Figure 3. Tumor-derived factors promote integrin α4β1 activation and myeloid cell adhesion to endothelium.
(A) Adhesion of chemoattractant-treated myeloid cells (fluorescence units, F.U.) to endothelium in the absence or presence of control IgG, anti-α4, anti-αM, anti-α5 and anti-αv integrin antibodies (n=3) or after transfection with α4 integrin and control siRNAs, *P<0.01 vs IgG. (B) Adhesion to endothelium of chemoattractant-treated WT and MyD88−/− myeloid cells and WT myeloid cells in the absence or presence of pertussis toxin (PTX) or AMD3100 (n=3). *P<0.01 vs WT. (C) Histogram of human myeloid cells stained to detect active (HUTS21, upper panel) and total (P4C10, lower panel) human integrin β1 in the absence (unstimulated, gray filled) or presence (stimulated, black line) of SDF-1α, IL-1β, or Mn2+, a positive control activator. IgG negative control appears as thin line (n=5).
(D) Images: clustering of integrin α4 (green, arrowheads) in human myeloid cells incubated with SDF-1α, IL-1β, or human serum albumin (HSA)-coated microspheres and counterstained for DNA (blue). Graph: Percentage of integrin α4β1 clustering per myeloid cell (n>15). *P<0.001 vs HSA.
(E) Percentage of integrin α4β1 clustering of WT, MyD88−/−, pertussis toxin (PTX) and AMD3100-treated WT myeloid cells after incubation with SDF-1α or IL-1β-coated microspheres (n>15). *P<0.001 vs WT.
Scale bars indicate 5 µm. Error bars represent SEM.