Low-dose CCL2 does not interfere with rapid LFA-1 or VLA-4 activation by CCL21 but impairs adhesion persistence in vitro. (A) Frequency and strength of adhesive tethers between human T cells (intact or CCL2-pretreated) interacting with ICAM-1 coated alone or coimmobilized with CCL21 or denaturated (den) CCL21 (control). Experiments were performed at a shear stress of 0.5 dyn/cm². (B) Human T cells (intact or CCL2 pre treated) were allowed to accumulate for 2 minutes at a shear stress of 0.5 dyn/cm² on ICAM-1/CCL21 or denaturated CCL21 (den) and their adhesion persistence (ability to resist detachment by a constant application of high shear stress (5 dyn/cm²) was assessed at the indicated time points. Results are shown as percentage of cells initially accumulated on the integrin ligands. The experiment shown is representative of 4 independent experiments. (C) Effect of pre-exposure of lymphocytes to CCL2 (0.1 ng/mL) on the CCL21-triggered induction of the high-affinity LFA-1 epitope 327C in PBL. FACS staining showing spontaneous and CCL21-triggered expression of the 327C epitope at the indicated time points is presented as mean fluorescence intensity (MFI). The experiment shown is representative of 3 independent experiments. (D) 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. Immunoprecipitates were separated by 10% (wt/vol) SDS-PAGE and p-Vav and Vav were analyzed as described in “Vav detection.” (E) Graph summarizing the inhibition of Vav phosphorylation by CCL2 in CCL21-treated or untreated cells from 4 different experiments.