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. Author manuscript; available in PMC: 2021 Feb 1.
Published in final edited form as: Crit Care Med. 2020 Feb;48(2):e123–e132. doi: 10.1097/CCM.0000000000004094

Figure 1. Effects of FPR1 antagonists on PMN function.

Figure 1.

A. Effects of FPR-1 antagonists on fMLF-induced PMN Ca2+depletion from ER. PMN were prepared from six healthy volunteers and stimulated with 100 nM fMLF. fMLF-induced Ca2+ store depletion was studied using Fura-2AM. Responses were assessed as the area under the intracellular Ca2+ concentration ([Ca2+]i) curve for 60 sec after stimulation (AUC60 – see Supplemental Digital Content 1). Dose-dependent inhibition of fMLF-induced Ca2+ depletion was seen in the presence of all three compounds, POL7200, POL7178, and CsH. Effect size was determined by comparing AUC60 for Ca2+ depletion in the presence of antagonists with vehicle (DMSO) assigned as 100%. Values are shown as mean ± SE.

B. Effects of FPR1 antagonists on mtDAMPs-induced Ca2+ depletion from ER. Experiments done as in panel A except that sonicated human MT (mtDAMPs, 30 μg/mL final) prepared from human liver were used as the stimulant. N=5 PMN preparations were used for POL7200 and POL7178. N=2 PMN samples were used for CsH. Mean ± SE values are shown. EC50 for both POL compounds was 0.001–0.01μM. EC50 for CsH was 0.01–0.1μM.

C. Effects of FPR1 antagonists on fMLF-induced chemotaxis (CTX). PMN isolated from healthy volunteers were pre-treated with POL7200 or POL7178 for 15 min at room temperature. 105 PMN were applied to the upper chamber of transwells. The bottom chamber contained 100 nM fMLF. After 60 min migrated PMN were collected and counted by loading them with CyQUANT dye (Materials and Methods, Supplemental Digital Content 1). Effects of FPR1 antagonists on PMN CTX were calculated by comparison to CTX of vehicle-only treated PMN to fMLF, which was established as 100%. Mean ± SE values are shown. N=5.

D. Effect of POL7200 on ND6-induced CTX. PMN were pre-treated with POL7200 as shown. 100 nM ND6 was used as a chemoattractant. Mean ± SE values are shown for six different PMN donors. For each donor each condition was assayed in quadruplicates. The EC50 was about 50 nM. All pairs except “0” and “10 nM” POL7200-treated PMN (ns, not significant) showed significant difference (p<0.001) by One-Way ANOVA with Tukey’s test.