Figure 7.
Figure 7. Chemokine-triggered adhesion strengthening of VLA-4 tethers is impaired in LAD lymphoblasts due to defective VLA-4 rearrangement despite conserved integrin clustering and affinity to soluble ligand. (Ai) FACS staining of α4 integrins on control and LAD-derived EBV-transformed B lymphoblasts with the α4 subunit–specific mAb HP1/2. Staining of the SDF-1 receptor CXCR4 was performed with the mAb 6H8. (ii) Induction of 15/7 LIBS epitope by soluble monovalent VLA-4 ligand (Bio1211). Dose-dependent induction of the epitope by increasing concentrations of the specific ligand is depicted for LAD cells (○) and control cells (▪). The 15/7 epitope staining, as detected with PE-antimouse IgG and analyzed by FACScan, is expressed in mean fluorescence intensity units (MFI). (B) Chemokine-triggered adhesion strengthening of VLA-4/VCAM-1 tethers at subsecond contacts is impaired in LAD EBV lymphoblasts. Immediate SDF-1–augmented VLA-4–mediated capture and arrest of control and patient cells on VCAM-1 under shear flow. The frequency of different types of cell tethers to purified sVCAM-1 (2 μg/mL) coimmobilized with functional (+) or heat-inactivated (-) SDF-1 (1 μg/mL) was determined as in Figure 4. Complete blocking of chemokine-triggered or spontaneous blast adhesion to VCAM-1 with α4 and β1 integrin mAbs but not with the α4β7-specific mAb suggested an exclusive role for VLA-4 in SDF-1–triggered lymphoblast tethering to VCAM-1 (not shown). □ indicates transient tethers; ▦, rolling; ▪, arrest. (C) Chemokine triggering of transient VLA-4–mediated tethers to low-density ligand is not defective in LAD blasts. The frequency of different types of cell tethers to purified sVCAM-1 (0.5 μg/mL) coimmobilized with functional (+) or heat-inactivated (-) SDF-1 (2 μg/mL) was determined as in Figure 4. □ indicates transient tethers; ▦, rolling; ▪, arrest. (D) VLA-4 is uniformly distributed both on normal and LAD lymphoblasts prior to contact with chemokine and ligand. Fluorescence microscopy of α4 integrins on normal and LAD EBV lymphoblasts. Fixed cells were incubated with HP1/2, stained with Alexa-488–conjugated secondary Ab, and analyzed by confocal microscopy as described in “Materials and methods.” White scale bar, 5 μm. In each experimental group, 3 representative cells are shown. (E) Capture of lymphoblasts on immobilized α4-specific mAb HP1/2 (0.2 μg/mL) triggered by coimmobilized SDF-1 (2 μg/mL; inactive, -; intact, +). All cell capture events resulted in immediate arrest in this experimental setting. No tethers were observed on SDF-1 alone or on control mAb anti-VCAM-1 (not shown). (F) Normal PMA stimulation (100 ng/mL, 2 minutes) of ERK1/2 phosphorylation in control and LAD EBV lymphoblasts. Immunoblotting with antiphosphospecific ERK1/2 (top panel) and anti-ERK (bottom panel) is depicted. Cell lysates were separated on reducing 10% SDS-PAGE. (G) Impaired PMA-triggered VLA-4–mediated adhesion of LAD-derived EBV-transformed B lymphoblasts to VCAM-1 at 1-minute stationary contacts. Resistance to detachment by incremented shear forces developed by cells, untreated or stimulated by PMA (100 ng/mL, 2 minutes), settled for 1 minute at stasis on sVCAM-1 (0.5 μg/mL). Results are given as mean ± range of determinations in 2 fields of view. PMA failed to stimulate VLA-4 avidity at subsecond contacts of both normal and LAD EBV lymphoblasts tethered to VCAM-1 under shear flow (not shown). Experiments depicted in panels B-C,E,G are each representative of 3 to 4 independent tests.

