Clara cell 10 kilodalton protein (CC10), the predominant product from nonciliated cells in the epithelial lining of bronchioles (Clara cells), has been shown to have immunomodulatory and anti-inflammatory activity, and may play roles in controlling inflammation in the airway. This study was designed to examine immunohistochemical expression of CC10 in epithelial cells in small airways (perimeter < 6 mm) of asthmatic and control nonsmokers who underwent lung resection because of peripheral lung carcinoma and to compare CC10-positive epithelial cell proportions with numbers of inflammatory cells in small airways of asthmatics. Significantly decreased proportions of CC10-positive epithelial cells and significantly increased numbers of T cells, activated eosinophils, and mast cells in small airways of asthmatics were found compared with those of control subjects. CC10-positive epithelial cell proportions inversely correlated with numbers of T cells and mast cells in small airways of asthmatics. Decreases of CC10-producing cells may give an accelerating cause for further aggravation of inflammatory responses in chronic asthma.
Clara cell 10 kilodalton protein (CC10) is the predominant product from Clara cells, the nonciliated cell population in the epithelial lining of bronchioles in the lung (1). Human CC10 has been proven identical with human urinary protein 1 (UP-1) (2) and human uteroglobin (UG) (3). CC10/UG possesses varied biochemical and biological properties including phospholipase A2 (PLA2)- (4) and phospholipase C (PLC)- inhibitory (5), and immunomodulatory (6)/anti-inflammatory activity (7, 8). Because of its potent PLA2-inhibitory and anti-inflammatory activity, and its high level of constitutive expression in the pulmonary system, it has been suggested that this protein may play critical roles in controlling inflammation in the airway.
Changes in CC10 concentrations in sera and bronchoalveolar lavage (BAL) fluids have been reported in various lung diseases (9-11). There has been only one report on CC10 levels in asthmatics, showing decreased CC10 concentrations in BAL fluids. Moreover, CC10 levels have been shown to be influenced by cigarette smoking (9, 12). This study was designed to examine immunohistochemical expression of CC10 in epithelial cells in small airways from asthmatic and control nonsmokers and to compare CC10-positive epithelial cell proportions with numbers of inflammatory cells in small airways of asthmatics. CC10-positive epithelial cells are significantly decreased in small airways of asthmatics compared with control subjects. The possible relationship between reduction of CC10-positive epithelial cells in small airways and pathophysiology of asthma is discussed.
Lung specimens were obtained from 15 subjects undergoing resection for a small peripheral carcinoma (< 3 cm in diameter). They were all lifelong nonsmokers. None had a central tumor or obstructive pneumonia. Pulmonary function tests of eight patients showed normal spirometric results and normal lung volume (control group) (Table 1). Seven patients had bronchial asthma (asthma group); the diagnosis of asthma was supported by recurrent episodes of airflow limitation. The airflow limitation was improved in FEV1.0 of greater than 15% after inhaled salbutamol (200 μg) in the asthmatics. Inflammation in large airways was histologically confirmed together with airway wall remodeling. Four asthma subjects were atopic and the remaining were nonatopic. Therapeutic periods in the asthmatics were more than 5 yr. All were treated with an inhaled β2-agonist only when needed, and with a stable daily dose of inhaled steroid for greater than 6 mo.
