Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages

The objectives of this investigation were (I) to determine the effects of h emoglobin on the production of reactive oxygen species by activated rat alv eolar 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 myrista te acetate (PMA)-stimulated cells was assessed by measuring luminol-enhance d chemiluminescence (CL). Hemoglobin enhances PMA-stimulated CL in a dose-d ependent manner. The effect is maximal at 0.5-1.0 mu M 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 hemo globin-induced enhancement of PMA-stimulated CL is inhibited by superoxide dismutase, catalase, dimethylthiourea, or deferoxamine. These results sugge st that hydroxyl radical may Be formed from hydrogen peroxide which is deri ved from superoxide anion. Measurements of electron spin resonance spectra following spin trapping of radicals verify that hydroxyl radicals are produ ced-by the cells in the presence of PMA and hemoglobin. The hemoglobin effe cts appear to require iron in a protoporphyrin complex, because hemin stimu lates PMA-induced CL, whereas neither ferrous nor ferric iron has any effec t. These findings taken together suggest that hemoglobin can act as a biolo gical Fenton reagent to enhance the production of reactive oxygen species f rom alveolar macrophages and potentially contribute to lung damage during l eakage of Blood into the alveolar spaces.