Michio Hirasawa

Serum and BAL Clara cell 10 kDa protein (CC10) levels and CC10-positive bronchiolar cells are decreased in smokers

Cigarette smoking has diverse effects on the structure and function of the lung. Smoking appears to reduce the levels of Clara cell 10 kDa protein (CC10) in the alveolar lining fluid, but the influence of smoking serum on CC10 levels is still debated, and it has not been clear whether smoking reduces the number of CC10-producing lung cells. The aims of this study were to clarify the influence of smoking on CC10 levels in the alveolar lining fluid and bloodstream, and on the number of CC10-producing lung cells. CC10 concentrations were measured in sera and bronchoalveolar lavage (BAL) fluids, by means of enzyme-linked immunosorbent assay using monoclonal and polyclonal antibody, and the immunohistochemical expression of CC10 was examined in the lungs of nonsmokers and smokers using the monoclonal antibody, TY-5, against CC10/human urinary protein-1. CC10 concentrations in sera and in BAL fluids from healthy smokers were significantly lower than in healthy nonsmokers. Immunohistochemical expression of CC10 was found exclusively in nonciliated bronchiolar epithelial cells. As compared to that of nonsmokers, the mean percentage of CC10-positive bronchiolar epithelial cells was significantly decreased in lung tissue specimens obtained from smokers who had normal results in pulmonary function tests. It was concluded that smoking reduces the proportion of Clara cell 10 kDa protein-producing bronchiolar epithelial cells, resulting in decreased levels of Clara cell 10 kDa protein in the lower respiratory tract and in the bloodstream. The protein is a new blood biochemical and immunohistochemical marker, reflecting structural changes in peripheral airways induced by cigarette smoking.

Serum Levels of Clara Cell 10-kDa Protein Are Decreased in Patients with Asthma

Abstract. Clara cell 10-kDa protein (CC10), the predominant product from nonciliated cells in the epithelial lining of bronchioles (Clara cells), has been shown to have immunomodulatory and antiinflammatory activity and may play a role in controlling airway inflammation. This study was designed to measure serum CC10 concentrations in healthy and asthmatic nonsmokers. Serum CC10 concentrations in asthmatic nonsmokers were significantly lower than in healthy nonsmokers. Asthmatic patients with a long duration of the disease (≥10 years) had significantly lower serum CC10 levels than those with a short duration of the disease (<10 years). There was no significant difference in serum CC10 levels in asthmatic patients between the time of the asthmatic attack and the stable condition. Serum CC10 levels may reflect decreased production of CC10 caused by remodeling of the small airways in asthma.

Circulating intercellular adhesion molecule-1 (ICAM-1) antigen in sera of patients with idiopathic pulmonary fibrosis

Intercellular adhesion molecule‐1 (ICAM‐1), a member of immunoglobulin supergene family with a five‐domain structure, is known to play an important role in inflammatory diseases. An ELISA was developed using two MoAbs against human ICAM‐1 in order to detect the soluble shedding ICAM‐1 antigen in sera. We measured levels of circulating ICAM‐1 antigen in sera of patients with idiopathic pulmonary fibrosis (IPF), pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial and mycoplasmal pneumonia, and inflammatory diseases of other organs. The results clearly demonstrated that IPF had significantly high levels of circulating ICAM‐1 in sera as compared with other disorders or normal controls. Moreover, immunohistochemical analysis with MoAb against human ICAM‐1 disclosed that in IPF, the expression of ICAM‐1 was intensively enhanced on alveolar epithelial cells. These results suggest that ICAM‐1 may contribute to the pathogenesis of IPF.

Pulmonary vascular involvement in sarcoidosis: granulomatous angiitis and microangiopathy in transbronchial lung biopsies.

