OXIDATION OF DESFERRIOXAMINE TO NITROXIDE FREE-RADICAL BY ACTIVATED HUMAN NEUTROPHILS

Human neutrophils activated by PMA were found to induce the formation of a nitroxide radical from DFO. The presence of SOD was necessary to per-mit the formation of the DFO radical. The inactive phorbol ester did not induce DFO radical, and DL-sphinganine suppressed the radical produced by the active phorbol ester. Other cell stimuli (Zymocel and the chemotactic peptide) also induced the formation of the DFO radical, although radical concentration was very much lower than with PMA. Participation of .NO, .OH or O-1(2) was ruled out by the inability of N(G)-methyl-L-arginine, N(G)-nitro-L-arginine, DMSO, mannitol, histidine, and methionine to inhibit the formation of DFO radical produced by PMA-activated cells. Furthermore, PMA-activated cells did not produce detectable levels of NO2-, a stable oxidation product of .NO, and D2O, which enhances the lifetime of singlet oxygen, did not modify the intensity or the lifetime of DFO radical. The involvement of cell MPO was suggested by the inhibition of the DFO radical observed after treatment with catalase or with antihuman MPO antibodies. Also, HOCl was found to induce the DFO radical in cell-free reactions, but our data indicate that the reaction leading to DFO radical formation by neutrophils involves the reduction of MPO compound II back to the active enzyme (ferric-MPO). Anti-inflammatory drugs strongly increased the DFO radical produced by activated neutrophils. On the contrary, none of these drugs was able to increase the DFO radical produced by HOCl. Histidine and methionine that inhibited the DFO radical intensity in cell-free reactions, were shown to act directly on HOCl. Experiments with MPO-H2O2 in SOD- and Cl--free conditions showed the formation of DFO radical and confirmed the hypothesis of the involvement of compound II. The conversion of compound II to ferric MPO by DFO optimized the enzymatic activity of neutrophils, and in the presence of monochlorodimedon (compound II promoting agent) we measured an increased HOCl production. When DFO was modified by conjugation with hydroxyethyl starch, it lost the ability to produce the radical either by neutrophils or by MPO-H2O2 and did not increase HOCl production. The inability of these DFO derivatives to produce potentially toxic species might explain their reported lower toxicity in vivo.