Distribution of vitamin B12 R-binder in lung tumors. Implications for cell differentiation

Expression of vitamin B12 R-binder, a specific binding protein for vitamin B12, was studied immunohistochemically in normal lung tissues and 107 lung tumors of various types. In normal tissues, vitamin B12 R-binder (R-binder) expression was restricted to the mucous cells of bronchial or bronchiolar epithelium and submucosal glands as well as to nonciliated bronchiolar (Clara) cells. Among lung carcinomas, 38% of squamous cell carcinomas, 42% of adenocarcinomas and 23% of large cell carcinomas showed positive staining for R-binder whereas small cell carcinomas did not. These findings offer the possibility that a majority of the histologic types of lung carcinoma have common histogenetical characteristics with mucous or Clara cells. Of the bronchial gland tumors, R-binder could be detected in a mucoepidermoid carcinoma but not in adenoid cystic carcinomas. Epithelial components in both pulmonary blastomas and hamartomas showed a reactivity for R-binder, suggesting that these tumors contained components composed of cells with bronchiolar cell differentiation. The immunohistochemical examination of lung tumors, using anti-R-binder antibody, may have some implications in the cell differentiation of lung tumors.     

Morphometric analysis of the immunohistochemical expression of Clara cell 10-kDa protein and surfactant apoproteins A and B in the developing bronchi and bronchioles of human fetuses and neonates

 Morphometric analyses of the immunohistochemical expression of the Clara cell secretory 10-kDa protein (CC10) and surfactant apoproteins A and B (SP-A and -B) were carried out on the developing bronchi and bronchioles of human fetuses and neonates. We analysed the ratio of the number of CC10-positive cells per subepithelial length of the bronchial or bronchiolar basement membrane and found that both the bronchial and the bronchiolar population of CC10-positive cells was significantly higher than that of either SP-A or SP-B. In addition, CC10 was found to be distributed mainly in the bronchiole. CC10-positive cells began to be recognized in the late pseudoglandular phase (15 weeks of gestation) and thereafter gradually increased in the canalicular and terminal sac phases, which correspond to the active development period of the acini or peripheral airways. The earliest expression of SP-A was also noted at 15 weeks of gestation, but its positive epithelial cells were present mainly in the larger bronchi. Double immunohistochemical staining for CC10 and SP-A revealed that the CC10-positive cells lining both the bronchi and bronchioles were different from the SP-A-positive cells. This finding suggests that CC10-positive cells are functionally and developmentally heterogeneous in both fetal and neonatal lungs in humans Received: 22 May 1997 / Accepted: 21 July 1997     

Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung.

The expression of the surfactant-associated proteins in bronchiolar cells remains to be defined. We used in situ hybridization to identify sites of message expression of the surfactant-associated proteins A, B, and C (SP-A, SP-B, and SP-C) in adult and fetal rat lung. The expression of these messages by in situ hybridization was also compared with the localization of SP-A by immunocytochemistry. The localization of SP-A was used to identify type II cells and nonciliated bronchiolar epithelial (Clara) cells in these sections. The cRNA antisense probes for SP-A, SP-B, and SP-C appeared to hybridize over type II cells. Sense probes showed no localization or apparent specific hybridization. Messages for both SP-A and SP-B were also found in nonciliated bronchiolar epithelial (Clara) cells. However, no message for SP-C was observed in these cells. Clara cells from terminal to large bronchioles lacked detectable mRNA for SP-C. Expression of surfactant protein mRNAs was not detectable in type I cells, alveolar macrophages, interstitial cells, or vascular cells. Similarly, in fetal rat lung the messages for SP-A and SP-B but not SP-C were detected in bronchiolar cells. We conclude that rat Clara cells do not express SP-C mRNA, and thus SP-C can be regarded as a specific marker for rat type II cells.     

Surfactant protein A detection in large cell carcinoma of the lung.

