Carbohydrate cytochemistry of rhesus monkey tracheal epithelium

Three types of nonciliated secretory epithelial cells contribute material to the mucous lining of pulmonary airways: mucous cells, serous cells, and Clara cells. Extensive interspecies variation exists, especially between humans and laboratory mammals, with regard to occurrence, distribution, and granule content of these secretory cells. This study was designed to characterize one aspect of these differences in one species of nonhuman primate, the rhesus monkey. The complex carbohydrates of secretory granules present in the tracheal epithelium were characterized cytochemically. The tracheas of seven monkeys were fixed by airway infusion, processed, and embedded for both light and transmission electron microscopy. Histochemical stains including Alcian blue-periodic acid Schiff, dialyzed iron, and high iron diamine-Alcian blue were applied to serial methacrylate sections. The mucous cells were the predominant secretory cell type of the trachea and contained periodate-reactive sulfated glycoconjugates. The mucous secretory granules, as resolved with the electron microscope, consisted of a mesh or matrix surrounding a biphasic core. The matrix was stained by all cytochemical reactions used, which included periodic acid-thiocarbohydrazide-silver proteinate, dialyzed iron, low iron diamine, and high iron diamine. The biphasic core also reacted with the four stains, but most intensely with high iron diamine. We conclude from this study that 1) the mucous secretory granule contains carbohydrate throughout all phases of the granule, 2) the mucous granule contains periodate-reactive sulfated glycoconjugates, with sulfate esters concentrated in the core of the granule, and 3) the mucous granules of rhesus trachea morphologically and cytochemically resemble those described in human airways.     

Substructure of granules from serous cells of porcine tracheal submucosal glands

The ultrastructure of serous cells from porcine tracheal submucosal glands was studied by conventional transmission electron microscopy (TEM), and by cytochemical methods to stain for complex carbohydrates. In tissue fixed and processed for TEM, and stained with uranyl acetate and lead citrate, the condensing granules of serous cells occasionally possessed a hexagonal and sometimes a lamellar substructure. Tissue fixed in paraformaldehyde-glutaraldehyde and stained with periodic acid-thiocarbohydrazide-silver proteinate (PTS) or with phosphotungstic acid (PTA) showed secretory granules stained for complex carbohydrates and revealed a substructure similar to that noted in the condensing granules. The dark staining substructure revealed by either the PTS or the PTA technique appeared to correspond to electron-lucent areas observed in the condensing granules by conventional TEM. The PTS staining probably demonstrated the presence of neutral glycoprotein, since the serous-cell granules did not react with a dialyzed iron stain for acidic glycoproteins. Treatment of periodic acid oxidized thin sections with pronase or pepsin prior to thiocarbohydrazide and silver proteinate treatment decreased the intensity of the PTS staining, but did not digest away any components of the granules. The substructure revealed by the carbohydrate stains may be a reflection of the mechanism of packaging or the macromolecular structure of the glycoproteins in the serous-cell granules.     

Substance P stimulates CFTR-dependent fluid secretion by mouse tracheal submucosal glands

The mucosa of the proximal airways defends itself and the lower airways from inhaled irritants such as capsaicinoids, allergens, and infections by several mechanisms. Sensory nerves monitor the luminal microenvironment and release the tachykinin substance P (SP) to stimulate mucus secretion. Here, we have studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in SP stimulation by comparing mouse airway submucosal gland responses in wild-type (WT) and CFTR−/− mice. Capsaicinoids (chili pepper oil) increased fluid secretion by glands from WT mice five-fold, and this response was abolished by exposing the basolateral aspect of the tracheas to L-732,138 (10 μmol/l), a specific antagonist of the neurokinin-1 receptor. Secretion was also stimulated 25-fold by basolateral application of SP, and this response was strongly inhibited by the CFTR inhibitor CFTR_inh172. In contrast, submucosal glands from CFTR knockout mice failed to secrete when stimulated by SP (1 μmol/l), although those from wild-type control littermates were responsive. SP stimulation of wild-type glands was also abolished by clotrimazole (25 μmol/l), a blocker of Ca²⁺-activated K⁺ channels. These results indicate that SP mediates local responses to capsaicinoids through a mechanism involving coordinated activation of CFTR and K⁺ channels. To our knowledge, this is the first study in which CFTR-dependent responses to substance P have been directly demonstrated. Since CFTR regulation is qualitatively similar in human and mouse glands, loss of this local regulation in CF may contribute to reduced innate defenses in CF airways.     

Unique radiosensitivity of serous cells in rhesus monkey submandibular glands.

