Figure 4.

Role of CalDAG-GEFI in LFA-1– and VLA-4–mediated T-cell adhesion. (A) Representative Western blots of Rap1-GTP and CalDAG-GEFI levels in T cells transfected with control or CalDAG-GEFI siRNA and stimulated with 100 nM SDF-1α for 10 seconds (left) or 1μg/mL PMA for 5 minutes (right). β-actin is shown as a loading control. Severely diminished activation of Rap1 was observed in CalDAG-GEFI knock-down cells. Densitometric analysis evaluated the optical density, in arbitrary units, of Rap1-GTP levels after stimulation with SDF-1α in control siRNA and CalDAG-GEFI siRNA cells. CalDAG-GEFI levels in the same samples were also quantified by densitometry. Extent of Rap1 inactivation correlated with CalDAG-GEFI knock-down. *P < .04, **P < .005. (B) PMA-stimulated cell adhesion to immobilized ICAM-1 (2.5 μg/mL) by control or CalDAG-GEFI siRNA-transfected cells shows impaired adhesion in cells deficient in CalDAG-GEFI. *P < .001. Inset, Mn²⁺ or Ca²⁺ ionophore (A23187) treatments of T cells transfected with CalDAG-GEFI siRNA resulted in normal adhesion to ICAM-1. (C) Silencing CalDAG-GEFI does not prevent PMA-stimulated T-cell adhesion to immobilized VCAM-1 (2 μg/mL), compared with cells transfected with control siRNA. (D) Effects of CalDAG-GEFI knock-down on chemokine-stimulated LFA-1 adhesion to ICAM-1 under shear flow. T cells transfected with control siRNA or CalDAG-GEFI siRNA were perfused over ICAM-1 coimmobilized with SDF-1α at shear stress rate of 0.75 dyne/cm². The number of adherent cells is expressed as the mean plus or minus SEM. T cells lacking CalDAG-GEFI show impaired SDF-1α-mediated adhesion to ICAM-1 compared with control cells. *P < .05. (E) T cells, treated as described in panel D, were perfused over VCAM-1 coimmobilized with SDF-1α. Both control siRNA cells and CalDAG-GEFI siRNA cells adhered to VCAM-1. Three to 5 independent experiments were done for each panel.