Large cell carcinomas of the lung are undifferentiated malignant epithelial tumors that lack cytologic features of small cell carcinoma, glandular cell carcinoma, or squamous cell differentiation. Lung surfactant protein A (SP-A) is produced by alveolar type II cells and Clara cells. Most bronchioloalveolar carcinomas of the lung react positively for SP-A. Positive SP-A staining of large cell carcinoma of the lung could indicate that at least part of these tumors have the same cellular origin or differentiation as bronchioloalveolar carcinoma. The authors determined the SP-A staining of 63 large cell carcinomas of the lung by IHC. In 20 of the 63 (32%), the tumors stained positive for SP-A. This may imply that about one third of large cell carcinomas of the lung have a similar cellular origin or differentiation as bronchioloalveolar carcinoma. The significance of this finding for prognosis and new forms of treatment remains to be determined.     

Peripheral airway cell marker expression in non-small cell lung carcinoma. Association with distinct clinicopathologic features.

Paraffin sections of 247 primary and metastatic non-small cell lung carcinomas, the corresponding non-neoplastic lungs, and 75 other specimens were examined by immunohistochemical procedures using a panel of antibodies against the specific products of peripheral airway cells: the major surfactant-associated protein and 10-kD Clara cell protein. Non-small cell lung carcinoma tumors most frequently positive for either peripheral airway cell marker were adenocarcinomas (41%), especially those with papillolepidic growth pattern (56%), followed by large cell carcinomas (25%), other adenocarcinomas (22%), and squamous cell carcinomas (16%). Immunoreactivity was mainly focal and the expression of the two peripheral airway cell markers was discordant. The incidence of marker expression was similar in metastatic and primary non-small cell lung carcinoma. Other organs and their tumors were negative, with few exceptions. Non-small cell lung carcinomas positive for peripheral airway cell markers were associated with younger age and less-intense smoking, and surfactant-associated protein reactivity was more common in women than in men. Peripheral airway cell markers were independent prognostic factors for survival and delayed development of metastases in patients with less-advanced disease. It is concluded that surfactant-associated protein and 10-kD Clara cell protein are specific markers for non-small cell lung carcinoma and peripheral airway cell differentiation and provide useful tools to study the pathogenesis, biology, and prognosis of non-small cell lung carcinoma.     

Immunohistochemical and ultrastructural studies of basal cells, Clara cells and bronchiolar cuboidal cells in normal human airways

Immunohlstochemical studies were made of the distribution of various cytokeratins (CK), Clara cell secretory protein (CC10), surfactant proteln A (SP-A) and type VII collagen in normal human alrways. Electron microscopic studies were made to Identify hemldesmosomes and anchoring fibrils on the basal surfaces of the epithelial cells. CK19 was detected In all epithelial cells, and CK17 in all basal cells. CK14 was coaxpressed in a few basal cells, and this coexpression was decreased in the distal airways. Two types of basal cells were recognized. One type, found mainly In large airways, was characterized by abundant intermediate filaments and well-developed hemidesmosomes and anchoring fibrils. The second type contained few intermediate filaments and poorly developed hemidesmosomes and anchoring fibrils. Reactivity for type VII collagen was found along the basement membrane throughout the airways, but not in the alveoli. Clara cells were reactive for CC10 and CK17, but not for CK14 and SP-A. The bronchiolar cuboidal cells in the respiratory bronchloles were positive only for CK19. Surfactant proteln A was present only in type II alveolar eplthellal cells. Thus, two types of basal cells are present in always, and the bronchiolar cuboidal cells appear distinct from these basal cells, Clara cells and type II alveolar epithelial cells.     

Mouse bronchiolar cell carcinogenesis. Histologic characterization and expression of Clara cell antigen in lesions induced by N-nitrosobis-(2-chloroethyl) ureas.

