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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 4. Receptor desensitization by FPs is FPR1-specific.

Figure 4.

A. fMLF-induced desensitization is FPR1-specific. Bone marrow-derived neutrophils (BM-PMN) from wild type (WT), Fpr1-KO and Fpr2-KO mice were pre-treated with vehicle (0.1% DMSO, black bars) or 1 μM fMLF (gray bars) for 45 min. Cells were then applied to the top chambers of transwells while the bottom chambers contained 100 ng/mL of the mouse CXC-chemokine KC. Experiments were done in quadruplicate. Closed and open circles represent raw data points. PMN were pre-treated with either vehicle (DMSO, closed circle) or with 1 μM fMLF (open circle). PMN migration toward KC for 60 min was compared to migration of vehicle (DMSO)-pretreated cells set as 100%. Mean ± SE values are shown. # denotes significant differences (p<0.05) compared to DMSO-KC by Student’s t-test. Wild type and Fpr2-KO cells (both express FPR1) were desensitized to KC by prior fMLF exposure. On the other hand, Fpr1-KO PMN were protected from chemokine desensitization by exposure to formyl peptides.

B and C. POL7200 rescues ND6-induced heterologous receptor desensitization.

Surface expression of CXCR2 and BLT1 was studied by flow cytometry using specific florescent antibodies and isotype-control antibodies. Human PMN were divided into four groups. 1. Untreated, 2. 1 μM POL7200-treated, 3. 100 nM ND6-treated, and 4. 1 μM POL7200-pretreated, then treated with 100 nM ND6. The mean florescent index (MFI) of unstimulated PMN treated with isotype-control antibody was subtracted from the specific MFI for CXCR2 or BLT1 in all groups. Expression of receptors was normalized to that in untreated PMN, estimated as 100%.

B: Exposure to ND6 significantly reduces expression of CXCR2. However, pretreatment of POL prevented ND6-induced CXCR2 internalization. Mean ± SE values are shown. *: P=0.0046 vs. POL. P=0.005 vs. untreated. P=0.0089 vs. POL+ND6. Statistical analysis was done by One-Way ANOVA with Tukey’s test.

C: Exposure to ND6 significantly reduces expression of BLT1. Similarly, exposure to ND6 significantly reduced expression of BLT1 that was rescued by pre-exposure to POL7200. (#: p=0.0007 vs. POL, p=0.0008 vs. untreated, p=0.0029 vs. POL+ND6). Mean ± SE is reported for 3 separate experiments. Statistical analysis was done by One-Way ANOVA with Tukey’s test.