Figure 7.
Effects of IAIPs on extracellular ROS production and p47phoxphosphorylation in neutrophils. (A) The purified human neutrophils were incubated with different concentrations of IAIPs (12.5, 25, and 100 μg/mL) or HBSS for 30 minutes and the ROS produced extracellularly was determined using isoluminol as a substrate. The chemiluminescence in the medium was determined by FlexStation 3. The time-course changes are shown. Results are expressed as means ± SEM of 6 experiments. (B) The ROS produced extracellularly was determined at 15 minutes in the presence of different concentrations of IAIPs. The results are expressed as means ± SEM of 6 experiments. **P < .01 vs HBSS. (C) The purified human neutrophils were incubated with 25 ng/mL GM-CSF (positive control), HBSS, HSA (1 μM), or IAIPs (100 μg/mL) for 20 minutes at 37°C. Cells were then lysed, and proteins from 4 × 106 cells were analyzed with SDS-PAGE. Western immunoblotting was performed with anti-p47phox and anti-phospho-Ser328 antibodies. (D) Western immunoblots from 3 different experiments were scanned; phosphorylated and total p47phox were quantified by densitometry. Results are expressed as means ± SEM (n = 3). **P < .01 vs HBSS. (E) IAIPs 6.25 μg/mL or 12.5 µg/mL were preincubated with control IgG1 20 µg/mL or IAIP Ab 20 µg/mL for 30 minutes at 37°C. Purified human neutrophils were then incubated with IAIPs for 30 minutes and the ROS produced extracellularly was determined using isoluminol as a substrate. The chemiluminescence in the medium was determined by FlexStation 3. Percentage suppression of ROS can be calculated using the following formula: ((chemiluminescence intensity of cells treated with HBSS or control IgG1 or IAIPs Ab alone [control] − chemiluminescence intensity of cells treated with IAIPs or IAIPs with IgG1 or IAIPs with IAIP Ab)/chemiluminescence intensity of control) × 100. The results are the means ± SEM of 3 experiments. ☥P < .05 IAIPs vs IAIPs + α-IAIP; &P < .05 IAIPs + control IgG vs IAIPs + α-IAIP.