Clara cell adenomas of the mouse lung. Interaction with alveolar type 2 cells.

Multiple pulmonary adenomas were induced in the offspring of pregnant Swiss-Webster mice by transplacental exposure to ethylnitrosourea (ENU) on the 15th day of gestation. Development and growth of tumors were followed for up to a year after birth. Morphologic assessment indicated that the majority of adenomas were of Clara-cell origin and were relatively normal on the basis of structural features. Histochemical studies, utilizing nitroblue tetrazolium reductase activity as a marker for normal Clara cells demonstrated that the Clara-cell-derived tumors possessed nearly normal enzyme activity. Microscopic studies of the tumors and adjacent parenchyma revealed a unique Type 2 cell response to the presence of Clara-cell adenomas occurring in the alveoli beyond the margins of the tumor. Otherwise normal-appearing Type 2 cells, in a narrow zone around the Clara-cell tumors, accumulated large amounts of surfactantlike osmiophilic lamellar material within cytoplasmic vacuoles as early as 30 days after birth. These changes were clearly a Clara-cell-tumor-related response, and not seen in association with other non-Clara-cell adenomas of the same lung. Furthermore, the alterations occurred exclusively in Type 2 cells. The extent of Type 2 cell change was correlated with tumor size and age. Autoradiographic studies with tritiated choline showed marked incorporation of the labeled precursor by the altered Type 2 cells. By electron microscopy, these inclusions were membrane-limited and contained osmiophilic lamellar structures similar to lamellar bodies in normal Type 2 cells. Because these Clara cell adenomas may act as a concentrated focus of normal Clara cells, the alterations seen in Type 2 cells may reflect an amplification of a normal interaction between bronchiolar Clara cells and alveolar Type 2 cells in the centriacinar and juxtabronchiolar alveoli.     

Clara cell carcinoma of the lung

A peripheral scar adenocarcinoma of the lung was found to be composed of cells withthe ultrastructural features of Clara cells. It is suggested that malignant change occurred during hyperplastic and regenerative changes of the bronchiolar epithelium in which the Clara cell was the progenitor.     

Beta-adrenergic regulation of rat parotid gland exocrine protein secretion during aging

beta-Adrenergic regulation of exocrine protein secretion from the parotid gland was studied over the adult rat life span. Enzymatically dispersed cell aggregates were prepared from 3-, 6-, 12-, and 24-month-old rats and exocrine protein secretion (amylase release) measured. No age differences were seen in the time course of amylase release following (-)-isoproterenol stimulation or in the (-)-isoproterenol dose-response curve. the beta-adrenergic antagonist (+/-)-propanolol inhibited (-)-isoproterenol-stimulated protein secretion from parotid cell aggregates of young and old animals equally. Similarly dibutyryl cyclic AMP induced comparable rates of protein secretion from cells of different aged rats. Direct examination of beta-adrenergic receptor characteristics in parotid gland membranes from 3- to 24-month-old rats revealed no differences in the equilibrium dissociation constant (Kd) or maximum specific ligand binding capacity (receptor number). These results suggest that the rat parotid beta-adrenergic system remains functionally intact throughout the animals' lifetime.     

Keratin expression in chemically induced mouse lung adenomas.

Chemically induced mouse lung tumors exhibit distinctive growth patterns, characterized by an alveolar or solid appearance, a papillary appearance, or a combination of the two. Lung tumors induced in strain A/J mice by either benzo(a)pyrene (BP) or by N-nitrosoethylurea (ENU) were examined for expression of low- and high-molecular-weight cytokeratins. Simple cytokeratins (low molecular weight) were found in all epithelial cells of the normal mouse lung and in all tumor types, whereas higher-molecular-weight cytokeratins were found only in normal bronchiolar cells and in papillary tumor cells. These data lend support to the hypothesis that chemically induced papillary lung tumors in strain A/J mice are derived from bronchiolar Clara cells.     

