Activation of NFκB by the respiratory burst of macrophages

H2O2 and other reduced oxygen species have been proposed as activators of the transcription factor, NFκB. Stimulated macrophages produce superoxide and H2O2 (the respiratory burst). We tested the hypothesis that production of these species could serve as part of the NFκB activation pathway in rat alveolar macrophages and the J774A. 1 mouse monocyte/macrophage cell line. Phorbol myristate acetate (PMA) and ADP, which stimulate the respiratory burst, caused NFκB activation in both cells. Catalase abolished NFκB activation, while superoxide dismutase produced little inhibition. Thus, H2O2 was the principal agent of respiratory burst-associated NFκB activation. Abolition of NFκB activation by catalase also suggested that intermediate signaling pathways, such as protein kinase C activation or intracellular free calcium elevation must not be involved. Exogenous H2O2 added as a bolus ≥ 50 μM (≥ 50 nmol/106 macrophages) also activated NFκB in macrophages. Nevertheless, the maximum endogenous production of H2O2 by stimulated alveolar macrophages during a 30-min incubation was ≤ 1.3 nmol H2O2/ 106 cells for PMA stimulation and ≤ 0.2 nmol H2O2/106 cells for ADP stimulation. Thus, relatively little endogenous H2O2 generation was required to produce NFκB activation compared to the required amount of exogenous H2O2. As H2O2 rapidly diffuses and is consumed, these results suggest that the site of action for endogenously generated H2O2 is probably close to its origin, the plasma membrane.