Chemokine-triggered adhesion strengthening of VLA-4 tethers is impaired in LAD lymphoblasts due to defective VLA-4 rearrangement despite conserved integrin clustering and affinity to soluble ligand. (Ai) FACS staining of α4 integrins on control and LAD-derived EBV-transformed B lymphoblasts with the α4 subunit–specific mAb HP1/2. Staining of the SDF-1 receptor CXCR4 was performed with the mAb 6H8. (ii) Induction of 15/7 LIBS epitope by soluble monovalent VLA-4 ligand (Bio1211). Dose-dependent induction of the epitope by increasing concentrations of the specific ligand is depicted for LAD cells (○) and control cells (▪). The 15/7 epitope staining, as detected with PE-antimouse IgG and analyzed by FACScan, is expressed in mean fluorescence intensity units (MFI). (B) Chemokine-triggered adhesion strengthening of VLA-4/VCAM-1 tethers at subsecond contacts is impaired in LAD EBV lymphoblasts. Immediate SDF-1–augmented VLA-4–mediated capture and arrest of control and patient cells on VCAM-1 under shear flow. The frequency of different types of cell tethers to purified sVCAM-1 (2 μg/mL) coimmobilized with functional (+) or heat-inactivated (-) SDF-1 (1 μg/mL) was determined as in Figure 4. Complete blocking of chemokine-triggered or spontaneous blast adhesion to VCAM-1 with α4 and β1 integrin mAbs but not with the α4β7-specific mAb suggested an exclusive role for VLA-4 in SDF-1–triggered lymphoblast tethering to VCAM-1 (not shown). □ indicates transient tethers; ▦, rolling; ▪, arrest. (C) Chemokine triggering of transient VLA-4–mediated tethers to low-density ligand is not defective in LAD blasts. The frequency of different types of cell tethers to purified sVCAM-1 (0.5 μg/mL) coimmobilized with functional (+) or heat-inactivated (-) SDF-1 (2 μg/mL) was determined as in Figure 4. □ indicates transient tethers; ▦, rolling; ▪, arrest. (D) VLA-4 is uniformly distributed both on normal and LAD lymphoblasts prior to contact with chemokine and ligand. Fluorescence microscopy of α4 integrins on normal and LAD EBV lymphoblasts. Fixed cells were incubated with HP1/2, stained with Alexa-488–conjugated secondary Ab, and analyzed by confocal microscopy as described in “Materials and methods.” White scale bar, 5 μm. In each experimental group, 3 representative cells are shown. (E) Capture of lymphoblasts on immobilized α4-specific mAb HP1/2 (0.2 μg/mL) triggered by coimmobilized SDF-1 (2 μg/mL; inactive, -; intact, +). All cell capture events resulted in immediate arrest in this experimental setting. No tethers were observed on SDF-1 alone or on control mAb anti-VCAM-1 (not shown). (F) Normal PMA stimulation (100 ng/mL, 2 minutes) of ERK1/2 phosphorylation in control and LAD EBV lymphoblasts. Immunoblotting with antiphosphospecific ERK1/2 (top panel) and anti-ERK (bottom panel) is depicted. Cell lysates were separated on reducing 10% SDS-PAGE. (G) Impaired PMA-triggered VLA-4–mediated adhesion of LAD-derived EBV-transformed B lymphoblasts to VCAM-1 at 1-minute stationary contacts. Resistance to detachment by incremented shear forces developed by cells, untreated or stimulated by PMA (100 ng/mL, 2 minutes), settled for 1 minute at stasis on sVCAM-1 (0.5 μg/mL). Results are given as mean ± range of determinations in 2 fields of view. PMA failed to stimulate VLA-4 avidity at subsecond contacts of both normal and LAD EBV lymphoblasts tethered to VCAM-1 under shear flow (not shown). Experiments depicted in panels B-C,E,G are each representative of 3 to 4 independent tests.

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