Jon A. Hotchkiss, Ph.D.
Department of Veterinary Pathology, College of Veterinary Medicine
Michigan State University, MI 48824-1302
The mucosal epithelium lining the upper and lower conducting airways provides a barrier against injury from inhaled toxicants, bacteria, and other biogenic agents (organic dusts, pollen, mold spores, etc.). The normal respiratory epithelium is coated with mucus which lubricates, insulates, and humidifies the epithelium and protects it by entrapping bacteria and other particulates for removal by mucociliary clearance. This review will briefly examine interspecies similarities and differences in upper (nasal) airway structure, the mucous apparatus, and alterations in mucous secretion in response to inhaled toxicants.
The nose is the primary point of entry for inhaled air entering the respiratory tract in mammals, therefore, the mucosal epithelium lining the nasopharyngeal airways are exposed to the highest concentrations of inhaled allergens, toxicants and particulate matter. The nose is a structurally and functionally complex organ that not only serves as the principal organ of olfaction, but also filters, warms, and humidifies the inhaled air prior to entry into the lung. The nose has been characterized as the "scrubbing tower" of the lower airways and lung (Brain, 1970). The nasal mucosa has been shown to metabolize and detoxify many inhaled compounds (Dahl and Hadley, 1991), however, reactive metabolites formed during metabolism may result in site-specific injury within the nasal airways. There is increasing interest in the study of nasal toxicology and in assessing the human risk of nasal injury from inhaled toxicants (Miller, 1995).
There are striking interspecies differences in nasal architecture (Harkema, 1991). Humans have relatively simple noses with three rudimentary turbinates (the superior, middle, and inferior). Turbinates are bony structures lined by a well-vascularized mucosal epithelium which project into the airway lumen from the lateral walls of the left and right nasal air passages. In contrast, the turbinates in most non-primate species (i.e., dog, rat, mouse, rabbit) have complex folding and branching patterns. Marked differences in nasal airflow patterns among mammalian species are primarily due to variation in shape of the nasal turbinates. The nasal and oral cavities of humans and some non-human primates are configured in a manner that allows both nasal and oronasal breathing. This is in contrast to laboratory rodents (e.g. rats, mice, hamsters, guinea pigs) and horses which are obligate nose breathers due to the close apposition of the epiglottis to the soft palate.
Cellular Composition of Nasal Airways
Although there are differences in size, architecture, and airflow among different species, there are four distinct epithelial cell populations that are present in most mammalian species. The squamous epithelium is primarily restricted to the nasal vestibule; ciliated respiratory epithelium is found in the main nasal chamber and nasopharynx; a non-ciliated transitional epithelium is found between the squamous and the respiratory epithelium in the anterior nasal cavity; and a neuroepithelial olfactory epithelium lines the dorsal and dorsoposterior regions of the nasal cavity.
Olfactory Epithelium. The percentage of the nasal airway lined by olfactory epithelium varies greatly between species (Gross et al., 1982; Sorokin, 1988). The olfactory epithelium is composed of sensory cells, sustentacular cells, and basal cells. The olfactory sensory cells are bipolar neurons interspersed between the sustentacular cells. Unlike other neurons in the body the olfactory sensory cells can regenerate. Basal cells are considered to be the progenitor cells which regenerate the olfactory epithelium following injury. Sustentacular cells form a supporting network for the sensory cells and have abundant smooth endoplasmic reticulum and xenobiotic-metabolizing enzymes which are important in detoxification of inhaled xenobiotics and in olfaction (Dahl and Hadley, 1991). Located within the lamina propria beneath the olfactory epithelium are Bowman's glands. These are simple tubular glands composed of small acini. Ducts from these glands extend through the olfactory epithelium to the luminal surface. These glands contain large amounts of neutral and acidic mucosubstances that contribute to the mucus layer covering the lumenal surface of the olfactory epithelium.
Squamous Epithelium. The naris and nasal vestibule is lined by a lightly keratinized, stratified, squamous epithelium. This region of the nasal mucosa probably functions like the epidermis of the skin to protect the underlying tissues from potentially harmful inhaled materials.
