CCR2 is expressed on naive T cells and inhibits their cytoskeleton rearrangement and migration. (A) CD4⁺ and CD8⁺ T cells were separated. RNA was isolated and levels of CCR2 and HPRT mRNA were analyzed by reverse transcription–polymerase chain reaction. The results presented are representative of 3 independent experiments. (B,C) Transwell migration. Control or CCR2^−/− naive T cells were pretreated for 30 minutes with medium or CCL2 (0.1 ng/mL). The cells were then placed in the upper well of a 24-well transwell plate in the presence or absence of CCL21 (0.1 μg/mL). After 3 hours, the number of migrating cells found in the lower chamber was evaluated by fluorescence-activated cell sorting (FACS) analysis. Percent migration was calculated as the number of migrating cells in the lower chamber as a fraction of the input cell number in the upper chamber. The results presented are representative of 3 different experiments. (D-F) Cytoskeleton rearrangement. Naive T cells from control (D-F) or CCR2^−/− mice (D) were stimulated with CCL21 (0.1 μg/mL) for 15 seconds in the presence or absence of CCL2 (0.1 ng/mL; D-F), various concentrations of CCL2 (E), or Rantes, Mip1b, or Eotoxin (0.1 ng/mL; F). The cells were fixed and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin. The change in polymerized actin was analyzed by FACS. Percentage increase in actin polymerization was calculated as the polymerization of actin in the presence of chemokine stimulation/polymerization of actin without chemokine ×100. The results presented are representative of 3 separate experiments. (G) Naive T cells were pretreated with or without CCL2 (0.1 ng/mL) for 30 minutes. The cells were then stimulated with CCL21 and lysed immediately. Lysates were separated on reducing 10% (wt/vol) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-phosphospecific ERK 1/2 (p-ERK). Immunoblots were stripped and reprobed with anti-[total] ERK 1/2. (H) Cytoskeleton rearrangement. Naive T cells were stimulated with medium or CCL21 (0.1 μg/mL) for 15 seconds in the presence or absence of CCL2 (0.1 ng/mL) or the MEK inhibitor, U0126 (5 μM). The cells were fixed and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin. The change in polymerized actin was analyzed by FACS and calculated as described above. (I) Transwell migration. Naive T cells were pretreated for 30 minutes with medium or CCL2 (0.1 ng/mL) in the presence or absence of the MEK inhibitor, U0126 (5 μM). After 3 hours, the number of migrating cells found in the lower chamber was evaluated by FACS analysis. Percentage migration was calculated as described above.