Skip to main content
. 2014 May 12;9(5):e97088. doi: 10.1371/journal.pone.0097088

Figure 4. Involvement of the GPCR-PLC pathway in IL-8 induced NETosis. A. Treatment with pertussis toxin inhibits IL-8 induced NETosis.

Figure 4

Isolated neutrophils were pretreated with pertussis toxin (PTX) (100 ng/ml) for 15 minutes and then cultured with activators, rIL-8 (100 ng/ml), PMA (50 nM), ionomycin (5 µM) for 1 hour. NETosis was detected by fluorescent microscopy for SYTOX Green stained extracellular DNA structures. Magnification: 10x. B. Fluorimetric detection of NETosis reduction by pertussis toxin treatment in IL-8 stimulated neutrophils. This figure illustrates a quantitative reduction in IL-8 induced NETosis by treatment with PTX (15 minute pre-treatment) (upper panel), while that triggered by PMA (lower panel), was largely unaffected. An analysis from 6 different experiments is shown, and the data presented as mean ± SD (standard deviation). NET formation was normalized to 100% as induced by treatment with rIL-8 (100 ng/ml) or PMA (50 nM) in the absence of any inhibitors. Unpaired t-test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: **, P<0.01. C. Influence of staurosporine treatment on IL-8 induced NETosis. This figure illustrates a quantitative reduction in IL-8 induced NETosis by treatment with the protein kinase C inhibitor, staurosporine (STS) (15 minute pre-treatment) (upper panel), and almost complete abolition of that triggered by PMA. An analysis from 6 different experiments is shown, and the data presented as mean ± SD (standard deviation). NET formation was normalized to 100% as induced by treatment with rIL-8 (100 ng/ml) or PMA (50 nM) in the absence of any inhibitors. Unpaired t test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: **, P<0.01. D. Inhibition of Phospholipase C reduced IL-8 induced NETosis. Following pre-treatment with U73122 for 15 minutes, an inhibitor of phospholipase C, neutrophils were cultured for 1 hour in the presence of 100 ng/ml IL-8, or 50 nM PMA, or 5 µM ionomycin. These data indicate that only IL-8 induced NETosis was significantly affected. NETs were quantified fluorimetrically after 1 hr culture using SYTOX Green dye and the data normalized as described. An analysis from 4 different experiments is shown, and the data presented as mean ± SD (standard deviation). Unpaired t-test was used to calculate two-tailed P value to estimate statistical significance of differences between two groups. Statistically significant P values are indicated in the graphs as follows: Statistically significant P values are indicated in the graphs as follows: ****, P<0.0001.