Glucocorticoid sensitivity of lipopolysaccharide-stimulated chronic obstructive pulmonary disease alveolar macrophages

Abstract

It has been reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) display glucocorticoid (Gc) resistance. The Gc sensitivity of inflammatory mediators released by COPD macrophages may vary. The objective of this study was to identify Gc-insensitive inflammatory mediators produced by lipopolysaccharide (LPS)-stimulated alveolar macrophages from COPD patients. LPS-stimulated alveolar macrophages from 15 COPD patients, nine smokers (S) and nine healthy non-smokers (HNS) were stimulated with LPS with or without dexamethasone (100 and 1000 nM). Luminex and enzyme-linked immunosorbent assay were used to measure 23 inflammatory mediators. After LPS stimulation there were lower levels of inflammatory mediators in COPD patients and S compared to HNS. There was no difference between groups for the effects of dexamethasone at either concentration (P > 0.05 for all comparisons). Tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and growth-related oncogene (GRO)-alpha displayed the greatest sensitivity to dexamethasone in COPD patients, while IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) were the least sensitive. COPD macrophages have a reduced response to LPS. Gc sensitivity was similar in COPD macrophages compared to controls. We identify some Gc-insensitive cytokines, including GM-CSF, G-CSF and IL-8, that may be involved in the progression of airway inflammation in COPD patients.

Fig. 2

Dose–response of lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-α production from chronic obstructive pulmonary disease (COPD) patients’ alveolar macrophages. Concentrations of TNF-α measured in alveolar macrophage cell culture supernatants show the effect of 0·01–10 µg/ml LPS stimulation. Alveolar macrophages from healthy non-smoker (HNS), smoker (S) and COPD groups are shown. Mean ± SEM shown. ***P < 0·001, **P < 0·01 and *P < 0·05 compared to unstimulated levels. TNF-α measured by enzyme-linked immunosorbent assay.

Fig. 1

Time-course of lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-α production from chronic obstructive pulmonary disease patients’ alveolar macrophages. Concentrations of TNF-α measured in alveolar macrophage (n = 4) cell culture supernatants show the effect of 1 and 10 µg/ml LPS stimulation. Non-parametric data shown as median and interquartile range. ***P < 0·001, **P < 0·01 and *P < 0·05 compared to unstimulated time-matched controls, of which most were lower than the level of detection of assay. TNF-α measured by enzyme-linked immunosorbent assay.

Fig. 3

Unstimulated and lipopolysaccharide (LPS, 1 µg/ml)-stimulated inflammatory mediator production from alveolar macrophages. Concentrations of 11 cytokines in (a) unstimulated and (b) 1 mg/ml LPS-stimulated alveolar macrophage cell culture supernatants. Alveolar macrophages from healthy non-smoker (HNS), smoker (S) and chronic obstructive pulmonary disease (COPD) groups are shown. Parametric data set shown as mean ± SEM. Granulocyte colony-stimulating factor (G-CSF) and interferon-inducible protein (IP)-10 were non-parametric data sets shown with median, interquartile range and range, as box-plots. Enzyme-linked immunosorbent assay was used to quantify growth-related oncogene (GRO)-α, interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1α. Luminex was used to measure IL-6, monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-10, regulated upon activation normal T cell expressed and secreted (RANTES), G-CSF and IP-10. ***P < 0·001, **P < 0·01 and *P < 0·05 between groups.

Fig. 4

Inhibition of inflammatory mediator production by dexamethasone in alveolar macrophages. (a) Per cent inhibition of inflammatory mediators in response to 1 µg/ml lipopolysaccharide (LPS) stimulation by 0·1 µM dexamethasone. (b) Per cent inhibition of inflammatory mediators in response to 1 µg/ml LPS stimulation by 1 µM dexamethasone. Parametric data set shown as mean ± 95% CI. Granulocyte colony-stimulating factor (G-CSF) and interferon-inducible protein (IP)-10 were non-parametric data sets shown with median, interquartile range and range as box-plots. All inhibition shown is statistically significant except for granulocyte–macrophage colony-stimulating factor (GM-CSF) and G-CSF. Enzyme-linked immunosorbent assay was used to quantify growth-related oncogene (GRO)-α, interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1. Luminex was used to measure IL-6, monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, GM-CSF, IL-10, regulated upon activation normal T cell expressed and secreted (RANTES), G-CSF and IP-10. HNS, healthy non-smokers; S, smokers; COPD, chronic obstructive pulmonary disease.

Fig. 5

Effects of inhaled corticosteroids (ICS) and smoking status in chronic obstructive pulmonary disease (COPD) patients’ alveolar macrophages. Concentrations of tumour necrosis factor (TNF)-α measured in alveolar macrophage cell culture supernatants show the effect of 0·01–10 µg/ml lipopolysaccharide (LPS) stimulation. TNF-α measured by enzyme-linked immunosorbent assay. Per cent inhibition of inflammatory mediators in response to 1 µg/ml LPS stimulation by 1 µM dexamethasone is also shown for interleukin (IL)-8 and TNF-α, measured by Luminex. Alveolar macrophages from (a) ICS +ve and −ve and (b) current and ex-smoking COPD patients are shown. Mean ± SEM is shown. NS, no significant difference at each concentration of LPS, or with dexamethasone.

Fig. 6

Inhibition of inflammatory mediator mRNA production by dexamethasone in alveolar macrophages. Relative mRNA levels of five cytokines were measured in alveolar macrophages from obstructive pulmonary disease (COPD) patients and smokers (S). Data were non-parametric and shown as median ± interquartile range, ***P < 0·001, **P < 0·01 and *P < 0·05 compared to lipopolysaccharide (LPS). GRO, growth-related oncogene; IL, interleukin; MIP, macrophage inflammatory protein; TNF, tumour necrosis factor.