Large intracytoplasmic body in lung cancer compared with Clara cell granule

Large intracytoplasmic bodies in tumor cells of different pulmonary neoplasms were morphologically studied. In 27 of 105 cases of surgically resected lung tumors, large intracytoplasmic bodies were found in 13 adenocarcinomas, 9 squamous cell carcinomas, and 5 large cell carcinomas. These bodies appeared most frequently in large cell carcinomas and in well-differentiated adenocarcinomas, particularly of the Clara cell type. In squamous cell carcinomas, they were seen only in nonkeratinizing polygonal tumor cells. Large intracytoplasmic bodies in different pulmonary tumors and Clara cell granules in Clara cell carcinoma showed similar histochemical staining reactions. Electron microscopically, both large intracytoplasmic bodies and Clara cell granules appeared membrane bound and contained electron-dense matrices. The results suggest that large intracytoplasmic bodies in lung carcinoma cells may be a variant of Clara cell granules, only differing in size.     

Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pancreastatin secretion by pituitary adenomas and regulation of chromogranin B mRNA expression.

Pancreastatin, a carboxyl-terminal amidated peptide derived from chromogranin (Cg)A, inhibits secretion of insulin and parathyroid hormone. Our recent studies found significant amounts of immunoreactive pancreastatin in all pituitary adenomas except prolactin adenomas. To analyze the effects of pancreastatin on pituitary cell function, 17 cultured pituitary adenomas were examined for immunoreactive pancreastatin and pancreastatin secretion by the tumors. The effects of pancreastatin on pituitary hormone secretion and on pituitary hormone (follicle-stimulating hormone and prolactin), CgA, and CgB mRNA levels were also examined. Immunoreactive pancreastatin and CgA were present diffusely in gonadotroph and null cell adenomas, but only a few prolactin adenoma cells expressed pancreastatin or CgA. When cells were treated with hypothalamic peptides, gonadotroph adenomas were the only group that released increased amounts of pancreastatin in response to gonadotropin-releasing hormone (10(-7) mol/L). Pancreastatin (10(-7) mol/L) treatment did not stimulate pituitary hormone secretion significantly. In situ hybridization analyses showed that gonadotropin-releasing hormone and pancreastatin treatment led to significant increases in CgB and follicle-stimulating hormone mRNAs in gonadotroph adenomas, whereas CgA mRNA levels did not change significantly. These results show that there is a differential distribution of pancreastatin secretion in pituitary adenomas and that the hypothalamic hormone gonadotropin-releasing hormone and the CgA-derived peptide pancreastatin can regulate CgB mRNA in gonadotroph adenomas, suggesting an autocrine effect of pancreastatin on pituitary tumor function.     

Beta-adrenergic modulation of mucin secretion in cat trachea

The mechanism of beta-adrenergic regulation of mucin secretion was investigated in cat trachea in vitro. beta-Adrenergic agonists increased the release of [35S]SO4-radiolabeled mucin and mucosa-submucosal adenosine 3',5'-cyclic monophosphate (cAMP) levels in a dose- and time-dependent manner. The relative potencies and efficacies of l-isoproterenol, l-epinephrine, l-norepinephrine, terbutaline, and dobutamine for physiological and biochemical events were similar. The effect of these agonists were blocked by d-l-propranolol. 3-Isobutyl-l-methylxanthine (IBMX) and 8-bromo-cAMP mimicked the effects of the agonists on mucin release. IBMX increased cAMP levels and potentiated the increase in cAMP levels effected by beta-adrenergic agonists. The half-maximal effects of l-isoproterenol on cAMP levels and mucin release were attained at 1.6 and 8.8 min, respectively. Three major mucosa-submucosal proteins of apparent molecular weights of 49,000, 54,000, and 59,000 daltons displayed reduced binding of the photoaffinity label 8-N3-[32P]cAMP when endogenous cAMP levels were increased with l-isoproterenol and/or IBMX. The first two proteins correspond in electrophoretic mobility to the regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. The 59,000-dalton cAMP binding protein may be the phosphorylated form of the regulatory subunit of type II cAMP-dependent protein kinase. These data are consistent with the hypothesis that beta-adrenergic modulation of tracheal mucin release is mediated by cAMP and suggest that activation of cAMP-dependent protein kinases may be involved in the neurohormonal effects.     

