Figure 3.

Effect of DF 2156A on IL-8 binding to cellular receptors, G-protein activation and chemokine-mediated cell migration. (A) For IL-8 receptor binding studies, human PMNs were pre-incubated for 15 min at 37°C with vehicle or 1 µM DF 2156A. After incubation, aliquots of 0.2 nM of [¹²⁵I]-IL-8 and serial dilution of unlabelled IL-8 were added to 10⁶ cells in 100 µL of binding medium and incubated at room temperature for 1 h under gentle agitation. Data are from one experiment representative of three performed. (B) Human PMNs were directly exposed to vehicle or different concentrations of DF 2156A and aliquots of 0.2 nM of [¹²⁵I]-IL-8 and vehicle (DF 2156A-treated PMNs) or serial dilution of unlabelled IL-8 (vehicle-treated PMNs) were added to 10⁶ cells in 100 µL of binding medium and incubated at room temperature for 1 h under gentle agitation. Data are from one experiment representative of three performed. (C) IL-8-induced G-protein activation. Purified human PMN plasma membranes were pre-incubated for 10 min at 30°C with vehicle or 1 µM DF 2156A and next stimulated with 100 nM IL-8 or medium. The time-resolved fluorescence was then read using the Victor Plate Reader. Data are expressed as resolved fluorescence units (RFU; mean ± SD of four independent experiments). *P < 0.05 of DF 2156 versus vehicle-pretreated group by Student's t-test. (D) Effect on cell migration. L1.2 transfectants were pre-incubated for 15 min at 37°C with vehicle (control group) or increasing concentrations of DF 2156A and then stimulated with the appropriate chemokines: 10 nM IL-8 for CXCR2 and CXCR1, 10 nM CCL19 for CCR7, 3 nM CCL22 for CCR4, 3 nM CCL5 for CCR5 and 10 nM CXCL12 for CXCR4. Data are expressed as % of control group migration (mean ± SD of three independent experiments). *P < 0.05 and **P < 0.01 versus cell migration in the absence of DF 2156A by Mann–Whitney U-test.