Internalization of proteinase 3 is concomitant with endothelial cell apoptosis and internalization of myeloperoxidase with generation of intracellular oxidants

The important issue addressed by the studies presented here is the mechanis m of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelia l cells may be involved in vascular damage in patients with inflammatory va scular diseases. We have determined whether granule proteins proteinase 3(P R3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, a s examined by UV light, confocal, and electron microscopy. Coincident induc tion of apoptosis and/or the generation of intracellular oxidants were moni tored. The results indicate that human endothelial cells (human umbilical v ein endothelial cells, human umbilical arterial endothelial cells, human lu ng microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epit helial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specifi c and different from that of MPO. Internalization of PR3, but not MPO, corr elated with activation of apoptosis, Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the pro teolytic activity of PR3 for the induction of apoptosis was examined by gen erating PR3-truncated fragments that did not contain the components of the catalytic triad, An apoptotic function was localized to the C-terminal port ion of PR3, These studies reveal novel mechanisms by which the neutrophil g ranule proteins PR3 and MPO contribute to tissue injury at sites of inflamm ation.