: Shi
authors.forename: L
authors.initials: L
authors.lastname: Bowman
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006454:Hemoglobins / Q000494:pharmacology*; D007501:Iron / Q000494:pharmacology; D008163:Luminescent Measurements; D008262:Macrophage Activation / Q000187:drug effects; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011524:Protoporphyrins / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D016166:Free Radical Scavengers; D006454:Hemoglobins; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; D013481:Superoxides; D006427:Hemin; D007501:Iron; D013755:Tetradecanoylphorbol Acetate