Ultrastructure of goblet-cell metaplasia from Clara cell in the allergic asthmatic airway inflammation in a mouse model of asthma in vivo

Mucus overproduction from goblet cells, a characteristic feature of the allergic asthmatic inflammation induced by ovalbumin (OVA) in mice, was examined morphologically. In OVA-untreated (normal) mice, there were no goblet cells in intrapulmonary bronchus and bronchiole. However, goblet cells with or without hyperplasia in the mucosa of inflamed bronchus–bronchiole were recognized in the allergic asthmatic mice. The non-ciliated epithelium containing electron lucent granules (mucus) showed many similarities to Clara cells, which have characteristic secretory granules and many mitochondria, except for the less-developed smooth endoplasmic reticulum seen in normal mice. Ciliated Clara cells with or without mucus were rarely recognized. In addition, mucus was found in neither ciliated nor basal epithelium. The present study suggests that goblet-cell metaplasia in the bronchus and bronchiole of inflamed mucosa may be derived, at least in part, from Clara cells.     

Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse.

Clara cells are primary targets for metabolically activated pulmonary toxicants because they contain an abundance of the cytochrome P450 monooxygenases required for generation of toxic metabolites. The factors that regulate bronchiolar regeneration after Clara cell injury are not known. Previous studies of naphthalene-induced bronchiolar injury and repair in the mouse have shown that epithelial cell proliferation is maximal 1 to 2 days after injury and complete 4 days after injury. Proliferation is followed by epithelial re-differentiation (4 to 14 days). In this study, mice were treated with the environmental pollutant naphthalene to induce massive Clara cell injury. The distribution and abundance of three growth-regulatory peptides (epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha) was determined immunochemically during repair of this acute bronchiolar injury. EGFR and its ligands were detected at low levels in cells throughout the lung including peribronchiolar interstitial cells, blood vessels, and conducting airway epithelium. Immediately after naphthalene injury (1 to 2 days), EGFR, EGF, and TGF-alpha are expressed in increased abundance in squamous epithelial cells of the injury target zone, distal bronchioles. These immunopositive squamous cells are detected in clumps in the distal bronchioles at the time when cell proliferation is maximal. EGFR protein expression is decreased slightly 4 to 7 days after injury and continues to decrease below control levels of abundance 14 to 21 days after injury. This down-regulation of EGFR is not reflected in a corresponding decrease in EGF and TGF-alpha protein expression, indicating that control of cell proliferation is regulated at the receptor level. Co-localization of EGFR and bromodeoxyuridine-positive proliferating cells in the same bronchiole indicates that EGFR is up-regulated within the proliferative microenvironment as well as in specific proliferating cells within the injury target zone. The coincident localization within terminal bronchioles of EGFR, EGF, and TGF-alpha to groups of squamous epithelial cells 2 days after naphthalene injury suggests that these peptides are important in up-regulating cell proliferation after Clara cell injury in the mouse.     

Androgen regulation of glycosidase secretion in epithelial cell cultures from human epididymis.

The human epididymis and its secretions actively promote sperm fertilizing capacity and provide protection for spermatozoa against harmful influences. Among epididymal secretions, glycosidases have been recently studied and associated with molecular changes on the sperm surface. In the present work, we studied the influence of different concentrations of testosterone, dihydrotestosterone and cyproterone acetate on the secretion of alpha-glucosidase, N-acetyl-glucosaminidase, beta-glucuronidase and alpha-mannosidase by isolated and cultured epithelial cells from human caput, corpus and cauda epididymides. Cell cultures were obtained from aggregates of isolated tubule fragments plated on extracellular matrix-covered multi-well plates. Activities of the glycosidases were measured in conditioned culture media and were higher in the distal regions of the epididymis. Testosterone and dihydrotestosterone significantly increase the enzyme secretion in a concentration-dependent manner. This increase was higher in corpus and/or cauda than in caput epididymis. Cyproterone acetate caused a dose-dependent decrease in glycosidase secretion in cultures from all epididymal regions. It is concluded that the secretion of epididymal glycosidases is regulated by androgen, being stimulated by dihydrotestosterone and testosterone and inhibited by the androgen antagonist cyproterone acetate.     

Histogenesis of the papillary Clara cell adenoma.