|Asthma (n = 7 )||Control (n = 8 )|
|Age, yr||57 ± 7||58 ± 8|
|TLC, % pred||108 ± 4||103 ± 7|
|FEV, % pred||94 ± 5||102 ± 5|
|RV, % pred||115 ± 5||98 ± 4|
|FEV1.0, % pred||82 ± 8||99 ± 5|
|Dl CO, % pred||81 ± 9||96 ± 9|
Lung specimens without lung cancer were fixed with 10% formalin, and embedded in paraffin; at least 5 blocks were made in each case. The sections of 5 μm thickness were deparaffinized with xylene and stained with hematoxylin–eosin and with periodic acid–Schiff/alcian blue; sections were also immunostained for CC10 and different inflammatory cell markers by using the avidin/biotin peroxidase complex method. The monoclonal antibodies used were anti-CD3 (pan T lymphocyte; Novacastra Laboratories, Newcastle, UK), EG2 (activated eosinophils, secreting eosinophil cationic protein [ECP]; Kabi Pharmacia, Uppsala, Sweden), antitryptase (mast cells; Dako, Glostrup, Denmark), and TY-5 (CC10/UP-1) (13). In order to retrieve antigen, sections were incubated with 0.1% trypsin and 0.1% CaCl2 2H2O in 50 mM Tris buffer at pH 7.4 at 37° C for 120 min in case of immunostaining of tryptase, and sections were pretreated with autoclave in 0.01 M sodium citrate buffer (pH 6.0) at 120° C for 20 min in case of immunostaining of CD3. The sections were soaked in absolute methanol containing 0.3% hydrogen peroxide, for 30 min at room temperature, to eliminate endogenous peroxidase activity. The sections were incubated with 1.5% nonimmunized goat serum for 30 min at room temperature, then incubated with each monoclonal antibody for 60 min at room temperature, and washed three times with phosphate-buffered saline (PBS) for 30 min. Thereafter, the sections were incubated with biotinylated goat anti-mouse Ig serum for 60 min. The biotinylated goat anti-mouse Ig serum was preapplied human-Ig- coupled Sepharose-4B to remove nonspecific binding to human tissue. After being washed with PBS, the sections were reacted with avidin/ biotin peroxidase complex (Vector, Burlingame, CA). The enzyme reaction was developed as described previously (12). Nuclei were lightly counterstained with hematoxylin. Normal mouse Ig was used as a negative control. No significant reaction occurred in this case.
We measured inner perimeter (Pi) defined by the luminal border of the airways and the area of small airways according to the method described by Carroll and coworkers (14) with modification. Briefly, using a light microscope, we took microphotographs of small airways that were cut in transverse section, and analyzed measurements of airways on a minicomputer using images of microphotographs. Airways with Pi < 6 mm were assessed as small airways. We evaluated immunoreactive cells (T cells, activated eosinophils, and mast cells) and CC10-positive epithelial cells in the small airways using serial sections. CC10-positive cells in the small airways were expressed as percentages of CC10-positive epithelial cells to total epithelial cells. In case goblet cell metaplasia greater than 5% in total epithelial cells assessed by sections staining with periodic acid–Schiff/alcian blue was found in the small airways, they were excluded in this analysis. Immunoreactive cells were expressed as numbers per square millimeter of the small airway wall.
Data were expressed as means ± SD. The Mann-Whitney U test was used to compare paired sets of data and the Pearson's least squares linear regression analysis was used to determine correlations. The level of critical significance was assigned at a p value less than 0.05.
Representative microphotographs of immunohistochemistry of CC10, CD3, secreting ECP and tryptase were shown in Figure 1. The percentages of CC10-positive epithelial cells were evaluated in 4 to 8 small airways of each case. The mean percentage of CC10-positive epithelial cells in small airways from asthmatics was significantly lower than that in control subjects (p = 0.0012) (Table 2). Small airways from asthmatics demonstrated an increase in the number of T cells (CD3) (p = 0.0026), activated eosinophils (secreting ECP) (p = 0.0151), and mast cells (tryptase) (p = 0.0092) compared with those from the control subjects.