To evaluate the occurrence of granulomatous angiitis and microangiopathy in the lung with sarcoidosis, transbronchial lung biopsy specimens were examined from 174 cases with sarcoidosis. Granulomatous angiitis was seen in 72 cases, which corresponded to 53% of the cases with granulomata. Granulomatous angiitis showed venous involvement (65%), both venous and arterial involvement (24%) or arterial involvement only (11%). There was no significant difference in occurrence of granulomatous angiitis between upper and lower lobes. The cases with granulomatous angiitis in the lung had a higher frequency of ophthalmic symptoms and elevated serum angiotensin converting enzyme level. Basal lamina layering in the microvasculature was more often observed in the bronchial mucosa than in the alveolar walls and is not exclusively related to granulomata. Endothelial proliferation and basal lamina alterations in granulomatous angiitis may be closely associated with granulomas. The present study revealed coexistence of granulomatous angiitis and microangiopathy in the lung with sarcoidosis and suggests that both may participate in the development of pulmonary sarcoidosis.

Integrin expression and ability to adhere to extracellular matrix proteins and endothelial cells in human lung cancer lines

We examined the integrin expression in 19 human lung cancer cell lines with monoclonal antibodies to the integrin subunits alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, beta 2, and beta 4. We measured their ability to adhere to the extracellular matrix (ECM) and human umbilical vein endothelial cells (HUVECs). Almost all lines expressed the beta 1 subunit and approximately half of the lines expressed the beta 4 subunit; by contrast, none expressed the beta 2 subunit. Subunits alpha 2, alpha 3, alpha 5 and alpha 6 were frequently expressed, whereas very few lines expressed alpha 1 and alpha 4. Most lines adhered strongly to ECM (type I collagen, laminin and fibronectin) in correspondence to their expression of integrins. Binding by most lines to fibronectin was completely inhibited by arginine-glycine-aspartic acid (RGD) peptide. Three lines that expressed few or no integrins had very weak ability to adhere to ECM. Strong binding to HUVECs was found in most lines, but the three lines had very little ability to adhere to HUVECs. Binding to HUVECs was strongly inhibited at 4 degrees C, under divalent cation-free conditions and by antibodies to the beta 1 subunit. These results suggest that lung cancer cells adhere to ECM and endothelial cells through integrins, especially the beta 1 subfamily.

Pulmonary surfactant protein A in pleural effusions.

Pulmonary surfactant protein A (SP‐A) is known to be a major phospholipid‐associated glycoprotein in pulmonary surfactant, which is specific to the lung. Immunohis‐tochemically, expression of SP‐A in tumor tissues is found in approximately 50% of patients with lung adeno‐carcinoma but not in the other histologic types of lung cancer or metastatic lung tumors. In this study, the SP‐A content of pleural effusions was determined using an enzyme‐linked immunosorbent assay. These results showed that approximately 40% of patients with lung ad‐enocarcinomas (27 of 67) had high levels of SP‐A (> 500 ng/ml) in their pleural effusions. By contrast, patients with other histologic types of lung cancers, adenocarci‐nomas of different primary sites, and tuberculosis had low levels of SP‐A in their pleural effusions. The determination of SP‐A in malignant effusions will contribute to distinguishing primary lung adenocarcinoma from ade‐nocarcinomas of miscellaneous origin.

Circulating γδ-T-Cell-Receptor-Positive Lymphocytes in Sarcoidosis

We investigated phenotypic surface markers of peripheral blood lymphocytes including expression of γδ T cell receptor (TCRγδ) in 185 patients with sarcoidosis and 42 normal subjects. The pr

Soluble intercellular adhesion molecule-1 (ICAM-1) in sera and bronchoalveolar lavage (BAL) fluids of extrinsic allergic alveolitis.