Nasal Respiratory Epithelium. The majority of the nonolfactory epithelium lining the nasal airways of laboratory animals and humans is ciliated respiratory epithelium. The pseudostratified columnar nasal respiratory epithelium is composed of six morphologically distinct cell types: mucous, ciliated, non-ciliated columnar, cuboidal, brush, and basal (Pop and Martin, 1984). These cells are unevenly distributed along the mucosal surface. In the normal rat, mucous (goblet) secretory cells are primarily located in respiratory epithelium lining the proximal nasal septum. Mucous cells are the primary source of high molecular weight mucin glycoproteins; principal components of airway mucus. Serous cells are the primary secretory cells in the remainder of the respiratory epithelium in rodents. They contribute to airway mucus by secreting proteoglycans, lysozyme, lactoferrin, defensins, and protease inhibitors into the airway lumen. (reviewed in Fahy, 1998).
Transitional Epithelium. Distal to the squamous epithelium and proximal to the respiratory epithelium is a narrow zone of non-ciliated epithelial cells with microvilli covering their apical (lumenal) surface, which has been termed the nasal transitional epithelium. Similarities in this nasal epithelium among all laboratory animal species and humans include: 1) the anatomic location in the anterior nasal cavity between the squamous and respiratory epithelium; 2) the presence on non-ciliated cuboidal or columnar surface cells and basal cells; and 3) few or no mucous (goblet) cells with little stored intraepithelial mucosubstances. Exposure to high ambient concentrations of ozone can cause a rapid appearance of mucous cells (i.e., mucous cell metaplasia) in the transitional epithelium of both monkeys and rodents.
The Nasal Mucociliary Apparatus
The nasal mucociliary apparatus can be defined as a functional unit that includes the secretory cells, their progenitors, and the associated biomolecular machinery that is required to synthesize and secrete the principle components of the mucus blanket which lines and protects nasal airways. Airway mucus consists of two layers; a low-viscoelasticity periciliary layer that envelops the cilia, and a more viscous layer (gel phase) which rides on top of the periciliary layer. Maintenance of a protective mucus layer is a dynamic process that involves specific (mucin) gene expression, the production, storage and secretion of mucins into the airway lumen, and its clearance via ciliated cells lining the airways. Nasal secretions are derived from anterior nasal glands, seromucous submucosal glands, serous and mucous secretory cells of the surface epithelium and exudation from blood vessels. The synchronized beating of surface cilia propel the mucus at different speeds and directions depending on the intranasal location. In rats the mucus covering the olfactory epithelium moves very slowly while that covering the transitional and respiratory epithelium is moved along at between 1 and 30 mm/min (Morgan et al., 1984). The response of the nasal mucociliary apparatus to inhaled xenobiotics can be a sensitive indicator of injury (Morgan et al., 1986).
Mucin Structure and Function
Epithelial mucins can be subdivided into secretory and membrane-associated forms. The secretory forms are synthesized, stored, and then released by exocytosis from the apical surface of secretory cells to help form the overlying mucus gel. In contrast, the membrane associated forms have a hydrophobic membrane-spanning domain that serves to anchor them to the plasma membrane. They have not been reported to form oligomeric complexes but have been detected as soluble monomers in tissue culture supernatants and bodily fluids. The development of immunochemical reagents directed to the deglycosylated protein core of mucins and the use of molecular techniques has resulted in a marked increase in our knowledge of the gene and protein structure of mucins. To date, 9 distinct human mucin genes have been cloned and sequenced and are designated as MUC1 - MUC4, MUC5AC, MUC5B, and MUC6 - MUC8 (reviewed in Rose, 1992; Gendler and Spicer, 1995; Jeffery and Li, 1997). The protein cores of theses glycoproteins represent the primary gene products (apomucins) of this family of mucin genes. Homologs to human mucins have been cloned and additional sequences are appearing in the literature at an ever increasing rate. By convention, human mucin genes are designated by MUC, mouse by Muc, and rat by rMuc.
A common feature of all epithelial mucins analyzed to date is a protein core with tandem repeat sequences that are rich in the amino acids serine and threonine. MUC1 is the only membrane-spanning epithelial mucin identified to date. MUC2, MUC5AC, MUC5B, and MUC6 have all been described as gel-forming mucins and all have similar cysteine rich regions at their amino- and carboxy-terminal regions. These cysteine rich regions lack the tandem repeat sequences and are believed to allow intermolecular crosslinking through disulfide bond formation. MUC7, a salivary mucin, lacks both a membrane-spanning domain and the cysteine-rich regions and is the only soluble secreted mucin identified to date. Molecular analyses using Northern blot, RT-PCR, and in situ hybridization techniques indicate that the mucin genes are expressed in a tissue and cell-specific manner. However, the regulation of mucin gene expression and mucin production within the microenvironment of specific tissues remains to be determined.