|Case Number||CC10-positive Cells||T Cells||Activated Eosinophils||Mast Cells|
|Asthma 1||13.4 ± 5.2||304 ± 169||13.8 ± 8.6||167 ± 101|
|Asthma 2||19.2 ± 3.6||119 ± 46.4||15.5 ± 8.0||136 ± 57.7|
|Asthma 3||12.5 ± 5.7||174 ± 104||10.5 ± 3.8||210 ± 92.5|
|Asthma 4||11.4 ± 3.2||210 ± 124||14.3 ± 7.8||230 ± 121|
|Asthma 5||13.5 ± 3.9||202 ± 84.9||21.3 ± 8.4||161 ± 82.0|
|Asthma 6||12.0 ± 5.2||205 ± 101||11.4 ± 3.6||189 ± 92.3|
|Asthma 7||13.1 ± 3.7||208 ± 114||10.0 ± 5.1||137 ± 62.0|
|Control 1||26.1 ± 4.9||112 ± 54.9||3.3 ± 1.1||137 ± 52.9|
|Control 2||30.6 ± 6.8||66.9 ± 25.3||1.6 ± 1.0||68.3 ± 42.1|
|Control 3||26.2 ± 7.3||78.9 ± 31.9||10.3 ± 4.1||78.9 ± 49.3|
|Control 4||26.6 ± 3.8||100 ± 54.6||11.7 ± 5.2||140 ± 64.7|
|Control 5||27.9 ± 6.1||126 ± 84.1||5.1 ± 2.9||99 ± 42.9|
|Control 6||30.4 ± 8.9||120 ± 75.4||1.2 ± 1.0||147 ± 86.2|
|Control 7||25.8 ± 5.9||70.6 ± 35.4||10.0 ± 4.1||136 ± 68.9|
|Control 8||25.9 ± 7.1||72.1 ± 34.9||0.8 ± 0.5||72.1 ± 42.5|
We next analyzed the relationship between CC10-positive epithelial cell proportions and inflammatory cells in small airways (n = 36) from asthmatics (Figure 2). There were significant negative correlations of CC10-positive epithelial cell proportions with numbers of T cells (r = −0.598, p < 0.0001) and mast cells (r = −0.612, p < 0.0001) in the small airways from asthmatics, whereas there was no significant correlation between CC10-positive epithelial cell proportions and activated eosinophil numbers (r = 0.183).
This study documents increased numbers of T cells, activated eosinophils, and mast cells in small airways of asthmatics. The present result is in accordance with the results of recent studies (14, 15). This study could exclude influence of cigarette smoking for inflammatory cell infiltration in small airways of asthmatics because this study selected all lifelong nonsmokers. However, asthmatic subjects in the present study were relatively old and inhaled steroids were administered in all of them. It must be stated that there would be the limitations of tissue specimens obtained from younger asthmatics and from asthmatics who did not receive inhaled steroid therapy.
CC10 concentrations have been reported in various lung diseases; decreases in BAL fluid CC10 concentrations have been found in asthma (11), idiopathic pulmonary fibrosis (10), and chronic obstructive pulmonary disease (9). We have reported that CC10-producing bronchiolar epithelial cells are decreased in smokers (12). This study documents significantly reduced CC10-positive epithelial cells in small airways of asthmatics. Oshika and coworkers (13) have reported that glucocorticoids upregulate CC10 messenger RNA (mRNA) in early embryonic rat lungs. The evidence suggests that inhaled steroids might not reduce CC10-positive epithelial cells in small airways. To our knowledge, this is the first report to clarify decreased CC10-positive epithelial cells in small airways of asthmatics.
This study also demonstrates significant negative correlations of CC10-positive epithelial cell proportions with numbers of T cells and mast cells in the small airways of asthmatics. Persistent chronic inflammation in small airways in asthmatics occurs in conjunction with decreases in CC10-positive epithelial cells, although we cannot find any cause or effect relationship between two types of cells. Bowton and associates (16) have reported that PLA2 and arachidonate increase in BAL fluids after inhaled antigen challenge in asthmatics, although there was no difference between normal and stable asthmatics in either BAL fluid PLA2 activity or arachidonate concentration at baseline. CC10/UG shows several biochemical and biological functions including PLA2- and PLC-inhibitory, and immunomodulatory/anti-inflammatory activity (4-8). Recent studies (17, 18) have also demonstrated that CC10 has inhibitory functions of proinflammatory cytokines. As a result of reduced CC10-producing cells in the small airways of asthmatics, one would expect the release of high levels of arachidonate and proinflammatory cytokines, causing migration of immunocytes into the pulmonary wet mucosa. In addition, release of a high level of arachidonate may eventually lead to increased production of proinflammatory lipid mediators such as platelet-activating factor, leukotrienes, prostaglandins, and thromboxans. Thus, a vicious cycle could ensue by initiating and propagating an inflammatory response in the respiratory tract or aggravating existing inflammatory processes.
We conclude that decreases of CC10-producing epithelial cells occur and their proportions inversely correlate with numbers of T cells and mast cells in small airways of asthmatics. The reduced CC10 production in the small airways of asthma may give an accelerating cause for further aggravation of inflammatory response in chronic asthma.
Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (N.S., No. 10670556) and (Y.I., No. 11672302).
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