Figure 2

Chemotactic effect of r Hm C1q on leech microglia migration. (A) rHmC1q supernatant was compared to control supernatant using similar volumes in respective chemotaxis assays (0.1, 1, 3, 8, 15 μl). The diagram illustrates the chemotactic activity of rHmC1q in a dose-dependent manner. Asterisks denote cell migration of the indicated sample that is significantly different (p <0.01) than the corresponding control value. (B) Inhibitory effects on rHmC1q-mediated leech microglia chemotaxis. Leech microglial migration was evaluated with either L15 medium, control Pichia pastoris supernatant or rHmC1q-containing supernatant as chemoattractant. An inhibitory effect was demonstrated by preincubation of cells either with rabbit polyclonal anti-HmC1q antibodies (black hatching in each condition) or with a rabbit polyclonal anti-human C1qBP (alias gC1qR) antibody (black dots in each condition). Negative controls consisted of cells preincubated with rabbit preimmune serum or rabbit IgG isotype respectively. Asterisks denote cell migration of the indicated sample that is significantly different (p <0.01) than the corresponding controls. (C) Chemotactic effect of rHmC1q on leech microglia in ex vivo assays. Microglial cells, which are the only circulating resident cells present in the connectives, were counterstained with Hoechst dye. The Hoechst dying was observed immediately after crush injury (see arrows) with injection of phosphate-buffered saline (PBS) (a) or 4 h after injury with injection of PBS (b) illustrating microglial accumulation at the lesion site. Injection of rHmC1q increased microglial cells recruitment (c) compared either to the presence of PBS only (b) or to control yeast supernatant (d). When tissues were injected with polyclonal anti-HmC1q antibody (f), the chemotactic activity of rHmC1q was not observed whereas the corresponding preimmune serum did not exert any neutralizing effect (e).