Figure 4.

Activase rt-PA modulates microglia function in vitro. Cell migration assay (a and d), gelatinase activity assay (b), western blot (c) and chemotaxis assay (e and f) analyses examining the behavior of BV2 microglial cells after stimulation with Activase rt-PA (0,1 nM), LPS (2 μg/ml), IL-4 (5 ng/ml), and RAP (200 nM). Cell migration assay shows (a) an enhanced migration of microglial cells 24 h after stimulation with rt-PA or IL-4 compared with control or LPS exposure. Gelatin activity assay shows that (b) Activase rt-PA does not induce MMP2/9 activation in any conditions. Western blot analysis confirms (c) the unchanged expression levels of LRP1. Cell migration assay (d) shows that rt-PA-induced mobility is LRP1 dependent, as LRP1 inhibition with RAP, decreases cell migration. The two-chamber chemotaxis assay (e) provided evidence that rt-PA induces microglial mobilization toward a gradient of rt-PA present in the lower chamber. Microglia cells (f) are mobilized by Activase rt-PA in a LPR1-dependent manner 3 h after stimulation. Optical densities were corrected with β-actin levels. Data are means±SEM (n=3–4 independent experiments). *p<0.05, **p<0.01, ****p<0.0001 compared with control group (standard two-tailed unpaired t-tests). Dark spheres represent BV2 cells. The descending arrow illustrates cell mobilization from the upper chamber toward the lower chamber. Scale bar=250 μm.