Clara-like Cells Research Articles

Functional Live Imaging of the Pulmonary Neuroepithelial Body Microenvironment

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.

Clara cell adhesion and migration to extracellular matrix

BackgroundClara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention.MethodsUsing a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells.ResultsWe observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices.ConclusionThus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.

Pulmonary expression of voltage-gated calcium channels: special reference to sensory airway receptors

Studying depolarisation induced calcium entry in our recently developed in situ lung slice model for molecular live cell imaging of selectively visualised pulmonary neuroepithelial bodies (NEBs), exemplified the need for information on the localisation of voltage-gated calcium channels (Ca(v)) in lungs in general, and related to sensory airway receptors more specifically. The present study therefore aimed at identifying the expression pattern of all major classes and subtypes of Ca(v) channels, using multiple immunostaining of rat lung cryosections. Ca(v) channel antibodies were combined with antibodies that selectively label NEBs, nerve fibre populations, smooth muscle, endothelium and Clara cells. Ca(v)2.1 (P/Q-type) was the only Ca(v) channel expressed in NEB cell membranes, and appeared to be restricted to the apical membrane of the slender NEB cell processes that reach the airway lumen. Subpopulations of the vagal but not the spinal sensory nerve fibres that contact NEBs showed immunoreactivity (IR) for Ca(v)1.2 (L-type) and Ca(v)2.1. Ca(v)2.3 (R-type) was selectively expressed by the so-called Clara-like cells that cover NEBs only, and appears to be a unique marker to discriminate this epithelial cell type from the much more extensive group of Clara cells in rat airways. The laminar nerve endings of smooth muscle-associated airway receptors (SMARs) revealed IR for both Ca(v)2.1 and Ca(v)2.2 (N-type). More generally, Ca(v)1.2 was seen to be expressed in vascular smooth muscle, Ca(v)2.3 and Ca(v)3.1 (T-type) in bronchial smooth muscle, Ca(v)3.1 and Ca(v)3.2 (T-type) in endothelial cells, and Ca(v)1.3 (L-type) in a limited number of epithelial cells. In conclusion, the present immunocytochemical study has demonstrated that the various subtypes of Ca(v) channels have distinct expression patterns in rat lungs. Special focus on morphologically/neurochemically characterised sensory airway receptors learned us that both NEBs and SMARs present Ca(v) channels. Knowledge of the identification and localisation of Ca(v) channels in airway receptors and surrounding tissues provides a solid basis for interpretation of the calcium mediated activation studied in our ex vivo lung slice model.

Detailed confocal fluorescent visualization of Ca 2+ and membrane potential changes in pulmonary neuroepithelial bodies (NEBs) in a murine lung slice model

Conclusive physiological data on potential stimuli for pulmonary NEBs are very scarce because of the lack of a reliable in vitro model. We recently developed a method for selective staining of NEBs by 4-Di-2-ASP in vibratome slices of live mouse lungs. Aim of the study was optimizing the model for live cell imaging, allowing detailed confocal visualization of changes in intracellular free calcium ([Ca2+]i) and membrane potential, using the fluorescent indicators Fluo-4 and DiBac4 (3), respectively. In all NEBs, experimental short-term elevation of extracellular potassium ([K+]o) was seen to evoke a reproducible rise in cytoplasmic fluorescence for both Fluo-4, corresponding to an increase in [Ca2+]i, and DiBac4 (3), indicative for membrane depolarization. So-called Clara-like cells, known to almost completely shield NEBs from the airway lumen, appeared to exhibit a slightly delayed increase in Fluo-4 fluorescence after high [K+]o, while DiBac4 (3) did not reveal changes. The Ca2+ response in NEBs and Clara-like cells was abolished in Ca2+ free solution. This study provides the first evidence that physiological activity of pulmonary NEBs, and surrounding tissues, can be visualized in live lung slices using fluorescent indicators. The delayed increase in [Ca2+]i in Clara-like cells might be explained by stimulation of Clara-like cells via depolarization-induced neurotransmitter release from NEB cells. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)

Confocal fluorescent imaging of the interactions of cholinergic agonists with pulmonary neuroepithelial bodies (NEBs) using an in situ lung slice model

Evidence strongly suggests that components of tobacco smoke induce changes in the pulmonary neuroendocrine system. In this study, potential receptor mediated cholinergic interactions with NEBs are postulated based on the expression of vesicular acetylcholine transporter (VAChT) in nerve endings between the NEB cells. Aim was to explore the possible direct effects of cholinergic agonists on NEBs via Ca2+ imaging in a mouse lung slice model. NEBs were visualized by 4-Di-2-ASP and Ca2+-dependent activation by Fluo-4. Application of high extracellular K+ (5s; 50mM) was used as a positive control for NEB activation and for the identification of Clara-like cells, which cover NEBs and show a characteristic slightly delayed Ca2+ rise. Surprisingly, no rise in Fluo-4 fluorescence could be detected in NEB cells after stimulation with 10–100μM nicotine, acetylcholine or carbachol. In Clara-like cells, however, the cholinergic stimulation resulted in a clear Ca2+ rise. In conclusion, NEBs in mouse lungs did not show a Ca2+-dependent activation by externally applied cholinergic agonists. Clara-like cells that intimately interact with NEBs did reveal a cholinergic activation and, considering their possible stem cell-like properties, may be more important players in the airways in health and disease than accounted for today. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.

