The percentage of released radioactivity was calculated from the full total radioactivity of loaded target cells per well. Analysis of T cell migration to inflammatory sites WT and LIME-/- mice were sensitized with DNFB on day 0 and 1, and CD4+ T cells were isolated from draining lymph nodes on day 5. accompanied by the impaired infiltration of leukocytes and T cells to the inflammatory sites of LIME-/- mice. More specifically, the absence of LIME in effector T cells resulted in the reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10. Consistently, LIME-/- effector T cells were found to be defective in chemokine-mediated activation of Rac1 and Rap1, and dysregulated phosphorylation of Pyk2 and Cas. Taken together, the present findings show that LIME is a critical regulator of inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites. (Difco Laboratories, USA). The immunized mice were injected intraperitoneally with 400 ng of pertussis toxin (List Biological Laboratories, USA) on day 0 and day 2 after immunization and were scored daily for clinical signs of EAE, as follows: 0, normal; 0.5, distal limp tail; 1, complete limp tail; 1.5, limp tail and hind limb weakness; 2, unilateral partial hind paralysis; 2.5, bilateral hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete bilateral hind limp paralysis and unilateral forelimb; 4, total paralysis of fore and hind limbs; 5, death. The mean onset day of EAE was calculated by averaging the first day of clinical signs for individual mice. The mean maximum score was calculated by adding the highest score for each mouse. Rabbit Polyclonal to Adrenergic Receptor alpha-2B Cytotoxicity assays The conventional standard [51Cr]-release assay was performed as Fmoc-Val-Cit-PAB follows. In brief, WT and LIME-/- mice were injected intraperitoneally with 106 plaque forming unit (PFU) of lymphocytic choriomeningitis virus (LCMV, Amstrong strain). Five days after immunization, CD8+ T cells were purified and mixed with gp33-41 peptide-pulsed, [51Cr]-loaded target cells (2 104 cells/well) in triplicate at the indicated effector/target cell ratio in 200 l complete RPMI medium. [51Cr]-labeling of target cells were performed by incubating EL4 lymphoma cells with 200 Ci (3.7 MBq) of Na2 51CrO4 (Perkin Elmer, USA) for 1 h at 37C. After 4 h of co-culture in U-bottomed 96 well plates (Corning, USA), 100 l of supernatant was collected and the released radioactivity was measured by -counter (Perkin Elmer). The percentage of released radioactivity was calculated from the total radioactivity of loaded target cells per well. Analysis of T cell migration to inflammatory sites WT and LIME-/- mice were sensitized with DNFB on day 0 and 1, and CD4+ T cells were isolated from draining lymph nodes on day 5. Subsequently, WT and LIME-/- CD4+ T cells were loaded with carboxyfluorescein diacetate-succinimidyl ester (CFSE; Molecular Probes, USA) or 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Molecular Probes), respectively, mixed at a 1:1 ratio, and intravenously transferred to the WT recipient mice (n = 3). Prior to transfer, recipient mice were sensitized with DNFB on their shaved abdomens at day -6 and -5 and rechallenged on their ears at day -1. Twenty four hours after adaptive transfer, distinctly labeled WT and LIME-/- T cells from frozen Fmoc-Val-Cit-PAB ear tissue sections were counted in at least 10 randomly selected fields Fmoc-Val-Cit-PAB and mean values were calculated from these counts. Analysis of chemokine-dependent migration migration assay was performed as described previously (Park et al., 2007). Briefly, a total of 1 1 106 T cells in 100 l of serum-free RPMI enriched with 0.3% BSA were loaded in the upper wells of 5-m pore-size, 24-well Transwell plates (Corning). The Transwell plates were then inserted into wells filled with medium containing CXCL12 (500 ng/ml), CXCL10 Fmoc-Val-Cit-PAB (50 ng/ml), or CCL5 (50 ng/ml) and incubated for 3 h at 37?C. Migration was quantified by counting the number of cells that migrated to the lower well. The experiments were performed at least three times in triplicate. Analysis of chemokine-mediated T cell polarization WT and LIME-/- Th1 cells were loaded with CFSE (0.5 M) or CMAC (2 M), alternatively, and mixed at a 1:1 ratio. Cells were treated with 20 ng/ml CXCL-10 and incubated at 37C for the indicated time period. After fixation with paraformaldehyde, cells were plated on poly-L-lysineCcoated glass slides and stained with allexa-568Clabeled phalloidin to visualize actin filaments. Slides were examined with a confocal microscope and up to 10 randomly selected pictures were evaluated in each group. Analysis of Rac1 and Rap1 activation Th1 cells were prepared as described above, but after 4 h of serum starvation, 1 107 Th1 cells were either left untreated or treated with 50 ng/ml of CXCL10 or CCL5 for 10 min, immediately harvested by centrifugation, and lysed in 500 l of lysis buffer. To pull down GTP-bound Rac1, cell lysates were incubated with 20 g of GST-PAK-RBD bound to glutathione-Sepharose beads.