The objectives of the present investigation were to study the effects of hemoglobin on tree radical production by alveolar macrophages. Alveolar macrophages were harvested from anesthetized male rats. Free radical production by alveolar macrophages was assessed by measuring integrated luminol-enhanced chemiluminescence responses (cpmx10^5/.5x10^6 cells) and electron-spin resonance profiles under basal and activated conditions. A soluble phorbol ester (PMA; 12-0- tetradecanoylphorboI-13-acetate) was used to initiate free radical production by alveolar macrphages. Hemoglobin (0.1-1.00 microM) significantly enhanced PMA-stimulated chemiluminescence from alveolar macrophages in a dose-related manner. The possibility that hemoglobin may act as a biological Fenton reagent to promote enhanced free radical responses by alveolar macrophages was explored next. Addition of either an iron chelator, the removal of hydrogen peroxide or superoxide anions, or the presence of hydroxyl radical scavengers significantly decreased the effect of hemoglobin. These findings suggest that hemoglobin enhances free radical production by alveolar macrophages by acting as a biological catalyst and that the pronounced increase in reactive species generation is the overall result of an iron-catalyzed HaberWeiss reaction. Thus, alveolar macrophages may be a cell type which contributes to the potentiation of lung damage when hemoglobin is present.
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