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

Abstract

The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory vascular diseases. We have determined whether granule proteins proteinase 3(PR3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, as examined by UV light, confocal, and electron microscopy. Coincident induction of apoptosis and/or the generation of intracellular oxidants were monitored. The results indicate that human endothelial cells (human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human lung microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epithelial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specific and different from that of MPO. Internalization of PR3, but not MPO, correlated with activation of apoptosis. Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the proteolytic activity of PR3 for the induction of apoptosis was examined by generating PR3-truncated fragments that did not contain the components of the catalytic triad. An apoptotic function was localized to the C-terminal portion of PR3. These studies reveal novel mechanisms by which the neutrophil granule proteins PR3 and MPO contribute to tissue injury at sites of inflammation.

Figure 1.

Assessment of internalization by immunofluorescence microscopy. Photomicrographs of HUVECs (A–C and E–G) and SAECs (D and H). Untreated HUVECs did not show staining with either anti-PR3 antibody (A) or anti-MPO antibody (E). HUVECs incubated with PR3 (B) (10 μg/ml) or MPO (F) (10 μg/ml) displayed weak intracellular staining by 10 minutes. HUVECs treated with PR3 (C) or MPO (G) for 2 hours exhibited prominent intracellular staining. SAECs treated with PR3 for 2 hours (D) were negative for intracellular staining; SAECs treated with MPO (H) showed prominent staining.

Figure 2.

Assessment of internalization by immunofluorescence confocal laser-scanning microscopy. Photomicrographs of HUVECs (A–C and E–G) and SAECs (D and H). HUVECs treated with medium alone did not show staining with either anti-PR3 antibody (A) or anti-MPO antibody (E). HUVECs treated with PR3 (10 μg/ml) (B) or MPO (10 μg/ml) (F) displayed surface staining and weak intracellular staining by 10 minutes. HUVECs treated with PR3 (C) or MPO (G) for 2 hours resulted in intracellular staining with prominent staining in the cytoplasm. SAECs treated with PR3 for 2 hours were negative for intracellular staining (D). SAECs treated with MPO for 2 hours showed strong cytoplasmic staining (H).

Figure 3.

Assessment of internalization by electron microscopy. Electron micrographs of HUVECs stained by immunogold labeling. PR3 (A) (gold particles, arrows) was present in cytoplasmic extensions by 10 minutes and in the nucleus associated with heterochromatin by 2 hours (C); MPO (B) (gold particles, arrows) was associated with nuclear heterochromatin by 10 minutes and by 2 hours was detected in the cytoplasm and in secondary lysosomes (D).

Figure 4.

PR3 internalization induces apoptosis. A: Flow cytometric analysis. HUVECs treated with PR3 showed a concentration-dependent increase in apoptotic cells (shown as a subG₀/G₁ peak). UV light microscopy of DAPI-stained cells showed uniform nuclei in the untreated group (B); PR3 treated cells (12 hours) showed an apoptotic morphology with nuclear fragmentation (C). Dose-response curve showing percentage of apoptotic HUVECs (24 hours), as assessed by UV light microscopy (D). Time-dependent response curve of cells that were treated with 10 μg/ml PR3 or 100 μg/ml MPO (E). *, Statistically different from control (P = 0.0001). Values are means ±SEM; n = 6. PR3, but not MPO, induces HUVEC apoptosis in a dose- and time-dependent manner.

Figure 5.

Identification of a noncatalytic domain of PR3 with apoptotic function. Ribbon model of PR3 molecule (A): N-terminal fragment (PR3-N, light gray), middle fragment (PR3-M, white), and C-terminal fragment (dark gray, PR3-C). Cos-7 cells (B) transfected with PR3-C vector, stained with DAPI, show nuclear fragmentation. Flow cytometric analysis (C and D) of TUNEL-labeled EA. Hy926 cells treated with conditioned medium containing PR3-C. Limits were set using the minus TdT (C) to control for background fluorescence. Plus TdT (D) shows increased fluorescence indicative of apoptotic cells.

Figure 6.

Internalization of MPO results in generation of intracellular oxidants. Untreated HUVECs (A) and SAECs (C) incubated with DHR alone for 6 hours showed no cytoplasmic fluorescence. HUVECs (B) and SAECs (D) incubated with MPO (25 μg/ml) plus DHR for 6 hours showed increased intracellular oxidant generation. Flow cytometric analysis (E) showed an increase in mean of fluorescence intensity in HUVECs treated with 5 or 25 μg/ml MPO. Flow cytometric analysis (F and G) showing a dose- and time-dependent increase in intracellular oxidants generated by MPO treatment. Addition of catalase resulted in decreased intracellular oxidant generation. *, Statistically different from DHR alone (P < 0.05). Values are means ±SEM; n = 3. MPO causes the increase of intracellular oxidant generation in HUVECs in a dose- and time-dependent manner and the increase can be blocked by catalase.