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Submucosal Glands and Airway Defense

Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California

Correspondence and requests for reprints should be addressed to Jeffrey J. Wine, M.D., Cystic Fibrosis Research Laboratory, Room 450, Building 420, Sierra Mall (Main Quad), Stanford University, Stanford, CA 94305-2130. E-mail: wine{at}stanford.edu; Web: http://www.stanford.edu/~wine/

	ABSTRACT

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Most airway mucus is produced by submucosal glands in response to neural signals. Gland mucus traps microbes, inhibits their replication, and clears them from the airways. In cystic fibrosis mucus clearance is compromised, allowing pathogens to persist in static mucus. These trigger an influx of inflammatory cells, but optimal effectiveness of inflammation, and especially its resolution, also requires effective mucus clearance. Our objective is to understand the basis for defective mucus clearance in cystic fibrosis. We discovered that in subjects with cystic fibrosis, submucosal gland secretion in response to agents that elevate intracellular cyclic AMP level is completely lost and mucus stimulated by elevating intracellular Ca²⁺ level is thicker. We hypothesize that loss of functional cystic fibrosis transmembrane conductance regulator from gland serous cells renders them unable to secrete anions and fluid in response to any stimulus, resulting in thickened gland mucus that can be tethered to the gland ducts. In primary ciliary dyskinesias, mucus is normal, but the dysfunctional cilia lining the gland ducts may also lead to inadequate clearance of mucus from glands. Thus, understanding of lung pathology in each disease may require that an improved understanding of gland structure and function be added to our rapidly growing understanding of surface epithelia.

Key Words: acetylcholine • cystic fibrosis transmembrane conductance regulator • pH • vasoactive intestinal peptide

	MUCUS CLEARANCE

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
This is an exciting time to be in cystic fibrosis (CF) research. After many detours, the focus is once again on mucus clearance as the primary mechanism used to maintain sterility of the airways. In this view, defective mucus clearance is the root cause of CF airway infections (1–6). In support of this proposal, Knowles and Boucher emphasize the following findings in a review (6): (1) mucin carbohydrate side chains provide a "combinatorial library" of binding sites for pathogens (7); (2) antimicrobials suppress growth transiently during clearance (8); (3) cough clearance is important; (4) the periciliary liquid (PCL) layer is a lubricant; (5) the PCL is important for cough clearance as well as mucociliary clearance; (6) the PCL layer moves; (7) PCL depth is adjusted by absorption via the epithelial sodium channel and secretion via cystic fibrosis transmembrane conductance regulator (CFTR); and (8) shear increases clearance, possibly via released nucleotides that induce secretion and ciliary beating.

Knowles and Boucher use two genetic diseases, CF and primary ciliary dyskinesia (PCD), to provide contrasting insights into the crucial role of mucus clearance in maintaining airway sterility (6). CF airway disease is explained by them approximately as follows. The loss of functional CFTR disinhibits the epithelial sodium channel and so increases absorption of isotonic liquid from the PCL layer (9). In normal airways, if the PCL layer thins beyond a critical point the surface epithelia somehow convert from absorptive to secretory (10). Presumably, the lack of CFTR prevents that in CF. Thus, in CF the gel-forming mucins that normally float above the cilia are brought into close contact with the airway surface, where they anneal to tethered mucins. The antimicrobials within these tethered mucus plaques soon become ineffective, and microbes proliferate. Conditions within the plaques, perhaps low O₂ tension, contribute to the formation of a mucoid phenotype among the resident Pseudomonas aeruginosa, which thus become resistant to killing by the adaptive immune system. For PCD, mucociliary clearance is absent, but effective cough clearance remains. This can account for the milder airway disease observed in patients with PCD (6).

