Neuroendocrine cells of the human lung express substance-P-like immunoreactivity.

The occurrence of substance P (SP) in the neuroendocrine population of human lungs was investigated by immunohistochemical methods. All individuals studied (n = 16) had SP-like immunoreactive cells, being more numerous in lungs of fetuses and newborn infants than in adults. These cells, both solitary and forming neuroepithelial bodies, were found at all levels of the respiratory mucosa. Solitary neuroendocrine cells and neuroepithelial bodies were found in the bronchial and bronchiolar mucosa, while at the alveolar level neuroepithelial bodies were also seen. A more intense SP-like immunoreactivity was found in the basal cytoplasm of these cells. Occasionally they show cytoplasmic prolongations which interdigitate with neighboring epithelial cells. These facts suggest that SP-like immunoreactive cells may have a paracrine or local secretion function, acting over surrounding epithelial cells or structures situated in the lamina propria. The evidence of great numbers of SP-like immunoreactive neuroendocrine cells in fetuses and infants might be the expression of a functional role of SP in lung development.     

Identification of the endocrine cells detected by the monoclonal antibody HISL-19 in human tissues.

The human endocrine cells reacting with the monoclonal antibody HISL-19 were identified with hormone antisera of proven specificity using a double immunostaining procedure. The epitope for HISL-19 was found in all types of pituitary cells except ACTH cells, in thyroid C cells, in all types of adrenal medullary and pancreatic islet cells and in somatostatin and pancreatic polypeptide cells of the gastrointestinal mucosa. No staining was found in parathyroid cells and in most gastrointestinal endocrine cells. Either paranuclear focal accumulation or diffuse cytoplasmic distribution of immunoreactive material were found. The spectrum of HISL-19 immunoreactive cells was found to be only in part complementary to that of cells immunoreactive for chromogranin A. Thus, it is concluded that the monoclonal antibody HISL-19 is a useful addition to other immunohistochemical markers for the detection of cells showing neuroendocrine features.     

“Tasting” the airway lining fluid

Specialized epithelial cells of the respiratory tract have been termed "solitary chemosensory cells" based upon the expression of components of the canonical sweet, umami and bitter taste transduction pathway, or "brush cells" based upon their characteristic morphological feature, i.e. an apical, brush-like tuft of rigid, villin containing microvilli. Cells defined by these criteria might not match one-to-one, and a generally accepted terminology is still lacking. With respect to cellular shape, ultrastructure, expression of elements of the taste transduction cascade, innervation and synapse formation, and effects evoked upon their stimulation, it appears that chemosensory/brush in the upper respiratory tract (nasal respiratory mucosa, vomeronasal duct, auditory tube), in the olfactory mucosa, in the larynx, in the lower airways (trachea, bronchi) and in the alveolar region (rat only) each represent distinct groups. Still, they have in common to monitor the chemical composition of the mucosal lining fluid. They serve as sentinels detecting bacterial colonization or the presence of other harmful components in the mucosal lining fluid, leading to the initiation of avoidance reflexes and/or local defense mechanisms which are adapted to their anatomical localization. Free nerve endings are also responsive to inhaled irritants and further work will be needed to discriminate between the contributions of such nerve endings and chemosensory cells in chemical monitoring and defense initiation. Interestingly, there is first emerging evidence that respiratory chemosensory cells may respond to more than one canonical taste quality so that they, in analogy to polymodal nociceptors, may serve as polymodal chemosensors of potentially dangerous signals.     

Morphology and chemical characteristics of subepithelial laminar nerve endings in the rat epiglottic mucosa

