Effect of epidermal growth factor on the fetal development of the tracheobronchial secretory apparatus in rhesus monkey.

Fetal rhesus monkeys were treated with recombinant human epidermal growth factor (EGF) to determine if EGF can induce maturation of the tracheobronchial secretory apparatus. At 75% of gestation, EGF was administered simultaneously into both the amniotic fluid and fetal abdominal cavity at an average dose of 66 micrograms/kg body weight, by each route over a 7-d period. At the end of the treatment period, the fetuses were delivered and either euthanized immediately or after maintenance on ventilatory support for 6 h. The lungs were removed, and the trachea and one lobe of the right lung was fixed and embedded for light microscopy. The left lung was lavaged with saline, and the collected fluid was used to quantify released secretory product. Secretory product was also measured in amniotic fluid as well as in situ on histologic sections of tracheal epithelium. When the tracheas of EGF-treated monkeys were examined, the epithelium was found to be taller and to contain a greater proportion of secretory cells and a smaller proportion of intermediate cells than the control group. However, there was no significant change in the total number of cells per millimeter of basal lamina or in the proportion of the epithelial population made up of basal or ciliated cells. There was more secretory product stored in the epithelium and submucosal glands and increased quantities of respiratory secretions in both lung lavage and amniotic fluid of EGF-treated monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical demonstration of cytochrome P-450 monooxygenase in Clara cells throughout the tracheobronchial airways of the rabbit.

The nonciliated bronchiolar epithelial (or Clara) cell is considered the primary pulmonary site of cytochrome P-450-dependent monooxygenase activity. Despite the general conception that Clara cells are restricted to bronchioles, we have previously shown that a nonciliated cell with the cytological features of the Clara cell predominates throughout rabbit tracheobronchial airways. The present study was designed to determine if the cytochrome P-450 monooxygenase system has the same distribution. Trachea, terminal bronchioles and 3 generations of bronchi were selected by microdissection from fixed lungs of adult specific-pathogen-free rabbits. Serial sections of paraffin-embedded tissue were stained with either Alcian blue/periodic acid Schiff's (AB/PAS) or with antisera to one of the following: cytochrome P-450 form 2, form 5, or NADPH-dependent cytochrome P-450 reductase. The majority of nonciliated epithelial cells lining all 5 airway generations were PAS+ and AB-. Nonciliated cells in all 5 airway generations reacted positively with all three antisera. The primary deposition site was the apical portion of nonciliated cells. Other sites included ciliated surfaces and vascular endothelium. Reaction products from all three antisera had the same localization pattern. We conclude that (1) the cytochrome P-450 monooxygenase system is distributed throughout the tracheobronchial airways of the rabbit and (2) the Clara cells of the trachea and bronchi are functionally, as well as structurally, similar to those of the bronchioles.

Carbohydrate cytochemistry of rhesus monkey tracheal submucosal glands.

This study was designed to characterize the ultrastructure and carbohydrate content of secretory cells in submucosal glands of rhesus monkey and to compare this information with that available for humans. The tracheas from five adult monkeys were fixed by airway infusion, processed, and embedded for both light and transmission electron microscopy. Histochemical strains including alcian blue-periodic acid-Schiff, dialyzed iron, and high-iron diamine-alcian blue were applied to serial glycol methacrylate sections. The cytochemical stains used included periodic acid-thiocarbohydrazide-silver proteinate, high-iron diamine, and low-iron diamine. The glandular secretory cells were divided into four categories based on ultrastructure and location within the gland. Cells in the first category resembled the mucous cell of the surface epithelium and were located in ducts most proximal to the tracheal lumen. The second category consisted of cells that were located in distal ducts and contained large electron-lucent granules. The granules in both of these cell groups contained material that was periodate-reactive and sulfated. Cells of the third category contained granules that were either electron-lucent or electron-dense. These cells, which were difficult to characterize as either serous or mucous, were located in secretory tubules and acini and contained periodate-reactive glycoconjugates that were either sulfated or nonsulfated. The last category consisted mainly of cells that contained electron-dense granules that were lightly periodate-reactive or a few that were unreactive with any of the cytochemical methods used here.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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: abundance of proximal cells with electron lucent granules increased; abundance of distal cells with electron dense granules increased; and abundance of distal cells with abundant glycogen and few organelles decreased. We conclude that submucosal gland development in the rhesus monkey: is primarily a prenatal process; occurs first over cartilage; continues into the postnatal period; and involves secretory cell maturation in a proximal to distal sequence with mucous cells differentiating before serous cells.

An immunohistochemical characterization of rhesus monkey respiratory secretions using monoclonal antibodies.

A series of 12 monoclonal antibodies was made against mucous and serous components of rhesus monkey trachea. These antibodies were used to localize secretory products in the respiratory epithelium by immunohistochemical methods. Mucous cells of the tracheal surface epithelium and submucosal glands were shown by histochemical methods to be a homogeneous population containing periodate-reactive sulfated glycoconjugates. Immunohistochemical staining using the monoclonal antibodies on glycol methacrylate sections serial to those stained histochemically revealed a more antigenically heterogeneous population of secretory cells. Four distinct staining patterns were observed with the 12 monoclonal antibodies. Eight antibodies reacted with most, but not all, mucous and serous cells. Two antibodies recognized subpopulations of secretory cells. One antibody stained tracheal mucous cells with a concentration of reaction product on the luminal border, and one antibody stained only serous cells within the glands. Ten of the 12 antibodies were shown to react in an ELISA with the high molecular weight void volume containing mucous glycoproteins separated on a Sepharose CL-4B gel filtration column. None of the antibodies recognized blood group antigens. We conclude: (1) that production of a large panel of monoclonal antibodies to respiratory tract mucins from primates is feasible, (2) that the monoclonal antibodies will recognize epitopes on biochemically identifiable glycoproteins, and (3) that the intracellular mucous products of tracheal secretory cells exhibit greater heterogeneity than is detectable by conventional histochemistry.