The hypothesis that defective mucus clearance accounts for CF lung disease was stated as early as 1945 by Farber, who coined the term mucoviscidosis (11). What differs in the modern reincarnation of the mucus clearance hypothesis is the insight that deficient water in the mucus, rather than altered mucin molecules, is the root cause of altered mucus clearance. Deficient water will increase the viscosity and tackiness of mucus, reduce the lubricating PCL layer, and so make it more difficult to clear mucus from gland ducts and airways. In this article, we provide additional data and arguments to support the hypothesis that defective mucus clearance is a critically important contributor to CF airway disease, and we propose that an important source of defective clearance is deficient fluid secretion from serous cells of submucosal glands. With the focus now sharpened, experimentation can be directed toward clarifying details of the system, for example, fluid secretion and absorption by surface and glandular cells, and the effects of an altered periciliary liquid layer and mucus gel. Our working hypothesis is that all of these elements are negatively affected by CFTR mutations, and it is their aggregate effects that lead to defective mucus clearance.

It is natural and useful for different groups to emphasize different aspects of the problem. Thus, while agreeing with the importance of the periciliary liquid (PCL) layer, in this review we emphasize the serous cell malfunction hypothesis: we propose that defects in the production and clearance of mucus from the glands themselves is a significant feature of cystic fibrosis airway disease (1). Evidence for this view comes from work by Engelhardt and coworkers (12), Inglis and coworkers (4), Yamaya and coworkers (2), Jiang and coworkers (3), and others. However, as requested by the editors, we emphasize our own work, which is aimed at determining exactly what submucosal glands are, how they work, what they have evolved to accomplish, and how they are affected in each disease state.

	MUCUS PRODUCTION BY SUBMUCOSAL GLANDS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Submucosal glands are complex organs that rapidly produce copious mucus in response to neural signals (13). In healthy humans, submucosal glands are estimated to provide more than 95% of upper airway mucus (14). Glands occur at a frequency of about 1 per mm² in the trachea (15) and are abundant down to about the 10th generation, when airway lumens are about 1–2 mm in diameter. Each gland comprises multiple tubules that feed into a collecting duct, which narrows into a ciliated duct that is continuous with the airway surface (Figure 1 and Meyrick and coworkers [16]). Tubules are lined with mucous cells in their proximal regions and serous cells in the distal acini (16). Normal glands are about 60% serous and 40% mucous cells by volume, and the abundant serous cells secrete water, electrolytes, and a rich mixture of "anti" compounds with antimicrobial, antiinflammatory, and antioxidant properties, while mucous cells provide most of the mucins (17, 18). Because of the key role of their secreted products in fighting mucosal infections, serous cells have been described as "immobilized neutrophils" (17). Of special relevance to CF is the observation that within airways, CFTR is most highly expressed in serous cells (12).

View larger version (160K): [in this window] [in a new window]   Figure 1. Living human submucosal gland. A bright-field image at high contrast of a human submucosal gland after stimulation with carbachol. The gland is viewed through an oil layer, the surface epithelium, and the lamina propria, but all deeper tissue was dissected away. The bubble of mucus secreted by the gland is visible in the oil layer (open arrow). The presumptive collecting duct (C.D.), mucous tubules (M.T.), and serous acini (S.A.) are labeled by reference to the gland reconstruction by Meyrick and Reid (18). Reproduced with permission from Joo and colleagues (1).

	METHODS FOR STUDYING GLAND SECRETION

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

	GENERAL FINDINGS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Stimulation
We have monitored the secretions of more than 2,500 individual glands in 2 cats, 12 sheep, 56 pigs, and 33 humans, including 8 subjects with CF. Secretion rates over time in response to various mediators were quantified. For all four species, carbachol produced a short latency response that peaked within minutes and then declined to a sustained secretion rate about one-third or less of the peak rate. In response to phenylephrine, cats produced a large, fast response just like their response to carbachol, but responses in the other three species were small and transient. None of the species responded well to isoproterenol.

Responses to vasoactive intestinal peptide (VIP) and forskolin were studied in pigs and humans. In pigs, responses to forskolin required 10–20 minutes to reach maximum and were then sustained. Responses for 231 glands measured in 26 pigs were 1.7 ± 0.2 nl · minute^–1 per gland, but the distribution showed marked kurtosis and was positively skewed. Responses to 1 µM VIP had a faster onset but were smaller than the response to 10 µM forskolin. For humans, responses to VIP/forskolin were quantified for a subset of glands in donor tracheas and in bronchi from subjects who were transplanted because of diseases other than CF. For 68 glands from donor tracheas, the secretion rate was 1.0 ± 0.2 nl · minute^–1 per gland. For 48 glands from subjects with non-CF diseases, the mean rate was 1.1 ± 0.4 nl · minute^–1 per gland.

