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. 2007 Jul 9;204(7):1571–1582. doi: 10.1084/jem.20070058

Figure 7.

Figure 7.

Defective chemokine triggering of LFA-1– and VLA-4–mediated PBL arrest on ICAM-1 and VCAM-1. (A) Western blot analysis of T lymphocyte lysates derived from a C57BL mouse, a healthy donor (control), and LAD patient A (LAD). CalDAG-GEFI (CDGI) levels were probed with an anti–CalDAG-GEFI mAb (top). Actin levels are shown as controls (bottom). (B) FACS staining of LFA-1, VLA-4, and CXCR4 on control and LAD PBLs using the αL subunit–specific mAb TS2.4, the α4 subunit-specific mAb HP1.2, and 12G5, respectively. (C) LFA-1 extension epitope detected by the reporter mAb KIM127 (top row) and high affinity LFA-1 epitope detected by the mAb 327C (bottom row) in intact (−) and CXCL12-treated (+) control and LAD PBLs, assessed by FACS staining. Background antibody stainings in B and C had fluorescence intensity values of <5. (D) Tethering and immediate arrest of control (left) and LAD (right) PBLs on ICAM-1-Fc (coated at 2 μg/ml) triggered by immobilized CXCL12 (2 μg/ml). Control or LAD PBLs were perfused over the substrates at a shear stress of 0.5 dyn/cm2, and both the frequency and strength of all tethers were determined in two fields. Results are given as the mean ± the range. All adhesive tethers were blocked in the presence of the LFA-1 blocking mAb, TS1.18, or EDTA. The experiment shown is representative of three. (E) Tethering and arrest of control and LAD PBLs measured at a shear stress of 0.5 dyn/cm2 on sVCAM-1 (coated at 2 μg/ml) triggered by immobilized CXCL12 (2 μg/ml). The lifetimes of the transient tethers are depicted above the bars. All tethers were blocked in the presence of the VLA-4 blocking mAb HP1.2, or the low mol wt ligand, Bio1211 (not depicted). The different tether categories were determined as in D.