Figure 8.

The hypermigration of SSc monocytes and TGF-β-treated Control monocytes toward CCR5 ligands is regulated by caveolin-1. (A) Migration experiments were performed as described in the Methods using Control or SSc monocytes. Cells were treated with TGF-β and CSD as indicated. Lower wells contained 100 nM of MIP1α or MIP1β. Cells that had migrated were counted in six high power fields per filter. Note that for both MIP1α and MIP1β, the induction of migration was extremely enhanced in both SSc monocytes and TGF-β-treated Control monocytes compared to untreated Control monocytes and that in both cases CSD completely blocked the enhanced migration. The data represent the average ± s.e.m. from four independent experiments using cells from different subjects. (B) CCR5 level is increased in SSc monocytes; (C) CSD decreases the level of CCR5 in SSc monocytes. Control and SSc monocytes were isolated as described in the Methods. SSc monocytes were treated with CSD as previously described (Tourkina et al., 2010). For Western blotting experiments, cells were extracted with boiling SDS-PAGE Sample Buffer. In (B), the Western blot of a typical experiment shows a large increase in the level of CCR5 in SSc monocytes compared to Control monocytes and no change in the level of the loading control GAPDH. In (C), the Western blot of a typical experiment shows that CSD treatment decreases the level of CCR5 in SSc monocytes without affecting GAPDH. The densitometric quantifications in (B,C) show the average ± s.e.m. in arbitrary units for four repeats of each experiment performed with cells from different subjects. The Control value in (B) and the CSD minus value in (C) are set to 1 arbitrary unit.