The laminar nerve endings are distributed in the laryngeal mucosa, and described as sensory receptors evoked by laryngeal pressure changes. The present study aimed to determine detailed morphological characteristics of the laryngeal laminar endings of the rat. Immunohistochemistry for Na(+)-K(+)-ATPase, α(3) subunit, showed that laminar endings were distributed in the entire laryngeal surface of the epiglottis. The parent axons of the endings were thick in diameter, and they were branched and continued to the endings. In some cases, several endings from different parent axons fused into a large complex structure of 500 μm in width. The laminar endings were also immunoreactive for vesicular glutamate transporter 1 (vGLUT1) and vGLUT2, but not for P2X(3) purinoceptor. Around the laminar endings, terminal Schwann cells with immunoreactivity for S-100 protein were closely associated with axon terminals. Use of scanning electron microscopy with alkaline maceration method showed that the terminal Schwann cells consisted of a rounded perinuclear region and lamellar cytoplasmic processes. Ultrastructurally, axon terminals with numerous mitochondria were partly covered with Schwann cell sheath, and some terminals intruded into the epithelial layer. Clear vesicles of 50 nm in diameter were also observed especially in small cytoplasmic processes of 400 nm to 1 μm in size. The results in the present study suggested that the laminar endings in epiglottic mucosa have morphological characteristics of slowly adapting mechanoreceptors and contribute to sensation of laryngeal pressure via mucosal tension.     

Immunohistochemical localization of some endocrine cells in the gastroenteropancreatic system of Erinaceus europaeus

The distribution of chromogranin A and neuron specific enolase (NSE) in the neuroendocrine gut system and the morphology and distribution of cells containing gastrin, somatostatin, neurotensin and VIP in the gastroenteropacreatic (GEP) apparatus of Erinaceus europaeus were investigated by immunohistochemical methods. Chromogranin A and somatostatin immunoreactive cells were present throughout the gastrointestinal mucosa, with the exception of the oesophagus and in the pancreas. Gastrin cells were peculiar of the pyloric glands and duodenal mucosa and neurotensin cells of the small intestine. No VIP immunoreactive endocrine cells were noticed in the GEP system. VIP and NSE immunoreactivities were detected both in nerve cell bodies and terminals of the wall of the GEP apparatus. NSE immunoreactivity was found in the endocrine cells of the fundic and pyloric mucosa.     

Immunocytochemical characterization of two types of microvillar cells in rodent olfactory epithelium

Microvillar cells (MCs) have been identified in the olfactory epithelium of various mammalian species from rodents to humans. Studies on properties and functions of MCs to date have yielded partially controversial results, supporting alternatively an epithelial or a neuronal nature of these cells. In the present study, single and double immunolabeling investigations were carried out using antibodies against cytoskeletal and integral membrane proteins in order to further characterize MCs in rat and mouse olfactory epithelium. Application of antibodies against ankyrin (ANK), a protein that links integral membrane proteins to the submembrane cytoskeleton, led to intense labeling of the basolateral membranes of numerous cells with characteristic MC morphology. ANK-immunoreactive (ir) cells bore an apical tuft of -actin-ir microvilli, were filled with cytokeratin 18 (CK18)-ir filamentous network, and extended a basal process that appeared to end above the basal membrane. Immunoreactions for villin, an actin-crosslinking protein particularly prominently expressed in brush cells in the gastrointestinal and respiratory tract epithelia, and for the -subunit of sodium-potassium ATPase (Na⁺, K⁺-ATPase), revealed that ANK-ir MCs fall into two subpopulations. The less frequent type I MCs displayed villin immunoreactivity in their apical microvilli and underneath the basolateral membranes; the more numerous type II MCs were negative for villin but possessed intense basolateral immunoreactivity for Na⁺, K⁺-ATPase. Strong reactivity for the epithelial-type integral membrane protein of adherens junctions, E-Cadherin, was localized in basolateral membranes of both types of MCs. Our results support an epithelial nature of ANK-ir MCs in rat and mouse olfactory epithelium. Type I MCs strongly resemble brush cells in their immunocytochemical characteristics, namely, their ANK reactivity, CK18 reactivity, and villin reactivity. The intense Na⁺, K⁺-ATPase reactivity of type II MCs implicates these cells in transport processes.     

