Fourteen-member macrolides suppress interleukin-8 production but do not promote apoptosis of activated neutrophils

Abstract

A 14-member macrolide was found to inhibit interleukin-8 (IL-8) synthesis in lipopolysaccharide-stimulated neutrophils but did not accelerate apoptosis in activated neutrophils. These data suggest that 14-member macrolides achieve clinical efficacy for chronic airway diseases partly by suppressing IL-8 production by activated neutrophils, but not by enhancing apoptosis in these cells.

FIG. 1.

Effects of 14-member macrolides and DEX on neutrophil apoptosis, as estimated by morphology (A) and annexin V binding (B), in the presence (solid bars) or absence (open bars) of LPS. Separated neutrophils were incubated with a 14-member macrolide (10 μg/ml) or DEX (10⁻⁷ M; 43 ng/ml), and LPS (1 μg/ml) was then added to the cell suspension. After a 24-h incubation, neutrophils were recovered and analyzed for apoptosis. The control (CTRL) contained 0.1% DMSO in IMDM. Each value is the mean ± standard deviation from three determinations. ∗∗, P < 0.01 (compared with the control in the absence of LPS).

FIG. 2.

Effects of 14-member macrolides, DEX, or MG-132 (10⁻⁶ M) on IL-8 production by human neutrophils in the presence of LPS after 24 (A) or 48 (B) h of incubation. Separated neutrophils were incubated with each reagent at the indicated concentrations, and LPS (1 μg/ml) was then added to the cell suspension. After a 24- or 48-h incubation, IL-8 levels in culture supernatants were determined. The control (CTRL) contained 0.1% DMSO in IMDM. Each value is the mean ± standard deviation from three determinations. ∗∗, P < 0.01; ∗, P < 0.05 (compared with the control in the presence of LPS).

FIG. 3.

Effects of EMA (10 μg/ml), CAM (10 μg/ml), and DEX (10⁻⁷ M; 43 ng/ml) on IL-8 gene expression in LPS-stimulated human neutrophils. IL-8 gene expression of uncultured neutrophils without LPS stimulation was also examined as a negative control. The control medium (CTRL) contained 0.1% DMSO in IMDM. Results shown are IL-8 mRNA transcript levels (upper panel) and control G3PDH mRNA transcript levels (lower panel) in human neutrophils. Bars in graph represent densitometric units obtained from an autoradiogram by Northern blot analysis and expressed as the ratios of IL-8 mRNA transcript levels to G3PDH mRNA transcript levels.