Female Swiss mice (Cr:NIH(S)) developed bronchiolar cell hyperplasia, dysplasia, metaplasia, and various morphologic types of bronchiolar cell tumors after topical (skin) application of N-nitroso-methyl-bis-chloroethylurea (NMBCU) or N-nitroso-tris-chloroethylurea (NTCU). These compounds are the first found to induce systemically bronchiolar cell tumors in mice in high incidence. Twice a week, with a 3-day interval, a 25-microliter drop of 0.04 mol/l (molar) NMBCU or NTCU in acetone was applied to the shaved interscapular integument for a maximum of 35 to 40 weeks. The earliest lung neoplasms were seen in mice that died after 23 weeks of treatment and affected 11 of 19 with NMBCU and 14 of 19 with NTCU treatment. Tumor growth pattern was nodular or the neoplastic tissue was frequently disseminated throughout the parenchyma, starting from multicentric peribronchiolar foci. The most common tumor types were squamous cell carcinomas and adenosquamous carcinomas, followed by adenocarcinomas with or without secretory cells, and a single ciliated-cell tumor. Histochemical and immunohistochemical studies were carried out on paraffin-embedded lungs using the avidin-biotin immunoperoxidase complex procedure and antisera against keratin, Clara cell antigen, surfactant apoprotein, neuron-specific enolase, bombesin, and chromogranin A. In several mice from both groups, hyperplasias and tumors were composed of cells expressing Clara cell antigen. No tumor cells were found expressing alveolar type II or neuroendocrine cell markers. It appeared that bronchiolar cells, in particular Clara cells, had migrated from terminal bronchioles or invaded bronchiolar walls to extend into the alveolar parenchyma. Squamous cell metaplasia with keratin expression was seen within airways or associated with glandular tumors, especially at the periphery. A unique cell type, with large eosinophilic globules and associated eosinophilic crystals, was seen lining airways or forming hyperplastic and neoplastic lesions. N-nitroso-methyl-bis-chloroethylurea- and NTCU-induced mouse bronchiolar cell alterations could be an interesting new model to study mechanisms of bronchiolar cell differentiation and tumor formation.     

Immunohistochemical demonstration of Clara cell antigen in lung tumors of bronchiolar origin induced by N-nitrosodiethylamine in Syrian golden hamsters.

Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronchioloalveolar lung carcinomas and other pulmonary neoplasms, based on the presence of cell specific markers identified by immunocytochemical methods. Alveolar type II cell origin of solid and papillary lung tumors of the mouse has been demonstrated, and Clara cells have been suggested as cell of origin for hamster pulmonary neoplasms. Therefore, chemically induced bronchiolar hyperplasias and pulmonary neoplasms of Syrian golden hamsters were analyzed by avidin-biotin immunohistochemistry to localize a hamster-specific Clara cell antigen (CCA) and keratin. The hamsters had been treated subcutaneously with multiple doses of N-nitrosodiethylamine (NDEA). Proliferative lesions of low cuboidal, tall columnar, or pleomorphic cells were present within bronchioles or adjacent to airways in the alveolar parenchyma. Frequently areas of squamous cell differentiation were present focally or diffusely that were immunoreactive for cytokeratin. Immunoreactivity for cytokeratin was also noted for hyperplastic bronchiolar neuroepithelial bodies. Cellular hyperplasias extending out into the alveolar parenchyma contained ciliated cells and frequently consisted of cells immunoreactive for CCA, showing them to be of bronchiolar Clara cell origin. Tumors developed from bronchiolar cell hyperplasias localized within bronchioles and from bronchiolar cells lining former alveolar walls. Neoplastic growth patterns were tubulo-papillary, forming loose networks or densely cellular areas. Immunoreactivity for cytoplasmic CCA was found in 50% of the tumors and was seen most frequently in small cuboidal cells and larger, vacuolated cells scattered throughout the neoplasms. In summary, evidence is presented that NDEA-induced pulmonary tumors of the Syrian golden hamster originated from cells lining bronchioles and from extrabronchiolar Clara cell hyperplasias of the terminal bronchioles. As the pulmonary tumors of the hamsters progressed towards a squamoid cell type, CCA was no longer detectable but cells became immunoreactive for keratin.     