The saliva of patients undergoing radiation therapy for head and neck cancer contains increased acidic mucosubstances associated with a reduced serous component. To assess the morphologic features of the acute radiation damage in serous versus mucous acinar cells, the mixed serous/mucous submandibular glands of 18 rhesus monkeys were studied 1-72 hours after irradiation with single doses of 2.5-15.0 Gy. Selective degeneration and necrosis of serous cells was observed with doses of 2.5-7.5 Gy. Doses of 10.0-15.0 Gy caused widespread destruction of whole serous acini, but only isolated mucous cells were affected. The lesions were clearly expressed by 24 hours. Transient exudation of neutrophils was replaced by plasma cells and lymphocytes. Examination at 16, 22 and at 40 weeks revealed that late atrophy was caused solely by loss of serous acini in glands treated with 7.5 and 10.0 Gy. Although both serous and mucous acini were reduced in glands treated with 12.5 and 15.0 Gy, the atrophy was mainly due to loss of serous acini. The finding that serous cells are more vulnerable to radiation injury than mucous cells provides a morphologic explanation for early and late changes in saliva composition after salivary gland irradiation.     

Intercellular growth factor signaling and the development of mouse tracheal submucosal glands.

To provide a genetic framework for investigating changes in airway submucosal gland function in human respiratory disease, we have investigated their counterparts in normal and mutant mice. We describe their morphogenesis in relation to the expression of genes encoding conserved intercellular signaling pathways. Submucosal glands are severely reduced in number and size in mice heterozygous for Fgf10. Glands are completely absent in mice lacking Ectodysplasin (Eda) and Edaradd (Eda receptor adaptor protein), members of the tumor necrosis (TNF) superfamily of signaling factors. Furthermore, components of the Eda and closely related pathways are transcribed throughout the respiratory system in the adult mouse. Finally, the temporal and spatial pattern of Bmp4 expression suggests that it may control submucosal gland development and homeostasis. Taken together, our observations have important implications for the better understanding of the submucosal gland remodeling that occurs in human respiratory disease.     

Differentiation of tracheal epithelium during fetal lung maturation in the rhesus monkey Macaca mulatta

Of the eight categories of epithelial cells identified in pulmonary conducting airways, four are found in the trachea of adult primates: basal, mucous goblet, intermediate, and ciliated cells. While their ultrastructure is well characterized, little is understood about their origin or differentiation. This study describes the pattern of differentiation of the tracheal luminal epithelium in a species of nonhuman primate, the rhesus monkey, Macaca mulatta. Tracheas of 57 fetal and postnatal rhesus were fixed with glutaraldehyde/paraformaldehyde: ten at 29–54 days gestational age (GA), ten at 59–80 days GA (pseudoglandular stage), sixteen at 82–130 days GA (canalicular stage), ten at 141–168 days GA (saccular stage), eight at 1–134 days postnatal, and three adults (2 yr 11 months to 11 yr 11 months). Slices taken proximal to the carina were processed for electron microscopy by a selective embedding procedure. In the youngest fetuses, essentially one population of cells lined the tracheal epithelial surface. These cells were columnar in shape with a central nucleus, few organelles, and large amounts of cytoplasmic glycogen. At 46 days GA, ciliated cells were observed on the membranous side of the trachea. Some nonciliated cells had concentrations of organelles in the most apical portion of their cytoplasm. At 59 days GA, membrane-bound cored granules were intermixed with organelles in the apices of some glycogen-filled cells. They were observed first on the cartilaginous side. Between 59 and 100 days GA, a large number of cell forms which appeared to be transitional between ciliated, secretory, basal, and undifferentiated cells were present. These included ciliated cells with electron-lucent inclusions resembling mucous granules. Mucous secretory cells were more numerous and had more granules and less glycogen in older fetuses. By 105 days GA, few of the secretory cells had significant amounts of glycogen and the cytoplasm was condensed. Secretory granules were very abundant in some cells and minimal in others. The Golgi apparatus was prominent. In animals 120 days GA and older, small mucous granule cells and basal cells resembling these cells in adults were present. By 134 days postnatal age, the epithelium resembled that in adults. We conclude that (1) most of the differentiation of tracheal epithelium in the rhesus monkey occurs prior to birth; (2) the cells differentiate in the following sequences: ciliated, mucous goblet, small mucous granule, basal; and (3) basal and small mucous granule cells do not play a role in ciliated and mucous cell formation in the fetus.     

