Role of α4-integrin in mediating glomerular neutrophil adhesion in response to high-dose anti-MPO. (A) Effect of anti–α4-integrin (PS/2, 2 mg/kg, n = 9) or anti–VCAM-1 (6C7.1, 180 μg, n = 6) versus an isotype control mAb (n = 6) on glomerular leukocyte adhesion induced by high-dose anti-MPO (200 μg), as assessed by intravital microscopy. Anti–adhesion molecule mAbs were administered prior to administration of anti-MPO. (**P < .05 vs isotype control mAb.) (B) Up-regulation of neutrophil α4-integrin expression in response to anti-MPO in vitro. Following erythrocyte lysis, leukocytes isolated from whole blood were treated with anti-MPO (shaded) or anti-OVA (filled; 50 μg/mL, 30 minutes), and α4-integrin expression was assessed via flow cytometry. Data from cells treated with the appropriate isotype control antibody for the anti–α4-integrin antibody are also shown (dotted line). Neutrophils were identified by high expression of Gr-1 and Mac-1 (M1/70). Similar procedures were performed to assess resting splenic neutrophils (not shown). The data shown are representative of 3 to 4 individual experiments using both blood and splenic neutrophils. (C) Alteration in α4-integrin affinity on splenic neutrophils, as assessed by binding of soluble VCAM-1, in response to in vivo administration of anti-MPO (200 μg intravenously, 5-30 minutes, shaded). Data are also shown for cells from mice treated with anti-OVA (open). The data shown are representative of 4 individual experiments using splenic neutrophils. Similar anti-MPO–induced affinity changes were observed when treating splenic neutrophils from naive mice with anti-MPO in vitro.