Differential accumulation of surfactant protein A, B, and C mRNAs in two epithelial cell types of hyperoxic lung

The physiologic response of the lung to oxygen toxicity is complex, and similar among all mammals studied. Acute exposure to 100% O2 results in severe decreases in respiratory function and is accompanied by alterations in pulmonary surfactant metabolism, including the regulation of surfactant proteins A, B, and C (SP-A, SP-B, SP-C). Because surfactant proteins and their mRNAs can be expressed in alveolar epithelial type II cells, and nonciliated bronchial epithelial (Clara) cells, we were interested in determining if alterations in the abundance of SP-A, SP-B, and SP-C mRNAs occurred differentially in these two cell types during hyperoxic lung injury. Using quantitative in situ hybridization, we found that hyperoxic lung injury resulted in nearly 20-fold increases in SP-A and SP-B mRNAs in Clara cells, with relatively small (2-fold or less) increases in type II cells. Immunohistochemical analysis suggested a commensurate increase in SP-A protein in Clara cells. SP-C mRNA was only detected in type II cells, and changed little in hyperoxic lung. Because Clara cells are not known to produce surfactant, and appear to lack SP-C mRNA, these observations suggest that increased SP-A and SP-B may serve nonsurfactant functions in hyperoxic lung.     

Immunohistochemical localization of the secretory products of rat Clara cells

We used proteins in rat lung lavage fluid to successfully produce an antiserum against Clara cell secretory products. When used with the immunoperoxidase method, the antiserum specifically stained cells of the bronchiolar lining, which are morphologically consistent with Clara cells, as well as a few columnar cells in the bronchial and tracheal mucosa. B-5-fixed lung tissue furthermore demonstrated the immunoreactive layer over the bronchiolar epithelium. The alveolar and bronchial lining layers, on the other hand, were not immunoreactive, although a trace of granular immunoreactivity was seen in the latter. It was suggested that Clara cells produce and secrete some proteinaceous materials, which are mainly localized in the bronchiolar area after secretion and are seldom transferred into the alveolar lining layer. Our antibody cross-reacted with the Clara cells of mice, but not with those of hamsters, guinea pigs, rabbits, dogs, cats, monkeys, and man. The high degree of specificity of this antisera to Clara cells in formalin-fixed materials should make it a valuable tool for identifying Clara cell change in non-neoplastic lung pathology and in obtaining some insights into cell origin in neoplastic diseases.     

High-level expression of CD109 is frequently detected in lung squamous cell carcinomas

CD109 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, which is a member of the alpha2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. It has been reported that CD109 is expressed in a subset of hematopoietic cells, endothelial cells and several kinds of human tumors. Herein it is reported that the CD109 protein is preferentially expressed in lung squamous cell carcinomas compared with other types of lung carcinoma including adenocarcinomas, large cell carcinomas and small cell carcinomas. Immunohistochemical staining of surgically resected lung specimens using an anti-CD109 antibody detected CD109 expression in basal cells of bronchial and bronchiolar epithelia and myoepithelial cells of bronchial secretary glands, but not in bronchial and bronchiolar apical epithelial cells and alveolar epithelial cells. Furthermore, the CD109 immunoreactivity was observed in squamous cell carcinomas at a high frequency compared with other types of lung carcinoma. Although the detailed function of CD109 protein is unclear, these results suggest that CD109 expression may play a role in the development of lung squamous cell carcinoma.     

Localization of the 92 kd gelatinase mRNA in squamous cell and adenocarcinomas of the lung using in situ hybridization.

