FIG 6.

(A) P. stomatis induces the exocytosis of secretory vesicles and gelatinase granules. Neutrophils were unchallenged (basal) or challenged with fMLF (5 min), with P. stomatis (30 min), or with P. gingivalis (30 min). (A and B) Secretory vesicle exocytosis was determined by the increase in plasma membrane expression of CD35 by flow cytometry. Data are expressed as the means ± SEM of the mean channel of fluorescence (mcf) from 3 independent experiments. (C) Gelatinase granule exocytosis was measured as levels of matrix metalloproteinase 9 (MMP-9) present in the supernatants collected from the different experimental conditions by ELISA. Data are expressed as means ± SEM of MMP-9 release (in nanograms per 4 × 10⁶ cells) from 4 independent experiments. (D) The activities of MMP-9 on the supernatants from the same experimental conditions were analyzed by gelatin zymogen assays. The supernatant from P. stomatis growth media (GS) was also analyzed. The protein standard is shown on the left. MMP-9 isoforms are indicated. (E and F) The densitometry profile of the degraded gelatin zymography 92-kDa band was analyzed by ImageJ software from the gels of 4 independent experiments (E) or from 3 independent experiments (F). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.