Identification of uteroglobin-related protein 1 and macrophage scavenger receptor with collagenous structure as a lung-specific ligand-receptor pair.

High in normal (HIN)-1 is a secreted protein highly expressed in normal breast epithelium and down-regulated in breast carcinomas. By searching GenBank expressed sequence tag databases, we identified HIN-2, a protein homologous to HIN-1. HIN-2 is identical with a recently identified protein called uteroglobin-related protein 1 (UGRP1). Northern blot analysis demonstrated that UGRP1 is specifically expressed by lung, but not by the other tissues examined. By in situ hybridization experiments, UGRP1 was shown to be expressed by lung Clara-like cells in the bronchial epithelium and to be up-regulated in cystic fibrosis. In a mammalian expression system, secreted recombinant UGRP1 was copurified with apolipoprotein A-I. Using a retroviral vector-mediated expression cloning approach, we identified macrophage scavenger receptor with collagenous structure (MARCO) as a receptor for UGRP1. Northern blot and in situ hybridization experiments indicated that MARCO is expressed by alveolar macrophages in the lung. UGRP1 also bound to bacteria and yeast. LPS, a previously identified MARCO ligand, competed with UGRP1 for binding to MARCO and bacteria. Our findings suggest that UGRP1-MARCO is a ligand-receptor pair that is probably involved in inflammation and pathogen clearance in the lung.

BMK1 (ERK5) regulates squamous differentiation marker SPRR1B transcription in Clara-like H441 cells.

Various toxicants and carcinogens upregulate the expression of small proline-rich protein 1B (SPRR1B), a squamous differentiation marker, in bronchial epithelial cells both in vivo and in vitro. We have recently shown that phorbol 13-myristate 12-acetate (PMA)-stimulated SPRR1B transcription in Clara-like H441 cells is mainly mediated by activator protein-1 (AP-1) and c-Jun N-terminal kinase-1 (JNK1). Though mitogen-activated protein kinase (MAPK) kinase (MEK)-1/2 pathway inhibitors strongly suppressed both basal and PMA-inducible SPRR1B transcription, overexpression of dominant negative (dn) forms of extracellular signal-regulated kinase (ERK)-1 and/or -2 did not have any significant effect indicating the involvement of another ERK-like MAPK in this pathway. Here, we report for the first time the involvement of ERK5 in PMA-inducible SPRR1B transcription in H441 cells. PMA significantly induced ERK5 activation in H441 cells. Overexpression of dn-ERK5 strongly suppressed both basal and PMA-inducible SPRR1B transcription, whereas wild-type ERK5 upregulated it. Consistent with this, a mutant form of MEK-5, an upstream activator of ERK5, strongly suppressed PMA-inducible promoter activity. However, coexpression of c-Jun restored promoter activation suppressed by dn-ERK5. Thus, in addition to JNK1, the activation of MEK5-ERK5 MAPK pathway probably plays a pivotal role in transcriptional regulation of AP-1-mediated SPRR1B expression in the distal bronchiolar region.

JNK1 and AP-1 regulate PMA-inducible squamous differentiation marker expression in Clara-like H441 cells.

Exposure of distal bronchiolar region to various toxicants and pollutants suppresses Clara cell differentiation marker expression and greatly enhances the induction of squamous cell differentiation (SCD). Here, we demonstrate for the first time phorbol 13-myristate 12-acetate (PMA)-inducible expression of SCD markers, SPRRs, in Clara-like H441 cells. The transcriptional stimulation of human SPRR1B expression is mainly mediated by a -150- to -84-bp region that harbors two critical activator protein (AP)-1 sites. In unstimulated cells, the -150- to -84-bp region is weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA prominently induced the binding of JunB and Fra1. Consistent with this, overexpression of wild-type Jun proteins upregulated the SPRR1B promoter activity. Conversely, a c-jun mutant suppressed both basal and PMA-inducible reporter gene expression. Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly enhanced basal level activity, indicating an opposing role for these proteins in SPRR1B expression in a manner similar to that observed in proximal tracheobronchial epithelial cells (BEAS-2B clone S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun NH(2)-terminal kinase (JNK) 1 mutant significantly reduced the PMA-inducible SPRR1B promoter activity in H441 cells. Thus either temporal expression and/or spatial activation of AP-1 proteins by JNK1 might contribute to the induction of SCD in Clara cells.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.

