Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages

Abstract

An inflammation of the airway of patients with diffuse panbronchiolitis (DPB), is characterized by dense neutrophil infiltration. Resolution of the inflammation can be achieved by the removal of apoptotic neutrophils by human alveolar macrophages (AM) without liberating neutrophil proteases in the airway. To understand clinical efficacy for the treatment of DPB by 14- or 15-member macrolides, their effects on the phagocytosis of apoptotic neutrophils by AM were examined. Treatment of AM with erythromycin (ERY) or clarithromycin at clinically achievable levels significantly increased the levels of phagocytosis of apoptotic neutrophils. A serum factor was not essential for the enhancement by these 14-member macrolides. Of the antibiotics tested, these effects were specific for the 14-member macrolides and a 15-member macrolide, azithromycin, but not for the 16-member macrolides, clindamycin or beta-lactam antibiotics. The enhanced phagocytosis of apoptotic neutrophils by ERY had no effect on the levels of interleukin-8 or tumor necrosis factor alpha production by lipopolysaccharide-stimulated AM after phagocytosis of the apoptotic neutrophils. The increased phagocytosis of apoptotic neutrophils by ERY was also found to be phosphatidylserine receptor-dependent for AM. These data indicate a novel anti-inflammatory action of 14-member and 15-member macrolides, and suggest that such antibiotics achieve clinical efficacy for patients with DPB, in part, through enhancing the nonphlogistic phagocytosis of apoptotic neutrophils by AM.

FIG. 1.

(A) Photomicrograph of human AM that contain phagocytosed apoptotic neutrophils. The arrowhead indicates the intracellular apoptotic neutrophils which are stained for MPO within the MPO-negative AM. (B) Complement-dependent phagocytosis of apoptotic neutrophils by AM cultured for 24 h. Autologous apoptotic neutrophils were exposed to AM in the presence of fresh human serum (closed circles) or heat-inactivated serum (open circles). Each data point represents the mean ± standard deviation (error bars) of three determinations. Statistical significance: *, P < 0.05(versus no serum); **, P < 0.01(versus no serum and heat-inactivated serum with the same concentration).

FIG. 2.

Effects of 14-member macrolides on the phagocytosis of apoptotic neutrophils by human alveolar macrophages (AM) in the presence of 10% fresh homologous serum (A) or medium alone (B). AM were treated with ERY or CLR at concentrations of 0.1 to 10 μg/ml, with JOS, AMP, or CEC at a concentration of 10 μg/ml for 72 h. Treatment of AM with DEX (10⁻⁷ M) was done for 24 h. The control (CTRL) represents the treatment of AM with medium containing 0.1% DMSO. Each value represents the mean ± standard deviation (error bars) of three determinations. *, P < 0.01 (versus CTRL).

FIG. 3.

Effects of 14-member and 15-member macrolides on the phagocytosis of apoptotic neutrophils by human AM in the presence of medium alone. AM was treated with ERY, RXM, OLM, AZM, JOS, SPM, or CLDM at concentrations of 10 μg/ml for 72 h. Treatment of AM with DEX (10⁻⁷ M) was done for 24 h. The control (CTRL) represents the treatment of AM with medium containing 0.1% DMSO. Each value represents the mean ± SD of three determinations. *, P < 0.01 (versus CTRL).

FIG. 4.

Effects of ERY or DEX on IL-8 production (A) or TNF-α (B) by unstimulated or LPS-stimulated human AM after the phagocytosis of apoptotic neutrophils. AM were treated with ERY at a concentration of 10 μg/ml, DEX at a concentration of 10⁻⁷ M or medium containing 0.1% DMSO (CTRL) for 72 h. AM were, then, incubated with LPS (10 ng/ml) or IMDM alone for 3 h, followed by exposure to apoptotic neutrophils or IMDM alone for 30 min. After removing unphagocytosed neutrophils, AM were cultured for 18 h. After incubation, the IL-8 or TNF-α levels in the cell culture supernatants were determined. Each bar denotes no phagocytosis without LPS (open bars), phagocytosis without LPS (slash bars), no phagocytosis with LPS (solid bars), and phagocytosis with LPS (gray bars), respectively. Each value represents the mean ± standard deviation (error bars) of three determinations. Statistical significance: *, P < 0.001 (versus LPS-stimulated AM without phagocytosis in CTRL treatment); **, P < 0.001 (versus LPS-stimulated AM with phagocytosis in CTRL treatment).

FIG. 5.

PS receptors are predominantly responsible for the ERY-induced phagocytosis of apoptotic neutrophils by human AM. AM were treated with ERY at a concentration of 10 μg/ml, DEX at a concentration of 10⁻⁷M or medium containing 0.1% DMSO (CTRL) for 72 h. AM were preincubated with each inhibitor at 37°C for 30 min, and then exposed to apoptotic neutrophils after washing the macrophages with medium. The inhibitors were as follows: 1, phosphatidylcholine (PC) liposomes at 0.1 mM; 2, PS liposomes at 0.1 mM; 3, MAb 217 at 100 μg/ml; 4, control IgM MAb at 100 μg/ml; 5, RGDS at 2 mM; 6, RGES at 2 mM. Each value represents the means ± standard deviations (error bars) of three determinations. Statistical significance: *, P < 0.01 (versus PC liposomes in each treatment); **, P < 0.01 (versus PC liposomes in CTRL treatment).