Cytoskeletal markers allowing discrimination between brush cells and other epithelial cells of the gut including enteroendocrine cells

Brush cells are specialised epithelial cells scattered throughout the simple epithelia of the respiratory and alimentary tracts. These cells have been suggested to serve a still unknown receptive function and use nitric oxide as a gaseous messenger molecule. At the light microscope level, brush cells can be identified by antibodies against the actin filament crosslinking proteins villin and fimbrin that not only stain the apical tuft of microvilli and their rootlets, but also label projections emanating from the basolateral surface of these cells. Since brush cells contain numerous intermediate filaments and microtubules and display a complicated basolateral cell morphology, we tested in this study whether antibodies against cytokeratin, tubulin and components of the membrane cytoskeleton might provide further markers for these cells at the light microscope level. Here we show that brush cells (identified by villin antibodies) can be discriminated from the neighbouring simple epithelium of the stomach, pancreatic duct and duodenum by particularly strong immunoreactivity with antibodies specific for cytokeratin 18. Tubulin antibodies reacted strongly with the upper half of brush cells in a pattern not observed in the other epithelial cells of these tissues, including enteroendocrine cells of the duodenum. Ankyrin, a protein that links the spectrin-based membrane cytoskeleton to integral proteins of the plasma membrane was revealed as a third cytoskeleton-associated protein, prominently expressed in brush cells where ankyrin is restricted to the basolateral membrane domain. The apparently high concentration of cytokeratin 18, tubulin and ankyrin in brush cells suggests that these cytoskeletal proteins might play a role in the mechanical stability and polarised organisation of these putative receptor cells.Dedicated to Prof. Dr. Drs. h.c. Andreas Oksche on the occasion of his 70th birthday     

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.     

Identification of cytoskeletal markers for the different microvilli and cell types of the rat vomeronasal sensory epithelium

The vomeronasal organ (VNO) of the mammal nose is specialized to detect pheromones. The presumed site of the chemosensory signal transduction of pheromones is the vomeronasal brush border of the VNO sensory epithelium, which has been shown to contain two different sets of microvilli: (i) the tall microvilli of supporting cells and (ii) the short microvilli of the chemoreceptive VNO neurons that branch and intermingle with the basal portions of the longer supporting cell microvilli. A key problem when studying the subcellular distribution of possible VNO signal transduction molecules at the light microscope level is the clear discrimination of immunosignals derived from dendritic microvilli of the VNO neurons and surrounding supporting cell structures. In the present study we therefore looked for cytoskeletal marker proteins, that might help to distinguish at the light microscope level between the two sets of microvilli. By immunostaining we found that the VNO dendritic microvilli can be selectively labelled with antibodies to the calcium-sensitive actin filament-bundling protein villin, whereas supporting cell microvilli contain the actin filament cross-linking protein fimbrin, but not villin. Useful cytoplasmic marker molecules for cellular discrimination were cytokeratin 18 for supporting cells and β-tubulin for dendrites of VNO neurons. A further finding was that the non-sensory epithelium of the rat VNO contains brush cells, a cell type that appears to be involved in certain aspects of chemoreception in the gut. Brush cells or other structures of the vomeronasal brush border did not contain α-gustducin.     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium

A quantitative immunohistochemical study was performed of the distribution of protein gene product 9.5 (PGP, a soluble protein localized in neurons and neuroendocrine cells as well as in some non-nervous cells) and ubiquitin along the rat epididymis. In the ductuli efferentes, PGP immunoreaction was observed in the whole cytoplasm of some columnar cells; a smaller number of columnar cells showed ubiquitin immunoreactivity with limited apical and basal cytoplasmic localization. In the proximal caput epididymidis, the whole cytoplasm of all columnar cells showed PGP immunoreactivity, ubiquitin immunostaining was negative in this region. In the middle and distal caput epididymidis and the distal cauda, the apical cytoplasm of some columnar cells and the whole cytoplasm of some basal cells showed immunoreactivity to PGP. In these regions, immunoreactivity to ubiquitin was positive in the supranuclear cytoplasm of some columnar cells but not in the basal cells. No immunoreactivity to PGP or ubiquitin was detected in the corpus epididymis and the proximal cauda. Double immunostaining revealed that all the epididymal ubiquitin immunoreactive cells were also PGP immunoreactive, whereas most PGP immunoreactive cells did not immunoreact to ubiquitin. In ubiquitin-PGP immunoreactive cells, the site of the PGP immunoreaction differed from that of the ubiquitin immunoreaction. PGP-ubiquitin immunoreactive cells also seemed to be immunoreactive to anti-AE1/AE3 keratin antibodies. The spermatozoal heads were immunoreactive to PGP antibodies in the epididymal regions from proximal caput to distal cauda but not in the ductuli efferentes. The findings suggest that non-ubiquitinated PGP immunoreactive proteins are secreted in the epididymis, mainly in the proximal caput, and attach to spermatozoa.     