For either kind of stimulation, rates of mucus secretion varied more than 10-fold among individual glands in all species. The tissue volumes of individual, microdissected glands also varied more than 10-fold, suggesting that gland size determines secretion rate. We have begun the tedious process of correlating, gland by gland, gland volume with secretory rate.

Inhibitors of Secretion and Effect on pH
Studies by Ballard and colleagues show that pig mucus secretion in response to acetylcholine (ACh) is inhibited by bumetanide or dimethyl amiloride, with the combination eliminating most secretion (4, 22–25). We confirmed the bumetanide effect for average secretion by individual glands, suggesting that about half the volume of secretion depends on the Na⁺,K⁺,2Cl^– cotransporter. In an attempt to determine the contribution of HCO₃^– secretion we removed all HCO₃^–, maintained the pH with N-2-hydroxyethylpiperazine-2',2-ethanesulfonic acid (HEPES), and pregassed with pure O₂. Under these conditions secretion was again reduced by about half, and the combination of HCO₃^– replacement plus bumetanide inhibited secretion by about 90%. For technical reasons we have not yet determined whether individual glands vary in their sensitivity to either inhibitor.

The Calu-3 model of serous cells secretes almost pure HCO₃^– in response to forskolin stimulation (26). Unexpectedly, we found that gland secretion in response to forskolin was inhibited at least as strongly by bumetanide as was secretion stimulated by carbachol, and inhibition of forskolin-stimulated secretion by HEPES replacement of HCO₃^– was no greater than that of bumetanide, and no greater than the HEPES effect on carbachol-stimulated secretion.

	SECRETION BY CYSTIC FIBROSIS SUBMUCOSAL GLANDS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Abnormal secretion by CF glands was expected on the basis of numerous findings: (1) CFTR is expressed in airway gland serous cells (12); (2) ion and fluid secretion is reduced in cultures of CF gland cells (2, 3) and in intact pig glands pharmacologically treated with CFTR inhibitors (27, 28); (3) patch-clamp studies of primary cultures of serous cells (29) and Calu-3 cells (30) (widely regarded as good models of serous cells) indicate that CFTR is the only physiologically relevant apical anion channel in those cells; (4) Using chamber studies of Calu-3 cells indicate that CFTR is required for secretion in response to agents that elevate intracellular Ca²⁺ level ([Ca²⁺]_i) as well as to agents that elevate intracellular cyclic AMP level ([cAMP]_i) (5, 31, 32). All of these findings suggest that serous cells in CF glands should be incapable of fluid secretion. However, nasal glands of subjects with CF, which are presumably identical to airway glands, still show copious reflex secretion (33). This could mean that in spite of all of the above arguments there is an alternative, non-CFTR pathway for anion secretion from serous cells, or it could mean that the serous component of gland secretion is too small to be missed.

We are now directly studying secretions of intact, individual CF submucosal glands to clarify these issues. Because glands contain mucous cells that do not appear to contain CFTR (12), the predicted defect in serous cell fluid secretion should be most easily detected if serous cells could be stimulated without activating mucous cell fluid secretion. Because CFTR is activated by elevations of [cAMP]_i, we used VIP and forskolin to assess gland secretion in normal and CF glands. We discovered a complete absence of secretion in response to these mediators by glands from subjects with CF (1). Glands for lung donors, and glands from lungs of patients with diseases other than CF, responded well (Figure 2). Because the glands failed to respond to forskolin, which bypasses receptor pathways and activates adenylate cyclase directly, the defect cannot be secondary to the loss of VIP receptors, a possibility that is raised by the finding that VIP-containing nerves around sweat glands are decreased in patients with CF (34). Also, because the CF submucosal glands still responded to carbachol, the defect cannot be the result of a generalized defect in gland function. However, response of CF glands to carbachol differs quantitatively from responses of normal glands in several ways that are still being analyzed. All differences are consistent with a diminished mucous fluid content. A straightforward analysis is complicated because CF glands have volumes two to three times greater than control glands (35–37).

