Figure 7.

Involvement of TLR2 and PTX-sensitive heterotrimeric G proteins in HP-NAP-induced secretion of IL-8 by ATRA-induced differentiated HL-60 cells and neutrophils. (A) TLR2-dependent secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 2 × 10⁶ cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam₃CSK₄, 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in Figure 6. Data are expressed as mean ± SD of four independent experiments. (B) TLR2-dependent secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10⁶ cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam₃CSK₄, 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. (C) PTX-sensitive secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP and Pam₃CSK₄. ATRA-induced differentiated HL-60 cells at a density of 2 × 10⁶ cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam₃CSK₄, or D-PBS as unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in A. Data are expressed as mean ± SD of three independent experiments. (D) PTX-sensitive secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10⁶ cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 4 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. *: p value < 0.05, **: p value < 0.01 as compared with unstimulated cells. #: p-value < 0.05, ##: p value < 0.01 as compared with stimulated cells in isotype control group or vehicle control group.