T1R3 is expressed in brush cells and ghrelin-producing cells of murine stomach

Various digestive and enteroendocrine signaling processes are constantly being adapted to the chemical composition and quantity of the chyme contained in the diverse compartments of the gastrointestinal tract. The chemosensory monitoring that underlies the adaptive capacity of the gut is thought to be performed by so-called brush cells that share morphological and molecular features with gustatory sensory cells. A substantial population of brush cells is localized in the gastric mucosa. However, no chemosensory receptors have been found to be expressed in these cells so far, challenging the concept that they serve a chemosensory function. The canonical chemoreceptors for the detection of macronutrients are taste receptors belonging to the T1R family; these have been identified in several tissues in addition to the gustatory system including the small intestine. We demonstrate the expression of the T1R subtype T1R3, which is essential for the detection of both sugars and amino acids in the gustatory system, in two distinct cell populations of the gastric mucosa. One population corresponds to open-type brush cells, emphasizing the notion that they are a chemosensory cell type; T1R3 immunoreactivity in these cells is restricted to the apical cell pole, which might provide the basis for the detection of luminal macronutrient compounds. The second gastric T1R3-positive population consists of closed-type endocrine cells that produce ghrelin. This finding suggests that ghrelin-releasing cells, which lack access to the stomach lumen, might receive chemosensory input from macronutrients in the circulation via T1R3.     

Immunohistochemical Studies of the Cellular Changes in the Peripheral Olfactory System After Zinc Sulfate Nasal Irrigation

The peripheral olfactory system has a remarkable capacity for repair. We have performed an immunohistochemical study of the cellular changes that occur after zinc sulfate irrigation of the nasal cavity. The rapid loss of epithelial cells was followed by the proliferation of basal cells and the restoration of the epithelium with olfactory tissue. Horizontal basal cell markers, anti-cytokeratin 5/6 (CK5/6), and the Bandeiraea simplicifolia (BS-1) lectin initially co-localized on day 1 after treatment but rapidly displayed a disparity in their staining profile, with CK5/6 immunoreactive cells having a profile more akin to cells expressing the sustentacular marker cytokeratin 18 (CK18). This suggests CK5/6 and BS-1 label a different subset of horizontal basal cells. Axonal degeneration and regeneration was studied with a panel of markers to olfactory receptor neurons, their terminals, and olfactory bulb dendrites. The glial cells of the peripheral olfactory system, olfactory ensheathing cells, remained in position, with little change in immunoreactivity to laminin, although an increase in the expression of glial fibrillary acidic protein was observed. The events and the extent of reconstitution of the olfactory system after degeneration serves as a foundation for future studies designed to understand the unique regenerative capacity of the olfactory system.     

Laryngeal endocrine cells: topographic distribution and adaptation to chronic hypercapnic hypoxia

The morphology, topographic distribution, effects of denervation, and exposure to hypercapnic hypoxia of endocrine cells were examined in rat larynx. The endocrine cells, which were immunoreactive for protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP), were observed within the epithelial layer of the laryngeal cavity and in the laryngeal gland, while solitary endocrine cells with apical and/or basal cytoplasmic processes appeared near the glottis. After denervation of the left cervical vagosympathetic trunk and the superior laryngeal nerve, the number of mucosal endocrine cells in the denervated side was not significantly different from that in the intact side. After exposure to hypercapnic hypoxia for 3 months, the number of endocrine cells with PGP 9.5 and CGRP was markedly increased. In conclusion, the secretion of laryngeal endocrine cells may be stimulated by CO2 rather than O2. Furthermore, the endocrine cells and the sensory and autonomic nervous system may regulate each other by an axon reflex mechanism. Endocrine cells appear to play a very important role in the local regulation of the laryngeal mucosa.     

