Both H2O2 (IC50 = 70 microM) and HOCl (IC50 = 8.5 microM) inhibited mitogen-induced MNL proliferation in a dose-dependent manner. This was found to be due to a depletion of intracellular ATP by at least two distinct mechanisms. HOCl and high concentrations (greater than 100 microM) of H2O2 inhibit ATP generation via sulfhydryl group oxidation on the active site of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) enzyme of the glycolytic pathway. On the other hand, low H2O2 concentrations cause ATP depletion by an activation of the DNA repair enzyme, poly(ADP-ribose)polymerase (pADPRP), leading to consumption of NAD+, an essential cofactor for G3PDH. The anti-oxidants ascorbate and cysteine protected MNL against the anti-proliferative effects of HOCl. Similar results were achieved with the HOCl-mediated inhibition of ATP production and G3PDH activity. However, ascorbate was unable to protect against H2O2-mediated inhibition of MNL functions, while cysteine protected against the inhibitory effects on ATP production and G3PDH activity, induced by this oxidant.
Hypochlorous acid (HOCl) at concentrations of 6.25 microM and greater caused statistically significant, dose-related inhibition of mitogen-activated proliferation of human mononuclear leucocytes (MNL). The anti-proliferative effects of HOCl, which were evident using both undepleted and adherent-cell depleted MNL, could not be attributed to decreased mitogen binding by HOCl-treated cells. The anti-oxidants ascorbate and cysteine (50 microM), when added to MNL prior to exposure to HOCl (25 microM), prevented the anti-proliferative effects of the oxidising agent. Likewise reversal of oxidant-mediated inhibition of the responsiveness of MNL to mitogens was observed when ascorbate and cysteine were added after HOCl treatment of the cells. These results suggest that HOCl, derived from activated phagocytes, is a potential mediator of immunosuppression, especially in the setting of abnormal host anti-oxidant defences.
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