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

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posted on 2016-05-06, 05:21 authored by Gabriele Zuchtriegel, Bernd Uhl, Daniel Puhr-Westerheide, Michaela Pörnbacher, Kirsten Lauber, Fritz Krombach, Christoph Andreas Reichel

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 CX3CR1GFP/+ 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).

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