Responses of the Nasal Mucociliary Apparatus of Rats to Toxicant Exposure
As the portal of entry into the respiratory tract the epithelial mucosa lining the nasal airways are exposed to the highest concentrations of toxicants in the inspired air. The epithelial response to an inhaled toxicant reflects the concentration of that agent, the dose to the epithelial cells, and the sensitivity of the cell population to the toxicant. We have examined the effects of two structurally dissimilar but common environmental toxicants, ozone and bacterial endotoxin. Both agents induce mucous cell metaplasia in the nasal airways of rats but in different anatomic sites.
Ozone, the principal oxidant gas in photochemical smog, induces mucous cell metaplasia in the nasal transitional epithelium (NTE) of both monkeys and rats (Harkema and Hotchkiss, 1994). A single 8 h exposure to 0.5 ppm ozone results in transient influx of neutrophils and a concomitant loss of transitional epithelial cells (17% less compared to controls) due to individual cell necrosis and exfoliation. A burst of DNA synthesis and cell proliferation returns the numeric cell density of the transitional epithelium to control levels 24 h postexposure (Hotchkiss et al., 1997). Repeated exposures result in a marked mucous cell metaplasia with increased amounts of stored mucosubstances in regions lined by transitional epithelium (Hotchkiss et al., 1991). Interestingly, blocking neutrophil influx attenuates the mucous metaplastic response with no effect on DNA synthesis, cell proliferation, or mucin gene (rMuc5AC) mRNA expression (Hotchkiss et al., 1998; Cho et al., 2000).
Endotoxins are lipopolysaccharide-protein molecules released from the walls of gram-negative bacteria. They are pro-inflammatory agents with a broad range of actions in the respiratory system. Inhalation exposure to endotoxin has been implicated as the principal pathogenic agent in several asthma-like or bronchitis-like diseases (Gordon and Harkema, 1995). Previous studies in our laboratory and those of others have demonstrated that laboratory rodents exposed by inhalation or instillation to endotoxin rapidly develop mucous cell metaplasia, with excessive amounts of intraluminal mucus, in nasal and pulmonary airways (Steiger et al., 1995; Shimizu et al., 1996). This metaplastic transformation occurs only in airways lined with respiratory epithelium containing primarily serous-type (not mucous) secretory cells. Intranasal instillation of endotoxin induces acute, transient, neutrophilic inflammation in both the nasal respiratory and transitional epithelium but only the respiratory epithelium undergoes mucous cell metaplasia. Endotoxin-induced increases in mucous cells and stored mucosubstances can also be attenuated by blocking neutrophilic inflammation.
These studies suggest that there are common cellular response pathways that can be activated in response to a broad range of toxicants to increase local mucus production in nasal airways. Our laboratory is currently using a biphasic culture system to examine the effects of toxicants and inflammatory cells on morphologically distinct nasal epithelial cell populations in vitro (Fanucchi et al., 1999). This system allows investigation of toxicant-dependent nasal airway epithelial remodeling in the absence of inflammatory cell influx or other potential systemic effects encountered in vivo. Inflammatory cells or their secreted products are then added to the culture system to determine their contribution to toxicant-induced mucous cell metaplasia. We hope that these studies proposal will provide insight into the cellular and molecular mechanisms regulating the expression, maintenance, and exacerbation of mucous cell metaplasia in the mucosal epithelium lining the nasal airways.
Brain, J.D. (1970) The uptake of inhaled gases by the nose. Ann. Oto. Rhinol. Laryngol. 79, 529-539.
Cho, H.Y., Hotchkiss, J.A., Bennett, C.B., and Harkema, J.R. (2000) Neutrophil-dependent and -independent alterations in the nasal epithelium of ozone-exposed rats. Am. J. Respir. Crit. Care Med. 162(2), 629-636.
Dahl, A.R., and Hadley, W.M. (1991) Nasal cavity enzy;mes involved in xenobioitic metabolism: Effects on the toxicity of inhalants. CRC Crit. Rev. Toxicol. 21, 345-372.
