Cellular penetration of fluorescently labeled superoxide dismutases of various origins

Background: Using fluorescently labeled superoxide dismutase (SOD) and now cytometry, we have shown previously that the enzyme CuZn SOD (EC 1.15.1.1) from bovine erythrocytes binds rapidly to the cell surface with slow uptake into the cell during the following hours. The degree of labeling was most important for monocytes in comparison to other blood cells (erythrocytes, l ymphocytes, and neutrophils) and fibroblasts. In agreement with the flow-cy tometric findings, the inhibition of superoxide production was more importa nt for SOD-pretreated monocytes than for neutrophils, as demonstrated with the cytochrome c reduction assay. It was thus of interest to confirm the ob served differences between monocytes and neutrophils with confocal laser mi croscopy, study in greater detail the kinetics of binding penetration, and intracellular localization of the enzyme, and compare the results obtained with bovine CuZn SOD with those from SODs of other origins and carrying dif ferent active sites. Materials and Methods: Recombinant human (rh), bovine, and equine CuZn SODs , as well as rh and E. coli Mn SODs, were studied before use with respect t o specific activity and purity (HPLC, SDS-PAGE electrophoresis). Fluorescei n isothiocyanate was covalently conjugated to the various SODs for study wi th high-resolution confocal scanning laser microscopy. Superoxide productio n by monocytes and neutrophils was measured with the cytochrome c assay. Results: As expected from our experiments with flow cytometry, only rare ne utrophils were labeled with FITC-SOD, even with the longest incubation time of 3 hr and the highest dose of 1500 units/ml. In addition, they showed a localized fluorescence pattern that was quite different from the diffuse pu nctate fluorescence pattern of monocytes. Lymphocytes were not labeled at a ll. The rapid binding to the cellular surface of monocytes was confirmed an d even after 5 min of preincubation, FITC-SOD was found on a small percenta ge of monocytes. This was correlated with a reduction in superoxide release after phorbolmyristate acetate (PMA) stimulation by 40%. An interesting fi nding was the perinuclear accumulation of the penetrated SOD after the long est pretreatment of 3 hr, suggesting a barrier against further progression. Indeed, through confocal microscopy we were able to exclude any fluorescen ce at the nuclear level. While the fluorescence labeling patterns and the k inetics of penetration were quite similar for bovine, equine, and rh CuZn S OD, the Mn SODs showed poor labeling, correlated with a weak inhibitory eff ect on cytochrome c reduction which was not statistically significant. Conclusions: The rapid binding of native CuZn SODs on the surface of monocy tes, leading to reduced superoxide release by these cells, explains the obs ervation that beneficial effects of injected SOD lasted for months despite rapid clearance of the enzyme from the bloodstream, according to pharmacody namic studies. The preferential binding to monocytes, in contrast to neutro phils, may play a role in chronic inflammatory diseases in which the monocy tes are in an activated state. The differences in binding capacity between CuZn SODs and Mn SODs, correlated with different inhibitory effects of supe roxide production by monocytes, may also have therapeutic significance.