A scanning and transmission electron microscopic study of the development of the surface structure of neuroepithelial bodies in the mouse lung

Neuroepithelial bodies (NEBs) are groups of neuroepithelial (NE) cells that are localized on mounds on the bronchiolar epithelium of the lung. The present study examined NEBs in mice ranging in age from 2 days before birth to 80 days after birth. The position and surface architecture of NEBs was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In foetal mice, 2 days before birth, NEBs were distinguished from the rest of the bronchiolar epithelium by a slight elevation of non-ciliated Clara-like cells arranged in a cobbestone-like pattern. The exposed surface of the NEB was identified by small protrusions with regular microvilli intermittenly located at the base of deep clefts between the Clara-like cells. The surface of the Clara-like cells had fewer and smaller microvilli and could be easily distinguished from the apical surface of the NEB. Before birth, the surface of all of the apical cells was covered by regularly placed microville, however after birth some of the more prominently positional apical cells revealed a bare patch at the centre of the portion of apical cell exposed to the lumen of the lung. As the mice aged there was an increase in the number of apical cell protusions observed with centrally positioned bare patches. These two morphologically distinct surfaces of apical cells may have separate specialized functions. The exposed surfaces of apical cells were often observed in pairs and this feature has been observed in various sensory organs providing support for chemoreceptive function. However small bright spheres resembling vesicles were frequently observed on the lumenal surface of apical cells of the centrally placed bare patch. Transmission electron microscopy confirmed the presence of vesicles on the surface of apical cells and due to their location these vesicles were thought to contain a substance secreted into the lumen of the lung by apical cells. The significance of the bare region on the apical cells is not clear in terms of the proposed chemoreceptive function usually attributed to NEBs. It may be possible that the morphological changes observed in apical cells after birth are more appropriate for secretion of than for chemoreception. This is supported by the observation in the present study of vesicles lying on the lumenal surface of the bare region of the apical cell, however the mechanism for secretion of whoel vesicles is not clear and requires further investigation.

Papillary Adenoma of the Lung

Two cases of papillary adenoma of the lung are presented along with results of histological and ultrastructural examinations. The tumors were encountered in two asymptomatic patients in a mass-survey chest X-ray examination. The chest X-ray films showed the tumors as well demarcated small lesions. Histologically, both tumors arose in the bronchioles and consisted of cuboidal cells resembling type II pneumocytes showing papillary growth with accompanying edematous connective tissue. Several tumor cells each possessed a large eosinophilic intranuclear inclusion. In case 1, ciliated cells and Clara-like cells were also present in the tumor. Ultrastructurally, most of the tumor cells had various numbers of lamellar bodies in their cytoplasm, indicative of type II pneumocytes, and some of case 1 showed features of Clara cells and ciliated cells. The intranuclear inclusions appeared as aggregates of tubular structures or had lamellar body-like features. These findings are identical to those of papillary adenoma arising from the bronchiole.

Phylogenetic aspects of the neuroepithelial bodies.

Neuroepithelial bodies (NEB) are small groups of cells bound together by desmosomes and which form part of the epithelium lining the bronchopulmonary ducts. The cells have an irregular, columnar shape often with laterally placed microvilli and unspecialized cytoplasmic protrusions into the intercellular spaces. Their most distinctive cytoplasmic feature is the numerous dense cored vesicles (DCV) located towards the basal region of each cell. A variety of peptides and the monoamine serotonin are distributed within these DCV. Evidence has been shown for the occurrence of more than one type of DCV for the co-existence of various peptides and serotonin in the same DCV and for morphological and functional variations in cells forming a NEB. In some animals the apical pole is in direct contact with the lumen of the duct, whereas in others the NEB is isolated from the lumen by either ciliated epithelial or Clara-like cells. When the apical pole contacts the lumen, the surface of each cell bears microvilli and occasionally a single cilium. The NEB of eutherian mammals are sensitive to a number of experimental procedures including hypoxia, hypercapnia and vagal stimulation. Experimental lesions above and below the nodose ganglion have shown that the major part of the innervation is afferent with collateral branches off the afferent forming efferent, cholinergic endings. A small efferent input is also derived directly from the central nervous system. These studies suggest that a minor component of the efferent innervation is derived contralaterally or from intrapulmonary ganglia. Other forms of innervation identified by transmission electron microscopy in some mammals and lower vertebrates include cholinergic reciprocal synapses, adrenergic and peptidergic nerve junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Repopulation of denuded tracheas by Clara cells isolated from the lungs of rabbits.