The origin of the endocrine cells in the respiratory tract and the gastrointestinal tract is still a matter of debate. In the original concept of the amine precursor uptake and decarboxylation (APUD) system, all APUD cells were considered to be derived from the neural crest. More recently it has been proposed that the APUD cell types of the gastrointestinal and respiratory tracts originate from neuroendocrine-programmed ectoblast. Still other investigators have reported observations that favor a direct endodermal origin of these cell types. Based on the assumption that in teratomas different tissue types which in normal embryogenesis are derived from the neuroectoderm might be expected to occur together, we investigated a series of cystic ovarian teratomas and testicular teratocarcinomas for the presence of brain tissue and of different types of APUD cells. In the ovarian teratomas, intestinal and respiratory APUD cell types were found almost exclusively without coexistence of brain tissue, whereas melanocytes, which are of neuroectodermal origin, occurred mostly together with brain tissue. In the testicular teratocarcinomas, intestinal types of APUD cells occurred without brain tissue. Peptide hormone production was found in appropriate tissues. It can therefore be concluded that in teratomas appropriate intestinal and respiratory APUD cells differentiate in and presumably descend directly from intestinal and respiratory epithelium.     

Postnatal lung development of rhesus monkey airways: Cellular expression of Clara cell secretory protein

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties. Evidence suggests that CCSP is involved in mitigating many lung disease states during development including asthma. This study's rationale is to define the distribution and abundance of CCSP in the airway epithelium of the rhesus monkey during postnatal lung development using carefully controlled site‐specific morphometric approaches in defined airway regions. Immunoreactive CCSP was found in nonciliated cells and mucous cells, including glands, throughout the airway epithelium at all ages, with proximal and mid‐level airways having the highest labeling. Overall airway CCSP levels were low at 1 week and 1 month, doubled between 1 and 3 months, and changed little from 3 months to 3 years. Thus, the critical developmental window for CCSP expression to reach adult levels in the rhesus conducting airways occurs between 1 and 3 months of age. Developmental Dynamics 238:3016–3024, 2009. © 2009 Wiley‐Liss, Inc.     

The TSH secretion in the human pituitary adenomas

TSH secretion by a pituitary tumor is very rare (2%) and it is often associated with another hormone: GH or PRL essentially. We present here nine tumors in which the TSH secretion was proved by immunocytochemistry (ICC) and by RIA in the tumor extracts, in the serum and in the culture medium. Four tumors secreted TSH only. Five tumors secreted TSH and GH predominantly. In 3 of them traces of other hormones (PRL and FSH) were also detected. The "pure" TSH adenomas were monomorphous with typical ultrastructural and immunocytochemical features. Plurihormonal TSH adenomas were bimorphous with different cells secreting GH and TSH or monomorphous with one type of cell which secreted TSH or GH or both TSH and GH. In a majority of the cases, the tumoral TSH secretion induced hyperthyroidism but in 2 patients with TSH adenoma there was euthyroidism and in another with TSH-GH adenoma there was no sign of acromegaly and GH serum levels were normal.     

Dendritic cell subsets and immune regulation in the lung

The lung is continuously exposed to inhaled particles, microbes and harmless antigens to which either immunity or tolerance is induced. Dendritic cells are mainly recognized for their extraordinary capacity to induce a primary immune response in the lung. Recent evidence suggests that particular subsets of DCs are essential in the decision between immunity or tolerance. Moreover, DCs play an essential role during secondary immune responses in the lung, where they control the inflammatory reaction. These novel concepts are of particular interest in understanding the pathogenesis of asthma, a disorder of aberrant immune reactivity to inhaled harmless allergens.     

Clara cell secretory protein (CC16): characteristics and perspectives as lung peripheral biomarker

Clara cell protein (CC16) is a 15.8-kDa homodimeric protein secreted in large amounts in airways by the non-ciliated bronchiolar Clara cells. This protein increasingly appears to protect the respiratory tract against oxidative stress and inflammation. In vitro, CC16 has been shown to modulate the production and/or the activity of various mediators of the inflammatory response including PLA2, interferon-gamma and tumour necrosis factor-alpha. CC16 has also been found to inhibit fibroblast migration or to bind various endogenous or exogenous substances such as polychlorobiphenyls (PCBs). This protective role is confirmed by studies on transgenic mice, showing that CC16 deficiency is associated with an increased susceptibility of the lung to viral infections and oxidative stress. In humans, a polymorphism of the CC16 gene, localized to a region linked to airway diseases, has recently been discovered in association with an increased risk of developing childhood asthma. Finally, CC16 also presents a major interest as a peripheral marker for assessing the integrity of the lung epithelium. The determination of CC16 in serum is a new non-invasive test to detect Clara cell damage or an increased epithelial permeability in various acute and chronic lung disorders.