FIGURE 5.

ICAM-1 deficient neutrophils or blockade of TREM-1 in WT neutrophils with LP17 produce less NETs following rmCIRP stimulation. A total of 10⁶ BMDN isolated from WT or ICAM-1^−/− mice were stimulated with rmCIRP for 4 hours. After stimulation with rmCIRP, isolated NETs were loaded in (A) MPO:DNA and (B) NE:DNA coated plates and assessed by ELISA. C, A total of 10⁶ BMDN isolated from WT or ICAM-1^−/− mice were stimulated with rmCIRP for 4 hours. 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. D, E, After 4 hours of injection of rmCIRP intratracheally (i.t.) in WT and ICAM-1^−/− mice lungs were perfused and harvested. Single cell suspensions of lung tissues were surface-stained with APC-rat anti-mouse Ly-6G Ab, FITC-mouse anti-MPO Ab, and rabbit anti-histone H3 (CitH3) Ab followed by staining with PE-donkey anti-rabbit IgG and NETs were determined by flow cytometry. D, The frequencies and (E) numbers of NET forming neutrophils in lungs are shown. Experiments were repeated at least three times. Data are expressed as mean ± SE or SD (n = 5–14 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs WT PBS; ^#P < .05 vs WT rmCIRP). F, A total of 10⁶ BMDN/mL in 24-well plate were treated with PBS or rmCIRP in the presence of vehicle (PBS), LP17 or scramble. After 4 hours of rmCIRP stimulation, NETs were collected from the cells and assessed NE contents by ELISA. Experiments were repeated at least three times. Data are expressed as mean ± SD (n = 6–11 samples/group). The groups were compared by one-way ANOVA and SNK method (*P < .05 vs PBS-treated group; ^#P < .05 vs vehicle (PBS) + rmCIRP-treated group; †P < .05 vs scramble + rmCIRP-treated group)