We have used in situ hybridization for RNA to localize cells containing mRNA for the 92 kd gelatinase in carcinomas of the lung. We used archival material to analyze sections from 12 cases of squamous cell carcinomas of the lung including six stage I and three stage II and from three cases of adenocarcinoma of the lung. Presence of mRNA in the tissue was verified by in situ hybridization for gamma actin. The 92 kd gelatinase mRNA was found in all 12 squamous cell carcinomas tumors and was highly expressed in the tumor cells themselves. In addition, it was found in host stromal cells surrounding the tumor, but not in normal lung fibroblasts. In contrast it was not found in the adenocarcinomas of the lung or in the stroma surrounding these tumors. The mRNA for the 92 kd gelatinase was present in normal pulmonary tissue, bronchial epithelium, basal cell hyperplasia of bronchial epithelium, alveolar macrophages, and focally in bronchial mucous glands. It was not present in normal alveoli, vascular cells, cartilage, or most lymphocytes. We corroborated the presence of the mRNA for the 92 kd gelatinase by ribonuclease protection assay. The levels of mRNA for the 92 kd gelatinase in two specimens of squamous cell carcinoma were 6- to 10-fold greater than in the nonneoplastic tissue and two adenocarcinoma specimens.     

Ultrastructure of Well-Differentiated Adenocarcinomas of the Lung with Special Reference to Bronchioloalveolar Carcinoma

The cytologic phenotypes of 20 well-differentiated pulmonary adenocarcinomas were determined by electron microscopy. On examination of more than 100 cells in each case, the tumors were classified according to the predominant cell types. Nine cases (45%) were of mucous cell type, further divided into 7 cases of bronchial surface epithelial cell type, 1 case of bronchial gland cell type, and 1 case of metaplastic bronchiolar goblet cell type. The remainder included 5 cases (25%) of Clara cell type, 2 cases (10%) of type II cell type, and 4 cases (20%) of mixed cell type. The predominant histologic pattern by light microscopy was “typically” bronchioloalveolar (Manning et al.'s type 1) in the metaplastic goblet cell tumor and papillary in most Clara cell-type tumors, while it was glandular in bronchial surface and bronchial gland cell types, although variable in type II cell or mixed cell type. Therefore, bronchioloalveolar carcinomas, when histologically defined inclusive of papillary tumors, present cytologic phenotypes also related to the bronchioloalveolar epithelium, i.e., metaplastic goblet or Clara or type II cell subtypes, which is in accordance with some previous reports. These tumors could be distinguished from the other (glandular) adenocarcinomas that show primarily bronchial mucous cell differentiation.     

Ultrastructure of well-differentiated adenocarcinomas of the lung with special reference to bronchioloalveolar carcinoma

The cytologic phenotypes of 20 well-differentiated pulmonary adenocarcinomas were determined by electron microscopy. On examination of more than 100 cells in each case, the tumors were classified according to the predominant cell types. Nine cases (45%) were of mucous cell type, further divided into 7 cases of bronchial surface epithelial cell type, 1 case of bronchial gland cell type, and 1 case of metaplastic bronchiolar goblet cell type. The remainder included 5 cases (25%) of Clara cell type, 2 cases (10%) of type II cell type, and 4 cases (20%) of mixed cell type. The predominant histologic pattern by light microscopy was "typically" bronchioloalveolar (Manning et al.'s type 1) in the metaplastic goblet cell tumor and papillary in most Clara cell-type tumors, while it was glandular in bronchial surface and bronchial gland cell types, although variable in type II cell or mixed cell type. Therefore, bronchioloalveolar carcinomas, when histologically defined inclusive of papillary tumors, present cytologic phenotypes also related to the bronchioloalveolar epithelium, i.e., metaplastic goblet or Clara or type II cell subtypes, which is in accordance with some previous reports. These tumors could be distinguished from the other (glandular) adenocarcinomas that show primarily bronchial mucous cell differentiation.     