Neuroendocrine lung structures and tumours: immunohistochemical study by specific markers

Out of 360 lungs or lobes surgically removed, 13 non neoplastic specimens and 16 neuroendocrine (NE) tumours are investigated with immunohistochemical methods, in order to evaluate the presence of NE structures in normal and pathological human lungs. The markers used are neuron specific enolase (NSE), chromogranin (CG) and the 80 kd antigen (80 kdAg) of NE secretory granules detected by the new monoclonal Phe-5 antibody. In non-neoplastic lung specimens, clearcut immunoreactivity for all three markers appears in NE cells, neuroepithelial bodies (NEB), NE cell-hyperplasias and dysplasias. In the same specimens 4 tumourlets with analogous clearcut immunoreactivities were also observed. The NE tumours show distinct immunoreactivity for all three antisera in the 8 well differentiated cases. The 8 poorly differentiated tumours are variably immunoreactive for NSE and present low to nil staining with antisera to CG and 80 kdAg. The immunohistochemical data are interpreted according to current views about a possible relationship between NE tumours and parent normal NE lung structures.     

Structural and compositional analysis of early stages in microvillus assembly in the enterocyte of the chick embryo

Morphological and immunocytochemical techniques were used to examine the distribution of villin, with respect to actin, during the early events of brush border morphogenesis in the embryonic chicken intestine. Immunolocalization studies indicate that actin and villin exist as a cortical array in the apical domain of embryonic enterocytes at a time when few surface microvilli are visible by scanning and transmission electron microscopic techniques. A population of villin is also localized at the level of the junctional complex. With time, the density of microvilli increases and the cells begin to flatten. In these cells, villin is detected in the newly formed microvilli and also in the subjacent cortex, where microvillar rootlets are beginning to appear. The significance of actin-villin associations in the process of brush border assembly is discussed in the light of the functional properties of villin.     

Adaptations to the air breathing in the posterior intestine of the catfish (Corydoras aeneus,Callichthyidae). A histological and ultrastructural study

A light and transmission electron microscopic study of the intestine of catfish C. aeneus shows that the anterior part of the intestine is a site of digestion and absorption and its structure is typical of that of other teleostean fishes. However, in this species the thin-walled posterior intestine is adapted to air breathing. In this region mucosa is smooth and lined with respiratory epithelium with capillary network. Several types of cells are observed in the epithelium: flattened respiratory epithelial cells with short microvili, goblet cells, scarce epithelial cells with numerous longer microvilli, and two types of endocrine cells (EC). The solitary brush cells with several long and thick microvilli described here are the first observation of such cells in the gastrointestinal tract of fishes. Bodies of respiratory epithelial cells lie between capillaries. Their cytoplasm, apart from typical organelles contains dense and lamellar bodies, which are a site of accumulation of surfactant. In regions where capillaries are covered by thin cytoplasmic sheets of respiratory epithelial cells, a thin (0.24-3.00 microm) air-blood barrier is formed, thus enabling gas exchange. Epithelial cells with longer microvilli do not participate in the formation of the air-blood barrier and are probably responsible for absorbtion. EC of the closed type are dispersed within the epithelium. Their cytoplasm contains characteristic round or oval dense core vesicles 69 to 230 nm in diameter. The role of EC and brush cells in the regulation of processes related to absorbtion, and to respiration, is disscused.     

Villin as a structural marker to study the assembly of the brush border

Summary Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocarcinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border.Villin is one of the actin binding proteins found in microvilli which compose brush borders. Villin is expressed in the adult and in the embryo before the appearance of the brush border. Villin can be used as a tissue-specific marker for normal diffentiated and undifferentiated cells derived from gastrointestinal tractus in the adult as well as in the embryo. Since villin is a good marker for intestinal cells and plays a structural role in the assembly of the brush border we have analysed its expression and its localization in HT 29 cells. In HT 29 cells, as in the tissue, villin is synthesized at low levels before the appearance of the brush border. The high rate of synthesis and the recruitement of villin at the apical pole of the cells can be correlated with the existence of a well developed brush border.