Figure 3.

VLA-4/VCAM-1-mediated tethering enhances B-cell activation under low shear stress. Naive B cells were perfused at low shear stress (0.1–0.05 dyn/cm²) over planar membranes containing VCAM-1 or ICAM-1 in the presence or absence of antigen. (A) Different time points of two representative experiments are shown. The upper panel in each horizontal pair shows DIC images in which tethered B cells are highlighted with a white circle. The bottom panels show their calcium response as fluo-4FF fluorescence in false colour scale. Scale bar, 5 μm. (B) The frequency of tethered B cells is represented as a percentage of the total number of perfused B cells under the different conditions analysed. The left panel shows 3-83 naive B cells on membranes loaded with the indicated densities of low-affinity p31 antigen, VCAM-1 or ICAM-1. The right panel shows the same but using MD4 B cells and HEL^RD as high-affinity antigen. The proportion of activated B cells was determined based on the increased levels of fluo-4FF fluorescence (filled bars: activated B cells; non-filled bars: non-activated B cells). (C) The fluo-4FF fluorescence intensity was measured every 4 s for at least 50 cells in random fields. The average intensity was plotted as a function of time for 3-83 B cells (left panel) and MD4 B cells (right panel) upon tethering on antigen-bearing membranes containing p31 and HEL^RD respectively, in the presence (blue line) or absence (green line) of VCAM-1. (D) Activation of the 3-83 naive B cells tethered (bound) versus the non-tethered B cells (non-bound) on membranes containing VCAM-1 and p31 as antigen. B-cell activation was evaluated after 24 h as the percentage of cells that upregulate CD86 and CD69, as described in Materials and methods. (E) As in (B), percentage of WT naive B cells or 3-83 naive B cells, which recognize H2-K^k as antigen, that tether and/or raise their calcium levels over L cells (VCAM-1⁺/H2-K^k), DCEK (VCAM-1⁻/H2-K^k) or DCEK transfectants (GPI-VCAM-1⁺/H2-K^k). (F) As in (C), calcium response of 3-83 B cells under the conditions specified in (E).