Fahy, J.V. (1998) Airway mucus and the mucociliary system. In Allergy: Principles and Practice. Eds: E. Middleton, C.E. Reed, E.F. Ellis, N.F. Adkinson, J.W. Yunginger, and W.W. Busse. Mosby, St. Louis, MO.
Fanucchi, M.V., Harkema, J.R., Plopper, C.G., and Hotchkiss, J.A. (1999) In Vitro culture of microdissected rat nasal airway tissues. Am.J.Respir.Cell Mol.Biol. 20, 1274-1285.
Gendler, S.J., and Spicer, A.P. (1995) Epithelial mucin genes. Annu. Rev. Physiol. 57, 607-34.
Gordon, T., and Harkema, J.R. (1995) Mucous cell metaplasia in the airways of rats exposed to machining fluids. Fundam.Appl.Toxicol. 28, 274-282.
Gross, E.A., Swenberg, J.A., Fields, S., and Popp, J.A. (1982) Comparative morphometry of the nasal cavity in rats and mice. J. Anat. 135, 83-88.
Harkema, J.R. (1991) Comparative aspects of nasal airway anatomy: Relevance to inhalation toxicology. Toxicol. Pathol. 19, 321-336.
Harkema, J.R., and Hotchkiss, J.A. (1994) Ozone-induced proliferative and metaplastic lesions in nasal transitional and respiratory epithelium: Comparative Pathology. In Nasal Toxicity and Dosimetry of Inhaled Xenobiotics: Implications for Human Health. Ed: F.J. Miller. Taylor & Francis, Washington, DC, pp 187-204.
Hotchkiss, J.A., Harkema, J.R., and Henderson, R.F. (1991) Effect of cumulative ozone exposure on ozone-induced nasal epithelial hyperplasia and secretory metaplasia in rats. Exp. Lung Res. 15, 589-600.
Hotchkiss, J.A., Harkema, J.R., and Johnson, N.F. (1997) Kinetics of nasal epithelial cell loss and proliferation in F344 rats following a single exposure to 0.5 ppm ozone. Toxicol. Appl. Pharmacol. 143, 75-82.
Hotchkiss, J.A., Hilaski, R., Cho, H.Y., Regan, K., Spencer, P., Slack, K., and. Harkema, J.R. (1998) Fluticasone propionate attenuates ozone-induced rhinitis and mucous cell metaplasia in rat nasal airway epithelium. Am.J.Respir.Cell Mol.Biol. 18, 91-99.
Jeffery, P.K. and Li, D. (1997) Airway mucosa: secretory cells, mucus, and mucin genes. Eur. Respir. J. 10, 1655-1662.
Miller, F.J. (editor) (1995) Nasal Toxicity and Dosimetry of Inhaled Xenobiotics: Implications for Human Health. Taylor & Francis, Washington, DC.
Morgan, K.T., Jaing, X.Z., Patterson, D.L., and Gross, E.A. (1984) The nasal mucociliary apparatus. Correlation of structure and function in the rat. Am. Rev. Respir. Dis. 130, 275-281.
Morgan, K.T., Patterson, D.L., and Gross, E.A. (1986) Responses of the nasal mucociliary apparatus of F344 rats to formaldehyde gas. Toxicol. Appl. Pharmacol. 82, 1-13.
Popp, J.A., and Martin, J.T. (1984) Surface topology and distribution of cell types in the rat nasal respiratory epithelium: Scanning electron microscopic observations. Am. J. Anat. 169, 425-436.
Rose, M.C. Mucins: structure, function, and role in pulmonary diseases (1992) Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7), L413-L429.
Shimizu, T., Takahashi, Y., Kawaguchi, S., and Sakakura, Y. (1996) Hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium induced by endotoxin. Am.J.Respir.Crit.Care Med. 153, 1412-1418.
Sorokin, S.P. (1988) The Respiratory System. In Cell and Tissue Biology: A Textbook of Histology. Ed.: L. Weiss. Urban & Schwarzenberg, Inc., Baltimore, MD.
Steiger, D., Hotchkiss, J.A.,. Bajaj, L, Harkema, J., and Basbaum, C. (1995) Concurrent increases in the storage and release of mucin-like molecules by rat airway epithelial cells in response to bacterial endotoxin. Am.J.Respir.Cell Mol.Biol. 12, 307-314.