rCXCL1 enhances NETosis in alcohol-challenged septic mice.

<p><b>A</b>, Western blotting of PAD-4, H3Cit and GAPDH in peritoneal cells (neutrophils) from alcohol-administered and control mice at 24 h post-CLP after rCXCL1 or BSA administration. This blot is representative of three independent experiments with identical results. <b>B</b>. Relative densities normalized against GAPDH are representative of the blots from 3 separate experiments (n = 3-5/mice/group). <b>C</b>, Peritoneal cells (neutrophils) harvested from alcohol-challenged and control mice after CLP were allowed to undergo NETosis after adding SYTOX Green for 8 h and then were fixed. Neutrophils were stained with citrullinated H3 Ab (α-H3Cit) to visualize citrullinated histone. Double-positive cells are indicated by arrowheads as evidence of NETosis. Images presented are representative of three independent experiments (n = 5-7/group). <b>D</b>, Twenty random images were selected from three experiments and quantified for the presence of NET-positive (double-positive; SYTOX Green- and H3Cit-positive) neutrophils from alcohol-administered and control mice. n = 5-8/group. <b>E</b>, Kinetic analysis of NETosis by peritoneal cells (neutrophils) harvested from alcohol-administered and control mice. Relative fluorescence intensity (RFU) was determined to evaluate NETosis each hour up until 8 h of culture. n = 6-9/group. Significance was calculated between rCXCL1 treated and BSA treated peritoneal cells from mice in the presence or absence of alcohol. <b>F</b>, Evaluation of NETosis by SEM. Peritoneal cells (neutrophils) harvested from alcohol-administered and control mice after CLP in rCXCL1 or BSA administration were analyzed by SEM. Presence of long thread-like structures (arrowhead) is evidence of NETosis. Scale bars, 20 μm. (n = 5–8 mice/group); NET forming neutrophils are indicated by the arrows and original magnification 20x; *, <i>p</i><0.05. AL, alcohol. Arrow indicates NET forming cells on merged images. SEM original magnification 8000x.