View larger version (29K): [in this window] [in a new window]   Figure 2. Control but not cystic fibrosis (CF) glands secrete in response to vasoactive intestinal peptide (VIP) or forskolin. (A) Responses to 1 µM VIP plus 10 µM forskolin of 20 glands from a disease control bronchial preparation, monitored by the large-field method. (B) Responses to VIP and then carbachol of two glands from a donor trachea, monitored by the small-field method. Note the long latency of response, variations in response profiles, and seven- to eightfold range of volumes secreted. Reproduced with permission from Joo and colleagues (1).

	IONIC COMPOSITION OF PURE GLAND MUCUS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

	WORKING MODEL FOR SUBMUCOSAL GLANDS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
On the basis of our experiments to date, we have proposed a working model of submucosal gland function (Figure 3) (1, 21). The model comprises four compartments based on gross structure and cell types (16, 18).

View larger version (36K): [in this window] [in a new window]   Figure 3. Schematic model of submucosal gland. Four functional compartments have been proposed on the basis of anatomic and immunohistochemical data, with cystic fibrosis transmembrane regulator (CFTR) located primarily in serous cells. Pathways that elevate intracellular Ca2+ level, such as acetylcholine (ACh), are hypothesized to activate fluid and macromolecular secretion from both serous and mucous cells. Pathways that elevate intracellular cyclic AMP level, such as VIP, are hypothesized to stimulate serous cells and mucin but not fluid secretion from mucous cells. The CFTR-dependent fluid-secreting pathway is deleted in CF glands. Reproduced with permission from Joo and colleagues (1).

Mucous cells line tubules just proximal to the serous acini. Little is known directly about their ion and fluid transport mechanisms, but because virtually all gland fluid secretion is eliminated by the combination of bumetanide and HCO₃^– replacement, we infer that mucous cells, like serous cells, generate fluid secretion by transporting Cl^– and HCO₃^– into the gland lumen. Mucous cells secrete the bulk of the mucin molecules, and MUC5B is the predominant type (39). Mucus secretion (mucins, other proteins, water, and ions) is partially preserved in CF glands, yet no known mechanism exists for non-CFTR, anion-mediated fluid secretion from serous cells. Therefore, we hypothesize that water and ion secretions from mucous tubules occur via non-CFTR-dependent mechanisms (20). We propose that mucous cells secrete fluid and mucin molecules in response to ACh, but in response to VIP they release only proteins via exocytosis. This conclusion is based on the complete absence of mucus secretion from CF glands exposed to VIP or forskolin.

The collecting duct epithelium consists of columnar cells up to 70 µm in height and containing abundant mitochondria (16). Their function is unknown, but Meyrick and Reid, who first described the collecting duct, pointed out that it is strategically located to monitor and condition all serous and mucous cell secretions on their way to the surface (16). We hypothesize that the collecting duct scavenges HCO₃^–, possibly converting it to CO₂ and water, that Na⁺ is also absorbed, and that either an osmolyte is added or the solution is able to remain hypotonic. This is based on the premise that serous cells, like Calu-3 cells, secrete a HCO₃^–-rich fluid in response to elevations of [cAMP]_i (26), yet the final mucus collected at the mouth of the duct has a pH between 7 and 7.2 versus a bath pH of 7.4 whether stimulated by ACh or VIP/forskolin (21). Furthermore, we find no difference in the pH of CF and control glands (20), in spite of the prediction that CF glands lack the HCO₃^– component from serous cells. Finally, secretions from bronchial submucosal glands (20) and nasal glands (33) of both control and CF subjects have significantly less Na⁺, Cl^–, and HCO₃^– than the bath. All of these findings indicate that the acinar fluid is modified extensively in the collecting and ciliated ducts, and is perhaps homeostatically controlled to bring it to a final common composition in spite of changes in the primary secretions.

The ciliated duct may simply be a continuation of the surface ciliated epithelial cells, in which case it would be primarily absorptive and able to acidify secretions via H⁺,K⁺-ATPase (43). However, the environment and functional role of the ductal cells differ considerably from those of the surface epithelium, and we hypothesize that the ciliated duct cells have a specialized phenotype.

