LTA induces slow internalization of cell-surface CCR5 molecules. Monocytes were preincubated on ice with MC-5 to label cell-surface CCR5 molecules before being transferred to 37°C for internalization assays. (A) The distribution of MC-5–labeled receptors after 1 hour of treatment was assessed by microscopy on fixed and permeabilized cells stained with a fluorescent secondary antibody (red) and DAPI (blue). (B-C) Internalization of labeled receptors was assessed by flow cytometry by measurement of cell-surface MC-5 fluorescence intensity on monocytes incubated for ≤ 30 minutes (B) in medium alone (□), with 100nM CCL5 (■), or 10 μg/mL LTA (▴), or an hour (C). Average CCR5 internalization values were determined from the results of 3 independent duplicate experiments; *P < .05, **P < .01, and ***P < .005. (D) MC-5–prelabeled LTA-treated cells were costained for TLR2. Single confocal sections are shown (scale bar = 5 μm).