Human SAA1(47-104) synergizes with CXCL8 in neutrophil chemotaxis via FPR2. (A) The chemotactic potencies of CXCL8 (0.2-3 ng/mL), SAA1(47-104) (300 and 1000 ng/mL), and a combination of CXCL8 and SAA1(47-104) were evaluated on neutrophils in the Boyden microchamber assay. Data represent the mean CI from 6 to 10 independent experiments. (B) Neutrophils were preincubated (10 minutes, 37°C) with desensitizing SAA1(47-104) (300-3000 ng/mL) or with control buffer and were subsequently added to the upper wells of the Boyden microchamber. Chemotaxis was induced by adding 3 ng/mL of CXCL8, 300 ng/mL of hu rSAA1α or a combination of CXCL8 and rSAA1α to the lower wells. Data are shown as mean CI ± SEM and are pooled from 5 to 6 independent experiments. (C) Neutrophils were treated with the FPR2 antagonist WRW4 (15 µg/mL) or were left untreated before loading the cells to the upper compartment of the Boyden microchamber. Chemotaxis was induced by adding 1 ng/mL of CXCL8, 300 ng/mL of SAA1(47-104), 10 ng/mL of the FPR2 agonist WKYMVm or a combination of CXCL8 and SAA1(47-104) to the lower wells. Data represent the mean CI ± SEM and are pooled from 5 independent experiments. (D) The chemotactic potencies of CXCL8 (0.2-3 ng/mL), SAA1(52-104) (300 and 1000 ng/mL), and a combination of CXCL8 and SAA1(52-104) were evaluated on neutrophils in the Boyden microchamber assay. Data represent the mean CI from 6 to 7 independent experiments. (A-D) Statistically significant synergy is indicated by daggers (compared with the sum of the net CI values when both chemotactic agents are tested separately; †P ≤ .05; ††P ≤ .01; Mann-Whitney U test) and statistically significant inhibition of synergy or chemotaxis by desensitizing agents or by antagonists is indicated by dollar signs (compared with synergy on buffer-treated cells; $P ≤ .05; $$P ≤ .01; Mann-Whitney U test).