Overexpression of pulmonary surfactant apoprotein A mRNA in alveolar type II cells and nonciliated bronchiolar (Clara) epithelial cells in streptozotocin-induced diabetic rats demonstrated by in situ hybridization.

Pulmonary surfactant is critical for gas exchange and is composed of both phospholipids and specific surfactant-associated proteins. The most abundant surfactant protein is termed surfactant apoprotein A (SP-A). This protein is thought to be important in the formation of tubular myelin, in absorption of surfactant to the air-liquid interface, in recycling of surfactant in alveolar type II cells, and in the regulation of secretion. We have examined the expression and localization of SP-A mRNA in streptozotocin-induced diabetic rats by in situ hybridization using a specific rat cDNA probe. Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin. After 10 wk, lungs were excised and examined by in situ hybridization and by light and electron microscopy. The ultrastructural examination demonstrated the marked changes of endoplasmic reticulum of alveolar type II cells, as reported previously. Immunohistostaining of SP-A in diabetic lungs was weak in alveolar type II cells. However, by autoradiographs of in situ hybridization, compared with the control lungs, a larger number of silver grains for the SP-A mRNA were shown in alveolar type II cells and also in some bronchiolar epithelial (Clara) cells from the diabetic lungs. Alveolar type II cells having high contents of silver grains were also increased in number. These results were confirmed by measurement of the SP-A content and by Northern blot analysis. The present study demonstrates an overexpression of SP-A mRNA despite the ultrastructural changes in the endoplasmic reticulum of alveolar type II cells in the diabetic lungs, which will provide new information on the regulatory mechanism of SP-A gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser capture microdissection and real-time reverse transcriptase/ polymerase chain reaction of bronchiolar epithelium after bleomycin

Terminal airways are affected in many lung diseases and toxic inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We used laser capture microdissection (LCM) to collect terminal bronchiolar epithelial cells from frozen sections of lungs of mice subjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymerase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRNAs in whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)-alpha mRNA was reduced but whole-lung TGF-alpha mRNA was not changed, whereas terminal bronchiolar epithelial epidermal growth factor (EGF) receptor mRNA was not changed but whole-lung EGF receptor was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene expression in terminal bronchiolar epithelium is not necessarily reflected in analysis of whole-lung gene expression.     

Effects of nitrogen dioxide exposure on Clara cell proliferation and morphology.

The present study was performed to relate Clara cell proliferation and Clara cell morphology to duration and intensity of nitrogen dioxide (NO2) exposure as a model of lung injury in rats. Male Sprague-Dawley rats were exposed to 5, 10 and 20 ppm NO2 for three (short term exposure) and 25 days (long term exposure). Epithelial proliferation was immunohistochemically assessed by BrdU-incorporation into the bronchial and bronchiolar epithelium. Clara cell specific 10 kD protein was detected using an antibody versus recombinant protein. Following short term exposure Clara cells showed a significant alteration of their morphology: the cells lost the apical intraluminal projections, and the damaged epithelium was covered by a layer of CC10 reactive material. After long term exposure the Clara cell morphology was indistinguishable from that of unexposed controls. In all short term exposure groups cellular proliferation was higher in the bronchiolar epithelium than in the bronchial epithelium. Whereas short term exposure to 5 ppm NO2 sufficed to induce a maximum epithelial proliferation of the bronchiolar epithelium, long term exposure to 5 ppm NO2 yielded no significant increase in the epithelial proliferation, a finding reflecting the adaptation of the airway epithelium to oxidative stress. Long term exposure to 10 and 20 ppm NO2 resulted in a dosilinear increase of cell proliferation in the bronchial and bronchiolar epithelium. Double labelling of CC10 and BrdU showed that BrdU uptake and hence proliferation were restricted to Clara cells. We conclude that changes of the Clara cell morphology are closely related to the extent of inhalative stress and functional status of the Clara cell. The results are in keeping with the assumption that the Clara cell exerts progenitor cell functions in the airway epithelium.