Fig 1.
Placental leukocytes remain viable and functional during culture.
A) XTT viability assay, time 0 was consider as 95% of viability according to trypan blue staining. B) IL-1b ELISA in the supernatants of placental leukocytes along the culture used as functionality assay. Placental leukocytes in culture remain viable (around 90%) and secreting IL-1b from 24 to 96 h, which was the culture period for all the experiments. Three independent experiments in duplicate are expressed as mean ± SD. *P≤0.05 compared to time 0 (A) or 24 h (B).
Fig 2.
Placental leukocytes in culture secrete pro-enzyme and active MMP-9.
A) Representative gelatin zymography (0.5 μg protein per lane) of placental leukocyte supernatants shows the 92 kDa pro-enzyme and the 82 kDa active form of MMP-9. B) The bars represent the optical densities (OD) of pro-enzyme and active MMP-9. While the pro-enzyme increased after 24h in supernatants, the active form was increased significantly only after 48h of culture. Seven independent experiments in duplicate are expressed as mean ± SD. *P≤0.05, **P≤0.005
Fig 3.
MMP-9 activity increases in placental leukocyte supernatants in a time-dependent manner.
Specific substrate assay showed a significant increase in MMP-9 activity in the placental leukocyte supernatants along the culture, reaching the major activity at 72h. Each point represents the mean ± SD of 3 independent experiments in duplicate. *P≤0.05, **P≤0.005
Fig 4.
A natural activator of proMMP-9 is present in supernatants of placental leukocyte cultures.
Representative western blot showing increasing activation of the exogenous recombinant proMMP-9 incubated in placental leukocyte supernatants. Three different supernatants were run in duplicate.
Fig 5.
MMP-3 increments in placental leukocyte supernatants along the culture.
Multiplex assay was performed in placental leukocyte supernatants to measure MMP-1, MMP-3, MMP-7 and MMP-9. MMP-3 showed a significant increase at 48 and 72h, while the other MMP-s did not significantly change. Bars represent mean ± SD of five independent experiments in duplicate. *P≤0.05, ***P≤0.001
Fig 6.
MMP-9 activation is inhibited by MMP-3.
Cultures of placental leukocytes were incubated in the presence or absence of a neutralizing anti MMP-3 or a specific MMP-3 inhibitor. Culture media analyzed by gelatin zymography shows that the 82 kDa active form of MMP-9 disappears in the presence of the anti MMP-3 antibody at 24, 48 and 72 h (panel A representative gel, n = 3). MMP-9 activation is abolished by the specific MMP-3 inhibitor; each point represents the mean ± SD (**P<0.005) (panel B, n = 3).
Fig 7.
MMP-9 and MMP-3 are produced in large amounts during labor.
Representative immunofluorescence images showing MMP-9 or MMP-3 immunolocalization in placental leukocytes obtained from term labor and non-labor women after 48 h of culture. Each panel shows Evans blue contrast staining (red) (upper left), MMP-9 or MMP-3 (green) (upper right), Nomarski interference contrast (lower left), and merge (lower right). Placental leukocytes from term labor produce more MMP-9 and MMP-3 compared to term non-labor cells. Confocal microscopy 40X.