Tracheal submucosal gland development in the rhesus monkey, Macaca mulatta: ultrastructure and histochemistry

Summary The submucosal glands are thought to be the primary source of the mucus overlying the primate trachea and conducting airways. This study characterizes the development of submucosal glands in the trachea of the rhesus monkey. Tracheas from 46 age-dated fetal, 8 postnatal and 3 adult rhesus were fixed in glutaraldehyde/paraformaldehyde and slices processed for electron microscopy. The earliest (70 days gestational age (DGA)) indication of gland development was the projection of a group of closely packed electron lucent cells with few organelles and small pockets of glycogen into the submucosa. This configuration was observed up to 110 DGA. In fetuses younger than 87 DGA it was present almost exclusively over cartilaginous areas. Between 80 and 140 DGA, a cylinder of electron lucent cells projected into the submucosal connective tissue perpendicular to the surface. In fetuses younger than 100 DGA, it was restricted to cartilaginous areas. By 90 DGA, some glycogen containing cells in proximal regions contained apical cored granules. By 106 DGA, cells in proximal areas contained apical electron lucent granules. More distal cells had abundant GER and electron dense granules. The most distal cells resembled the undifferentiated cells at younger ages. Ciliated cells were present in the most proximal portions of glands at 120 DGA. This glandular organization was found in older animals, including adults, with the following changes: (1) abundance of proximal cells with electron lucent granules increased; (2) abundance of distal cells with electron dense granules increased; and (3) abundance of distal cells with abundant glycogen and few organelles decreased. We conclude that submucosal gland development in the rhesus monkey: (1) is primarily a prenatal process; (2) occurs first over cartilage; (3) continues into the postnatal period; and (4) involves secretory cell maturation in a proximal to distal sequence with mucous cells differentiating before serous cells.     

Quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants.

The development of the tracheal submucosal glands has been determined quantitatively in 22 infants with cystic fibrosis and in 25 control infants, all under 4 months of age. In cross-sections of normal trachea significant relationships were found between postconceptional age (PCA) and gland area (P less than 0.001), submucosal area (P less than 0.02), tracheal airway diameter (P less than 0.05), and acinar diameter (P less than 0.001). In infants with cystic fibrosis the pattern of development was similar to that of the control infants. No statistically significant differences were found between three subgroups of infants with cystic fibrosis, which included those with meconium ileus with no lung infection, those with meconium ileus with lung infection, and those with lung infection and no history of meconium ileus. The normal pattern of development of tracheal submucosal glands in infants with cystic fibrosis was in contrast to the deficiency of normal maturation seen in the exocrine pancreas of these infants. The lumen fraction, an index of dilatation of acinar lumina, showed no significant relationship with PCA in either the control group or the group with cystic fibrosis. However, statistically significant dilatation of acini was observed in the tracheal submucosal glands of infants with cystic fibrosis (0.14, P less than 0.005).     

Carbohydrate histochemistry of rat respiratory glands

Cytochemical methods applied to examination of rat respiratory tract glands have revealed diversity of secretory complex carbohydrates. With the Alcian blue-periodic acid Schiff (AB-PAS) and the high iron diamine (HID) techniques at the light microscopic level, certain patterns of glycoprotein content were noted at various levels of the respiratory tract. Serous tubules and demilunes found in abundance in laryngeal and tracheal glands contained neutral glycoprotein. Mucous tubules found in abundance in epiglottal and laryngeal glands and in lesser number in tracheal glands most often produced sulfated glycoprotein. However, mucous tubules in epiglottal and tracheal glands contained a few cells with sialylated glycoprotein, and mucous tubules in some areas in laryngeal glands contained mainly cells producing sialylated glycoprotein. Mucous ducts found in abundance in lower laryngeal and tracheal glands formed mainly sialylated glycoprotein and contained infrequent cells with sulfated glycoprotein. The type of glycoprotein found in each cell type by light microscopy was confirmed at the ultrastructural level by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP), dialyzed iron (DI) and high iron diamine (HID) methods. Serous cell granules displayed light reactivity with the PA-TCH-SP method and no DI or HID affinity and were judged to contain sparse neutral glycoprotein. Serous granules disclosed negative central foci with the PA-TCH-SP method. Granules of most mucous tubule cells stained strongly with the PA-TCH-SP and HID procedures and contained abundant sulfated glycoprotein. Occasional mucous tubules stained with the PA-TCH-SP but not with the HID method and apparently corresponded with cells judged to form sialylated glycoprotein from their blue staining with the HID-AB sequence. Two zones within individual granules in some cells revealed different HID staining intensity and appeared to differ in the amount or kind of sulfated glycoprotein they contained. Some cells exhibited granule heterogeneity containing both HID-positive and unstained granules. Spherical cores present in granules of mucous tubules below the upper laryngeal level occasionally appeared bizonal and invariably lacked reactivity demonstrative of complex carbohydrate. Mucous duct cell granules stained heavily with the PA-TCH-SP and DI methods and reacted infrequently with the HID procedure and were considered generally to contain sialylated glycoprotein, and occasionally to form sulfated glycoprotein. The three carbohydrate stains distinguished a heavily and a moderately reactive zone in the cortex outside the monophasic or biphasic, carbohydrate-free cores in granules of some mucous duct cells.     

