Figure 3. Specific FPR1 inhibition protects function of chemokine receptors.

A. POL7200 specifically inhibits PMN migration to fMLF but not to CXCL1 or LTB₄. PMN pre-treated with vehicle (0.1% DMSO; presented as (–)) or POL7200 (1 μM) were placed in the upper chambers of transwells and allowed to migrate toward 100 nM fMLF, 50 nM CXCL1 or 100 nM LTB₄ placed in the lower chambers for 60 min. Chemotaxis of vehicle-treated PMN toward each chemoattractant was designated as 100%. All experiments were done in quadruplicate. Blank values (spontaneous migration /chemokinesis) were used to correct the results. Data are shown as mean ± SE and are representative of at least three different experiments. POL7200 completely inhibits chemotaxis to fMLF (black bars) but has no effect on chemotaxis toward CXCL1 (white bars) or LTB₄ (grey bars).

*: denotes significant difference analyzed by Student’s t-test, p<0.05.

B. Pre-treatment with FPR1 antagonists protects PMN from fMLF-induced heterologous desensitization of BLT1 as analyzed by intracellular calcium depletion. As described in Supplemental Digital Content 1, PMN loaded with Fura-2 were applied to a cuvette and pretreated with 0.1% DMSO (vehicle), 1 μM of POL7200, POL7178, or CsH for 1 min with a constant stirring. Then collection of data was initiated (time 0). At 30 sec, 10 nM fMLF was applied followed by 100 nM LTB₄ at 200 sec in the absence of calcium to detect calcium depletion from endoplasmic reticulum. Area under curve for 60 sec (AUC₆₀) after application of LTB₄ was calculated and normalized as AUC₆₀ of LTB4-induced depletion from untreated PMN as 100%. Prior treatment with fMLF markedly inhibits Ca²⁺ flux response to LTB₄ but FPR1 blockade by any of the FPR antagonists rescued LTB₄ suppression completely. Second column is significantly different from all other columns.

*: denotes significant difference by One-Way ANOVA with Tukey’s test, p<0.001.

C. Pre-treatment with FPR1 antagonists protects PMN from fMLF-induced heterologous desensitization of CXCR2 by intracellular calcium depletion. Similar experiments (as described in B) were done using 5 nM CXCL1 (GRO-α) that also induces brisk PMN Ca²⁺ flux. Pre-treatment with fMLF strongly inhibits that response. Again, treatment with all the FPR1 antagonists rescued the CXCR2 response. Second column is significantly different from all other columns. *: denotes significant difference by On-Way ANOVA with Tukey’s test, p<0.001.

D. Effect of POL7200 on active mtFPs-induced calcium depletion. Similarly to B and C, PMN loaded with Fura-2 in cuvette were pretreated with 1 μM POL7200 for 1 min. Then the four most potent mtFPs, ND4, ND5, ND6, and Cox1 (7) were applied successively at 100 nM concentrations. Pre-treatment of PMN with POL7200 abolished Ca²⁺ flux responses to all the active mtFPs but subsequent Ca²⁺ response to 10 nM LTB₄ was still well-preserved.