Consequences of interactions of iMOs with platelets and neutrophils for the extravasation of iMOs.

Interactions of intravascularly adherent neutrophils and iMOs were analyzed by multichannel in vivo microscopy. Panel shows quantitative data for interactions (>30 s) between intravascularly adherent neutrophils and iMOs in animals receiving blocking mAbs directed against CD62L or PSGL-1/CD162, or istoype control antibodies (A; mean ± SEM; n = 4 per group; *p < 0.05 versus isotype control; scale bar 10 μm). Binding of ICAM-1/CD54 or VCAM-1/CD106 to iMOs isolated from the peripheral blood of WT mice was assessed upon exposure to recombinant murine CD40L/CD154, P-selectin/CD62P, L-selectin/CD62L, or PSGL-1/CD162 by flow cytometry (B; mean ± SEM; n = 4–6 per group; #p < 0.05 versus unstimulated; *p < 0.05 versus isotype control or vehicle). P-selectin-elicited binding of ICAM-1/CD54 to murine iMOs was quantified upon blockade of PSGL-1/CD162 or inhibition of MAPK (B, upper panels). Conformational changes of β2 integrins in human iMOs were analyzed by using conformation-specific mAbs (mean ± SEM; n = 4 per group; #p < 0.05 versus unstimulated; B, lower panels). Using multichannel in vivo microscopy on the CCL2-stimulated cremaster muscle of CX₃CR1^GFP/+ mice, intravascular rolling flux and firm adherence of iMOs were analyzed. Extravasation of iMOs was evaluated by using a peritonitis assay. Panels (C) show quantitative data for CCL2-challenged animals receiving blocking mAbs directed against Mac-1/CD11b, LFA-1/CD11a, VLA-4/CD49d, ICAM-1/CD54, ICAM-2/CD102, VCAM-1/CD106, PECAM-1/CD31, or JAM-A, or isotype control antibodies (mean ± SEM; n = 4–6 per group; *p < 0.05 versus isotype control).