C3a enhances SDF-1–dependenttrans-Matrigel migration and secretion of MMP-9.

(A) Stimulation of SDF-1–dependent trans-Matrigel migration of human CD34⁺ cells by C3a. BM CD34⁺ (▪) and mobilized PB CD34⁺ cells (░) prestimulated or not with C3a (1 μg/mL) were loaded onto Boyden chambers and allowed to migrate across Matrigel toward media alone (control) or toward an SDF-1 gradient (20 or 300 ng/mL). Each experiment was performed using at least 4 chambers for each set of conditions and cell counts were done in duplicate. *P < .002. (Inset) Stimulation of MMP-9 secretion by C3a. Media conditioned by BM- or PB-derived CD34⁺ cells preincubated or not with C3a (1 μg/mL) were analyzed by zymography. The bands indicating MMP-9 activity were quantitated by densitometric analysis, and fold stimulation of MMP-9 secretion in the presence of C3a was calculated relative to control. The data shown are pooled from experiments using 3 to 4 samples of BM and PB CD34⁺ cells. (B) Inhibition of SDF-1–dependent trans-Matrigel migration by MMP inhibitors. PB CD34⁺ cells were preincubated with C3a (1 μg/mL) with or without 10 μg/mL each of rhTIMP-1 or anti–MMP-9 monoclonal antibody for 2 hours or with 0.5 mMo-phenanthroline for 30 minutes. The bottom chambers contained 20 ng/mL SDF-1 except for the control (media alone). Each experiment was performed using at least 4 chambers for each set of conditions and cell counts were done in duplicate. *P < .0001.