Increased expression of the calcium-activated chloride channel hCLCA 1 in airways of patients with obstructive chronic bronchitis

1Meakins-Christie Laboratories, McGill University, Montreal, Quebec; 2Institut Pasteur, Lille, France; 3Genaera Corporation, Plymouth Meeting, Pennsylvania, USA Correspondence: Dr Qutayba Hamid, Meakins-Christie Laboratories, McGill University, 3626 St-Urbain Street, Montreal, Quebec H2X 2P2. Telephone 514-398-3864, fax 514-398-7483, e-mail qutayba.hamid@mcgill.ca H-P Hauber, C Bergeron, A Tsicopoulos, et al. Increased expression of the calcium-activated chloride channel hCLCA1 in airways of patients with obstructive chronic bronchitis. Can Respir J 2005;12(3):143-146.

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Subjects
Six patients with COPD fulfilling the diagnosis criteria for chronic bronchitis with airway obstruction according to the American Thoracic Society guideline (8) (all men; mean age 62.5 years, ranging from 49 to 69 years) and six healthy, nonsmoking control subjects (five men, one woman; mean age 35.8 years, ranging from 19 to 51 years) were recruited.Patients with COPD were diagnosed with chronic bronchitis if they had a history of cough and sputum production on most days for a minimum of three months/year for the past two years.Fixed airway limitation was defined as a forced expiratory volume in 1 s less than 80% of predicted, with less than 15% improvement in forced expiratory volume in 1 s after inhalation of 400 µg of salbutamol.All patients with COPD had a past history of cigarette smoking.All subjects were nonatopic (determined by skin prick testing) and had no clinical histories of allergy.Exclusion criteria included the presence of other lung diseases, systemic disease affecting the lungs, oral or inhaled corticosteroid use, exacerbations in the 12 weeks preceding the study and respiratory tract infections in the six weeks before the study.Approval was obtained from McGill University's (Montreal, Quebec) and from Institut Pasteur's (Lille, France) ethics committes, and informed consent was obtained from all subjects before recruitment into the study.

Tissue collection and preparation
Endoscopic bronchial biopsies were taken from the subcarinae of the lower and middle lobes.Three to four biopsies were obtained from all subjects.Biopsy specimens were either blocked in optimal cutting temperature medium by snap freezing in isopentanecooled liquid nitrogen for immunocytochemistry or blocked in 4% paraformaldehyde for in situ hybridization.

In situ hybridization
In situ hybridization with a radiolabelled complementary RNA probe was used to detect hCLCA1 messenger RNA (mRNA) in bronchial epithelium, as previously described (10,11).In brief, complementary DNA was inserted into an expression vector, linearized and transcribed in vitro in both directions (antisense and sense probes) in the presence of S 35 -UTP, T7 and SP6 polymerase.After permeabilization, sections were prehybridized with 50% formamide in 2× standard sodium citrate for 15 min at 37°C.Hybridization was carried out overnight at 40°C with a hybridization mixture containing hCLCA1, S 35 -labelled sense or antisense probe (0.75×10 6 cpm/slide).Posthybridization involved a series of washes with decreasing concentrations of 2× standard sodium citrate at 42°C.Samples were washed with ribonuclease solution for 20 min at 42°C to remove any unbound RNA probes.Hybridization signals were detected with standard autoradiography.

Quantification
All slides were read blinded (coded) and positive cells were counted.Epithelial cells with positive PAS staining, positive IL-9R cells and positive hCLCA1 cells were expressed as the percentage of positive cells per total number of epithelial cells.IL-9 immunoreactive cells were expressed as the number of positive cells per square millimetre of submucosa.Each slide was counted by two experienced observers.The intraobserver coefficient of variation for repeated measures was less than 5%.

Statistics
The differences in the number of cells expressing IL-9, IL-9R, hCLCA1 proteins, hCLCA1 mRNA or staining positive for mucin (PAS) were compared using the nonparametric Kruskal-Wallis test.Statistically significant differences between groups were subsequently analyzed with the Mann-Whitney U test (Systat version 7.0; SPSS Inc, USA).The results are expressed as mean ± SEM, if not otherwise stated.P<0.05 was considered statistically significant.

Increased expression of hCLCA1 mRNA and protein in patients with COPD
The percentage of cells expressing hCLCA1 mRNA and protein was significantly higher in the bronchial epithelium of patients with COPD (64.6±9.4% and 54.2±12.4%,respectively) than in samples from control subjects (37.5±8.5% and 22.9±9.4%,respectively; P<0.05) (Figures 1 and 2).

Increased expression of IL-9, IL-9R and PAS staining in patients with COPD
The number of IL-9 immunoreactive cells was significantly higher in the submucosa of patients with COPD than in the submucosa of control subjects (14.1±4.9 cells/mm 2 versus 7.0±2.6cells/mm 2 , respectively; P<0.05).Similarly, the percentage of cells expressing IL-9R in the bronchial epithelium of patients with COPD was significantly higher than in the bronchial epithelium of control subjects (41.7±11.0%versus 17.5±3.1%,respectively; P<0.05).Also, a significantly higher number of PAS-positive cells were observed in the bronchial epithelium of patients with COPD than in the bronchial epithelium of control subjects (52.1±5.9%versus 20.8±10.0%,respectively; P<0.05) (Figure 2).

DISCUSSION
In the present study, we found an increased expression of IL-9 in the airways of patients with obstructive chronic bronchitis, which is in agreement with previous data (7).Furthermore, the expression of IL-9R, hCLCA1 and mucin protein (PAS staining) were elevated in the bronchial epithelium of patients with obstructive chronic bronchitis (COPD).These data correlate with previous findings which found similar increases in the airways of patients with asthma or cystic fibrosis (5,6).
IL-9 has been reported to be produced by T cells in the submucosa of patients with chronic bronchitis (7).Furthermore, IL-9 is able to induce hCLCA1 and mucin protein expression in human primary lung cells (2,3).There was a strong correlation between the protein expression of hCLCA1 and the IL-9R (r=0.74),suggesting induction of hCLCA1 via the IL-9R.However, the correlation between hCLCA1 protein expression and PAS staining was weaker (r=0.54).This is not surprising because other mediators such as neutrophil elastase and lipopolysaccharide from Gram-negative bacteria have also been implicated in mucin production (12,13).
The subjects in the control group in the current study were significantly younger than the patients with chronic bronchitis.Although we cannot exclude an age confounder, we do not believe that aging, by itself, can trigger an inflammatory and pathological profile as that observed in smoking-induced chronic bronchitis.In addition, it was also difficult to have an age-matched control group.

CONCLUSION
Our results suggest that mucus hypersecretion in the chronic bronchitis type of COPD may be due to increased expression of hCLCA1, triggered by IL-9.

FUNDING:
This study was supported by Canadian Institutes of Health Research and Genaera Corporation (Plymouth Meeting, USA).

Figure 1 )Figure 2 )
Figure 1) Representative example of immunohistochemistry for human calcium-activated chloride channel 1 in (A) the bronchial mucosa of a control subject and (B) a patient with chronic obstructive pulmonary disease with chronic bronchitis.C and D are representative examples of negative isotype control in a control subject and a patient with chronic obstructive pulmonary disease, respectively.Positive epithelial cells stain red.Original magnification ×400