An experimental approach was examined to study the growth and differentiation potential of different epithelial cell types isolated from the airways of adult rabbits. Clara cells isolated from the lungs of rabbits and a mixed cell population obtained from rabbit tracheas were injected (separately) into denuded rat tracheas which were then grafted into nude mice. Epithelial cells were obtained from rabbit tracheas by digestion of tracheal epithelium with proteases. The resulting cell suspension contained mucous cells, Claralike cells, ciliated cells, basal cells and a small number of inflammatory and unidentified cells. This mixed cell suspension was inoculated into rat tracheas denuded of their own epithelium which were then grafted subcutaneously onto the backs of nude mice. The tracheas were recovered two weeks later and were prepared for light and electron microscopy. These grafts were found to be lined by a tall columnar pseudostratified mucociliary epithelium, indicating that the rat tracheal grafts transplanted into nude mice supported the growth and differentiation of tracheal epithelial cells. Similarly Clara cells, isolated and purified to 85.9 +/- 2.8% (n = 9) purity from rabbit lungs by a combination of centrifugation and elutriation procedures, were inoculated into rat tracheas which were transplanted into nude mice. Two weeks later the grafts were recovered and prepared for histological and ultrastructural examination. These tracheas were lined with a low cuboidal epithelium reminiscent of bronchiolar epithelium. The epithelium was composed of ciliated cells and Claralike cells rich in smooth endoplasmic reticulum but containing only a few secretory granules. These results indicate that the tracheal graft model is suited to examine the differentiative potential of specific cell types isolated from the airways. Furthermore, they show that Clara cell isolates are able to establish an epithelium resembling bronchiolar epithelium in denuded tracheas while tracheal cells containing at least four different epithelial cell types are able to establish a mucociliary epithelium resembling the pseudostratified tracheal lining.

Three-dimensional reconstruction of a small-granule paracrine cell cluster in an adult hamster bronchus.

Amine precursor uptake and decarboxylation (APUD) small-granule cells were stained by periodic acid-Schiff (PAS)-lead hematoxylin in 0.5-micron etched Epon sections of adult hamster lung fixed for transmission electron microscopy. The leading edge of a small-granule cell cluster was identified in a segmental bronchus as a single PAS-positive cell. From 256 serial thin sections through its entirety, a three-dimensional wooden reconstruction of the cluster and morphometric estimates of the apical and basal surfaces, cell volume, and intracytoplasmic distribution of mitochondria and small granules was made. Of moderate size, the body consisted of 16 small-granule cells, 11 forming its ovoid core with five outlying cells diverging at the margin; these were pyramidal, possessing wide bases and thin apical processes. At the bronchial surface, processes from the 11-cell core emerged together, whereas the divergent cells emerged in groups of two and three. Ten Clara-like cells and one ciliated cell encircled the core. Altogether they formed a pseudostratified epithelium in contrast to the surrounding simple columnar epithelium. Deeper in the cluster, numerous cytoplasmic extensions interdigitated with those from adjacent cells, and toward the base the Clara-like and APUD cells were increasingly interposed. In marked contrast to the apical cytoplasm, the infranuclear cytoplasm of the latter was densely packed with ca. 1,000 A electron-dense granules; and the basal, presumptively secretory face of each cell was five to six times greater than the area exposed to the bronchial lumen. Judged by granule size and ultrastructure, only one APUD cell type was recognized in the reconstructed cluster. Beneath it many fibrocytic processes were separated from the APUD cells by only the thickness of the basal lamina. Two fascicles of smooth muscle approached the cluster within 0.4-0.8 micron. Unmyelinated nerve fibers came as close but contacted only the muscle. Capillaries, in contrast, came no closer than 15 micron from the base of the body. Evidently, 1) fibrocytes and smooth muscle are more likely targets for secretions from such a paracrine body than cells reached through the blood-stream, and 2) not all small-granule cell clusters are innervated.

Pulmonary lesions induced by long-term exposure to ozone. II. Ultrastructure observations of proliferative and regressive lesions.

A tumor-resistant strain of 10-week-old Swiss-Webster mice was exposed to an ozone concentration of 2.5 ppm for two hours daily for 120 days. Groups of ten animals were killed periodically and the tracheobronchial tree was studied by light and electron microscopy. After the 45th day of exposure the trachea showed progressive metaplasia toward a squamoid epithelium containing many cytoplasmic secretory vesicles, with accompanying decrease in number of ciliated cells. Papillomatous proliferation of the bronchiolar epithelium occurred, together with abnormal cilia and micronodular hyperplasia of Clara-like cells. The tracheal and bronchiolar changes appeared to be reversible 120 days after the last exposure. However, micronodular hyperplasia of Clara-like cells remained unchanged in frequency and morphologic appearance.