ICAM‐1 plays AN important role in inflammatory diseases. We analysed ICAM‐1 expression on BAL fluid cells and measured soluble ICAM‐1 (sICAM‐l) concentrations in sera and BAL fluids from patients with extrinsic allergic alveolitis (EAA). We found significantly increased cellular ICAM‐1 expression on BAL fluid lymphocytes and alveolar marcrophages, and significantly increased values of circulating and BAL fluid sICAM‐l in EAA patients compared with controls. Successive measurement showed prompt decrease of both sICAM‐1 values in EAA patients during periods when antigen exposure was prevented. In BAL fluids from EAA patients. sICAM‐l values significantly correlated to neutrophil and ICAM‐1+ lymphocyte counts. In EAA patients, circulating and BAL fluid sICAM‐l values has significant negative correlations to values of carbon monoxide diffusing capacity and to time intervals between last episode and sample collection. However, these values had significant positive correlation to values of alveolar‐arterial oxygen pressure difference. In EAA, antigen exposure appears to induce cellular ICAM‐1 expression on BAL fluid cells, and also appears to up‐regulate shedding of ICAM‐1 in the alveolar lining fluid and in the circulation. The sICAM values appear to reflect disease activity of EAA.

Lipid Analysis and Surfactant-Associated Protein Expression in Lung Adenocarcinoma Cells from Pleural Effusion

Primary lung adenocarcinomas originate from the progenitor cells of peripheral airway cells. Alveolar type II cells and Clara cells are the major progenitor cells of peripheral airway cells. Alveolar type II cells produce a lipid-protein complex called surfactant, which contains surfactant proteins SP-A, SP-B, SP-C and SP-D. Phosphatidylcholine (PC) and phosphatidylglycerol (PG) are believed to be essential for the surfactant function. Clara cells also express SP-A, SP-B and SP-D but not SP-C. In this study we examined the properties of the cancer cells isolated from the pleural effusion of a patient with primary lung adenocarcinoma by analyzing lipids, proteins and mRNAs. The cancer cells, designated as LC117 cells, were isolated from the pleural effusion of a patient with primary lung adenocarcinoma. The percent distributions of [14C]-acetate incorporated into PC and PG in the cancer cells were 55.7 and 1.1%, respectively. The disaturated species in total PC was 46.2%. Immunoblotting analysis using anti-SP-D monoclonal antibody revealed that the pleural effusion from a patient with lung adenocarcinoma contained SP-D. We determined the concentrations of SP-A and SP-D by enzyme-linked immunosorbent assay. The pleural effusions from this patient and the media incubated with cancer cells exhibited significant levels of SP-D as well as SP-A. Reverse transcriptase-polymerase chain reaction demonstrated that the tumor cells expressed mRNAs for SP-C as well as the other surfactant proteins. The results demonstrate that tumor cells from lung adenocarcinoma express all of surfactant-associated proteins, indicating that LC117 cells originate from alveolar type II cells. This study indicates that the combination of analyses of lipids, proteins and mRNAs in the cancer cells isolated from pleural effusion is useful to understand the property of lung adenocarcinoma.

Immunolocalization of extracellular matrix proteins and integrins in sarcoid lymph nodes.

Abstract To improve our understanding of the role of extracellular matrix (ECM) proteins and integrins during the processes of granuloma formation in sarcoidosis, we examined the distribution of ECM proteins and the expression of integrins in sarcoid lymph nodes by immunohistochemical methods. We also examined the expression of transforming growth factor-β1 (TGF-β1), which is one of major regulators for synthesis of ECM proteins. Most ECM proteins were detected in the periphery of the granulomas in a concentric pattern, and fibronectin was diffusely detected from an early to a regressive stage. Compared with normal lymph nodes, most β1-integrin subfamilies (α1, α4, α5 and α6) were more strongly expressed on lymphocytes around the granulomas. Epithelioid cells exhibited strong expression of the α5 molecule. Fibroblasts exhibited the expression of the α2 and α5 molecules surrounding ECM proteins. The α5β1 molecule had a distribution similar to that of fibronectin. TGF-β1 was detected in epithelioid cells throughout the various evolutional stages and its expression was especially marked in mature granulomas. Interaction of fibronectin and the α5β1 molecule may have an important role in the process of formation of sarcoid granuloma. The expression of TGF-β1 may be involved in the regression of sarcoid granuloma by initiating fibrosis and atrophy of epithelioid cells.