Figure 8.

MC-4 inhibits CCR5 endocytosis mediated by chemokines. (A and B) CHO-K1 cells expressing CCR5 were preincubated with medium (▪), 10 μg/ml MC-4 (●), or MC-4 F(ab) (▵) for 30 min on ice and then incubated with RANTES (A) or AOP-RANTES (B) for 30 min at 37°C. Surface expression of CCR5 was measured by flow cytometry with the antibody MC-4 or MC-4 F(ab) (15 μg/ml, on ice) and FITC-conjugated secondary antibodies. Fluorescence was normalized as 100% in the absence of chemokines and 0% for background fluorescence. All experiments were repeated at least three times with similar results. Cells expressing CCR5-GFP are shown before (C), and 30 min (D) after MC-4 addition (10 μg/ml) at 37°C. Cells were then stimulated with 100 nM RANTES, and the subsequent dynamics of CCR5-GFP was recorded every 3 min by confocal microscopy. Responses are shown 15 (E), 30 (F), and 45 min (H) after RANTES addition. Arrows point to agonist-stimulated receptor patches. Arrowheads point to remaining cell surface CCR5-GFP after 45 min of RANTES stimulation. This session is representative of at least four similar experiments. Videos of the dynamics of CCR5-GFP trafficking in response to RANTES stimulation, as described in this figure, are available in the online version of this article. CCR5-GFP–expressing cells did not show differences in cell surface receptor levels before (H) and 30 min after control Ig addition (I), and did not inhibit the ability of RANTES to induce CCR5 endocytosis after 45 min of observation (J).