High-dose dexamethasone induced LPS-stimulated rat alveolar macrophages apoptosis

Abstract

Prolonged administration of an excessive dose of corticosteroids proved to be harmful for patients with acute lung injury (ALI). A previous study has found that repeated administration of an excessive dose of methylprednisolone reduced alveolar macrophages (AMs) in bronchoalveolar lavage fluid (BALF) with an unknown mechanism. This study aimed to investigate the effect of excessive use of dexamethasone (Dex) on BALF AMs in vitro. Transmission electron microscopy and DNA fragmentation analysis demonstrated that 10^-4 and 10^-5 M Dex induced lipopolysaccharide-stimulated rat AMs apoptosis with downregulation of tumor necrosis factor-α, interleukin (IL)-12 and upregulation of IL-10, transforming growth factor-β. These results indicated that apoptosis might be a novel contribution involved in the detrimental effect of excessive dose of Dex clinically used to treat ALI.

Keywords: acute lung injury; alveolar macrophage; apoptosis; dexamethasone; inflammatory cytokines; lipopolysaccharide.

Figure 1

Morphological changes of rat AM. Notes: Cells (1×10⁶/well in 6-well culture plates) were stimulated with the same volume of PBS (control group, A), LPS group (1 μg/mL, B), LPS and Dex (10⁻⁴–10⁻⁷ M) (C–F), respectively, for 48 hours. Original magnification ×200. Abbreviations: AM, alveolar macrophage; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone.

Figure 2

Effect of Dex on cell viability. Notes: Cells (1×10⁵/well in 96-well culture plates) were stimulated with the same volume of PBS (control group), LPS (1 μg/mL), LPS and Dex (10⁻⁴–10⁻⁷ M), respectively, for 6, 24, and 48 hours. Cell viability was assessed by the MTT assay, as described in the “Materials and methods” section. Data were expressed as mean ± SD (n=5). *P<0.05 versus LPS. ^#P<0.05 versus PBS. ^ΔP<0.05 versus LPS + Dex 10⁻⁴ M. Abbreviations: h, hours; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone; SD, standard deviation; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.

Figure 3

Transmission electron microscopy. Notes: (A) Cells in the control group showed integrated nuclear membrane, relatively homogeneous chromatin, and extensive membrane interdigitations and microvilli (arrows). Magnification ×5,000. (B) Devour vesicles (arrows) in the LPS group. Magnification ×5,000. (C) Apoptosis bodies (arrows) in the LPS + Dex 10⁻⁴ M group. Magnification ×5,000. (D) In the LPS + Dex 10⁻⁵ M group, stimulation of Dex induced nuclear chromatin concentration and edge accumulation (arrows). Magnification ×10,000. Abbreviations: LPS, lipopolysaccharide; Dex, dexamethasone.

Figure 4

The apoptotic DNA ladder induced by different doses of Dex. Notes: Cells (2×10⁶/well in 6-well culture plates) were stimulated with the same volume of PBS (control group, A), LPS (1 μg/mL, B), LPS and Dex (10⁻⁴–10⁻⁷ M) (C–F), respectively, for 48 hours. Then, DNA was extracted and electrophoresed on agarose gel. Abbreviations: PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone.

Figure 5

Effect of Dex on TNF-α synthesis. Notes: Cells (2×10⁶/well in 6-well culture plates) were stimulated with the same volume of PBS (control group), LPS (1 μg/mL), LPS and Dex (10⁻⁴–10⁻⁷ M), respectively, for 6, 24, and 48 hours. Expression of TNF-α in the culture medium was determined using ELISA assay. Data were expressed as mean ± SD (n=3). *P<0.05 versus LPS. ^#P<0.05 versus PBS. Abbreviations: h, hours; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone; TNF, tumor necrosis factor; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay.

Figure 6

Effect of Dex on IL-12 synthesis. Notes: Rat AMs (2×10⁶/well in 6-well culture plates) were stimulated with the same volume of PBS (control group), LPS (1 μg/mL), LPS and Dex (10⁻⁴–10⁻⁷ M), respectively, for 6, 24, and 48 hours. Production of IL-12 in the culture medium was determined using an enzyme-linked immunosorbent assay. Data were expressed as mean ± SD (n=3). *P<0.05 versus LPS. ^#P<0.05 versus PBS. Abbreviations: h, hours; AM, alveolar macrophage; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone; IL, interleukin; SD, standard deviation.

Figure 7

Effect of Dex on IL-10 synthesis. Notes: Rat AMs (2×10⁶/well in 6-well culture plates) were stimulated with the same volume of PBS (control group), LPS (1 μg/mL), LPS and Dex (10⁻⁴–10⁻⁷ M), respectively, for 6, 24, and 48 hours. Production of IL-10 in the culture medium was determined using an enzyme-linked immunosorbent assay. Data were expressed as mean ± SD (n=3). *P<0.05 versus LPS. ^#P<0.05 versus PBS. Abbreviations: h, hours; AM, alveolar macrophage; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone; IL, interleukin; SD, standard deviation.

Figure 8

Effect of Dex on TGF-β synthesis. Notes: Rat AMs (2×10⁶/well in 6-well culture plates) were stimulated with the same volume of PBS (control group), LPS (1 μg/mL), LPS and Dex (10⁻⁴–10⁻⁷ M), respectively, for 6, 24, and 48 hours. Production of TGF-β in the culture medium was determined using an enzyme-linked immunosorbent assay. Data were expressed as mean ± SD (n=3). *P<0.05 versus LPS. ^#P<0.05 versus PBS. Abbreviations: h, hours; AM, alveolar macrophage; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; Dex, dexamethasone; SD, standard deviation; TGF, transforming growth factor.