Cloning of rhesus monkey LILRs

Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.     

Liquid secretion properties of airway submucosal glands

The tracheobronchial submucosal glands secrete liquid that is important for hydrating airway surfaces, supporting mucociliary transport, and serving as a fluid matrix for numerous secreted macromolecules including the gel-forming mucins. This review details the essential structural elements of airway glands and summarizes what is currently known regarding the ion transport processes responsible for producing the liquid component of gland secretion. Liquid secretion most likely arises from serous cells and is principally under neural control with muscarinic agonists, substance P, and vasoactive intestinal peptide (VIP) functioning as effective secretogogues. Liquid secretion is driven by the active transepithelial secretion of both Cl⁻ and HCO₃⁻ and at least a portion of this process is mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), which is highly expressed in glands. The potential role of submucosal glands in cystic fibrosis lung disease is discussed.     

Acute and late radiation injury in rhesus monkey parotid glands. Evidence of interphase cell death.

Acute and chronic salivary gland dysfunction are common sequelae of radiotherapy for head and neck cancer; but the associated morphologic changes, especially of the acute damage, have received relatively little study. For investigation of the morphologic characteristics of acute radiation injury to parotid glands, rhesus monkeys were studied 1-72 hours after parotid irradiation with single doses of 2.5-15.0 Gy. The acute damage from all doses was clearly expressed by 24 hours. Histologically, parotid glands irradiated with 2.5 or 5.0 Gy had random degeneration and necrosis of the serous acinar cells. Doses of 7.5-15.0 Gy produced widespread degeneration along with necrosis of whole acini. Serous cell damage was accompanied by neutrophilic inflammation that subsided after 24 hours to become replaced by plasma cell and lymphocytic infiltrates. Parotid glands receiving 7.5-15.0 Gy were atrophic at 16-22 weeks after irradiation and showed no recovery by 40 weeks. Although parotid acinar cells are well-differentiated nondividing cells, these observations show that they express lethal radiation injury in interphase within hours of receiving a radiation dose as low as 2.5 Gy. This is unlike most mammalian cells that express radiation injury during mitosis. Chronic atrophy is a consequence of this direct, irreversible, and early injury, rather than the result of radiation-induced changes in the vasculature.     

Re-engineering primary epithelial cells from rhesus monkey parotid glands for use in developing an artificial salivary gland

There is no satisfactory conventional treatment for patients who experience irreversible salivary gland damage after therapeutic radiation for head and neck cancer or because of Sj?gren's syndrome. Additionally, if most parenchyma is lost, these patients also are not candidates for evolving gene transfer strategies. To help such patients, several years ago we began to develop an artificial salivary gland. In the present study, we used a non-human primate tissue source, parotid glands from rhesus monkeys, to obtain potential autologous graft cells for development of a prototype device for in situ testing. Herein, we present 3 major findings. First, we show that primary cultures of rhesus parotid gland (RPG) cells are capable of attaining a polarized orientation, with Na(+)/K(+)-adenosine triphosphatase, zonula occludens-1, and claudin-1 distributed in specific domains appropriate for epithelial cells. Second, we show that RPG cells exhibit 2 essential epithelial functions required for graft cells in an artificial salivary gland device (i.e., an effective barrier to paracellular water flow and the generation of a moderate transepithelial electrical resistance). Third, we show that RPG cells can express functional water channels, capable of mediating directional fluid movement, after transduction by adenoviral and adeno-associated virus type 2 vectors. Together these results demonstrate that it is feasible to individually prepare RPG cells for eventual use in a prototype artificial salivary gland.     

Spontaneous rhabdomyosarcoma in a rhesus monkey

In an adult male rhesus monkey, a large pelvic mass causing lysis of the ilium and destruction of pelvic musculature was diagnosed as a spontaneous rhabdomyosarcoma by the use of histological, ultrastructural and immunohistochemical techniques.