Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Abstract

Background: The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Methods: Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.

Results: We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.

Conclusions: CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.

Figure 1

Chronic exposure to cigarette smoke (CS) decreases airway surface liquid (ASL) height. Primary human airway epithelial cells from 4 donors (n = 8) were exposed to 30 puffs of whole cigarette smoke (2 cigarettes) every day for 5 days (120 hrs). (A) ASL height was measured one hour after each exposure to CS. ASL height was undisturbed over the course of the reading. *p < 0.05. (B) CFTR present at the plasma membrane was detected by immunoblotting after biotinylation of cell surface proteins (see Methods).

Figure 2

Cigarette smoke extract (CSE) decreases the expression of CFTR but not Na⁺/K⁺-ATPase in human bronchial epithelial cells. 16HBE14o- cells were treated with 10% CSE for up to 48 hours (A) or increasing concentrations of CSE prepared from commercial grade cigarettes (Camel) for 48 hours (B). CFTR and Na⁺/K⁺-ATPase were detected by immunoblotting. The same amount of protein was loaded in each lane as indicated by detection of β-actin. The blots are representative of at least three independent experiments. (C) Detection of CFTR mRNA transcript levels using quantitative RT-PCR analysis after treatment of 16HBE14o- cells with 10% CSE for 24 hours. Results are expressed as fold change and are representative of three independent experiments. *p < 0.05.

Figure 3

CFTR is decreased in the lung of GOLD 4 COPD patients. (A) CFTR protein was detected in the lung of GOLD 0 (Control 1 and 2) and GOLD 4 (Patient 1 and 2) patients. Formalin fixed paraffin embedded lung tissue sections from GOLD 0 and GOLD 4 patients were immunostained using a specific CFTR antibody (red) (A) or non-immune control (B). (C) Intensity of CFTR signal was scored as described in the Methods section. (D) The CFTR mRNA level was measured by quantitative RT-PCR and expressed as Relative Copy Number (RCN). N = 7 for number of patients GOLD 0 and N = 8 for number of patients COPD GOLD 4. Statistically significant differences were assessed using Mann–Whitney U test.

Figure 4

Metal analysis of lung samples from GOLD 0 and GOLD 4 COPD patients. The amount of aluminum (A), cadmium (B), chromium (C), copper (D), manganese (E), and zinc (F) were measured in lung biopsies from GOLD 0 and GOLD 4 patients. Data are expressed in μg/mg dry weight tissue. N = 8 for number of patients GOLD 0 (the never smoker patient was excluded) and N = 11 for number of patients COPD GOLD 4.

Figure 5

Metals present in CSE regulate CFTR expression. 16HBE14o- cells were incubated with 10% CSE before and after incubation with Chelex-100 beads, in absence or presence of 10 μM cadmium chloride. CFTR protein was detected by immunoblotting 48 hours after treatment. Blots are representative of at least three independent experiments. *p < 0.05.

Figure 6

Manganese and cadmium decrease the expression of CFTR in bronchial epithelial cells. 16HBE14o- cells were incubated with cadmium chloride (CdCl₂) or manganese chloride (MnCl₂) at the doses indicated for 24 hours. CFTR protein was detected by immunobloting using a monoclonal antibody as described in Materials and Methods.