Effects of PSGL-1 ligation on αMβ2. (A) Neutrophils were incubated with mAb IB4 or CBRM1/5 and Alexa Fluor 488–conjugated goat antibody to mouse IgG without (control) or with P-selectin Ig chimera (P-sel Ig) or PMA. They were then centrifuged through a cushion of FCS and fixed in 1% paraformaldehyde in PBS for flow cytometric analysis. Results are the representative of 3 independent experiments. (B) Neutrophils were incubated with Alexa Fluor 488–conjugated fibrinogen, in the absence (control) or presence of human IgG (hIgG), P-selectin Ig chimera (P-sel Ig), and PMA. The samples were processed as described in panel A. Results are the representative of 3 independent experiments. (C) Neutrophils were incubated with PMA or P-selectin Ig chimera (P-sel Ig) in the presence of an increasing concentration of soluble fibrinogen and then transferred to the wells coated with fibrinogen. The results of cell adhesion assays are expressed as the mean ± SD values of the adherent cells determined in triplicate measurements of 3 separate experiments. (D) Neutrophils were incubated with human IgG (hIgG) and P-selectin Ig chimera (P-sel Ig) in the presence of G1 or PS1 F(ab′)2 prior to adding them to the wells immobilized with moAb CBRM1/5. After washing, the bound neutrophils were quantified by MPO activities. All results are expressed as the mean ± SD values of the adherent cells determined in triplicate measurements of 3 separate experiments.
