FIGURE 7.

Inhibition of Rho activation reduces NET formation in BMDN. A, BMDN (2.5 × 10⁶) were stimulated with PBS or rmCIRP in presence of ICAM-1 neutralizing Ab for 120 minutes. After stimulating the BMDN with rmCIRP, cells were lysed in lysis buffer and Rho activity was determined by GTP pull down process, followed by western blot assays using anti-Rho Ab. A fraction of each lysate was retained to determine the total Rho and β-actin by western blot using rabbit anti-Rho and β-actin Abs. Representative western blots for active Rho, total Rho, and β-actin are shown. Active Rho in each sample was normalized to total Rho expression and the mean values of PBS-treated group was standardized as one for comparison. Experiment was repeated twice. Data are expressed as mean ± SD (n = 5–6 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs PBS; ^#P < .05 vs rmCIRP). B, C, A total of 10⁶ BMDN were stimulated with rmCIRP for 4 hours in presence of vehicle control (PBS) or Rho inhibitor. After stimulation with rmCIRP, the neutrophils were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG. NETs (MPO⁺citH3⁺ neutrophils) were assessed by flow cytometry. B, Representative dot plots of the NETs⁺ BMDN are shown. C, Bar diagram showing the frequencies of NETs⁺ neutrophils in the lungs are shown. Data are expressed as mean ± SE (n = 4 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs PBS; ^#P < .05 vs vehicle + rmCIRP)