To summarize, in this model ion-mediated water secretion from serous cells depends absolutely on CFTR, whereas ion-mediated water secretion from mucous cells is CFTR independent. ACh and other transmitters that elevate [Ca²⁺]_i activate ion, water, and macromolecular secretion from both serous and mucous cells, which are then mixed and conditioned in the ducts. Transmitters that elevate [cAMP]_i stimulate exocytosis and fluid secretion from serous cells, but only exocytosis of mucin from mucous cells. The CFTR-dependent, fluid-secreting serous cell pathway is deleted in cystic fibrosis glands. Present goals are to test all aspects of the model, and to formulate testable hypotheses to explain how a lack of functional CFTR in the gland contributes to CF airway disease. Like all models, this one is incomplete and is likely to be wrong in important ways. For a different model of submucosal glands see Shimura (44).

	CYSTIC FIBROSIS GLAND DEFECT: TOO LITTLE WATER, TOO LATE

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Normal mucus is about 98% water. Our data indicate that the major defect in CF glands is inadequate initial hydration of exocytosed mucins and peptides, but that remains to be established. We hypothesize that exocytosis of serous and mucous cell granules is unaffected in CF, but that the granules expand in an abnormally low volume of water. Although such an effect may seem trivial we hypothesize that it is crucial, because the viscoelastic properties of rapidly formed, tangled-polymer gels like mucus are determined by the concentration of polymers during initial formation. If the mucin molecules entangle while in a dilute solution a more dispersed tangle will form, and the gel viscosity will be lower than that of a more concentrated gel. Once formed, the properties of gels are quite stable. Subsequent addition of water does not disperse the gel for many hours or days; the only rapid effect is a modest swelling of the gel. That is why mucous layers persist on the external surfaces of aquatic animals and on the apical surfaces of epithelia in fluid-filled lumens.

Ballard and colleagues have shown the consequences of stimulating gland secretion in the almost complete absence of fluid secretion. They used inhibitors to block about 90% of fluid secretion from glands and surface epithelia, and then stimulated mucin secretion with ACh. That resulted in extremely viscous mucus that plugged the glands (23, 25, 27). It is fortunate that the CF phenotype is not so extreme. In CF airways, partial fluid secretion continues via non-CFTR pathways (1), which we propose are localized in mucous cells and possibly surface goblet cells; alternate pathways in CFTR-containing cells may also exist or be induced. Such residual secretion prevents a catastrophic failure of mucus clearance in CF airways. Indeed, many authors have made the point that mucociliary clearance in CF airways is slowed, not absent. In an important paper, Regnis and colleagues showed that slower clearance in patients with CF was in part secondary to their lung infections, with more severe infections associated with slower clearance. In patients with CF with milder infections, average clearance was about 60% of the normal rate (45).

Fortunately, in spite of a the total loss of secretion to VIP and forskolin in CF glands, glands retain the ability to secrete partially in response to ACh, which is the most important physiological transmitter. The model of gland function shown in Figure 3 predicts that all fluid secretion from CF glands originates from mucous cells. Because CF submucosal glands are two to three times larger than normal glands (35–37), they can maintain a near-normal volume of mucus secretion, but viscosity is increased (20) and the disposition of serous cell antimicrobials within the mucus may be suboptimal.

	CYSTIC FIBROSIS AIRWAY DISEASE: MULTIFACTORIAL HYPOTHESIS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Most people with cystic fibrosis die because their lungs become chronically infected with bacteria that are easily cleared from normal lungs. Importantly, bacteria in CF airways grow within mucus plugs (46).

It seems necessary to propose a multifactorial hypothesis to account for CF airway disease. We propose that loss of CFTR-mediated fluid secretion by gland serous cells and surface epithelial cells leads to underhydration of airway mucus and thinning of the periciliary liquid layer. This is exacerbated by increased fluid absorption caused by disinhibition of the epithelial sodium channel. Viscous mucus and the shallower periciliary liquid layer cause a modest slowing of mucociliary transport and probably also cause a modest decrease in the overall efficiency of cough clearance. However, we suggest that these effects are insufficient to explain the early onset, heterogeneous distribution, and life-long persistence of CF airway bacterial infections.

What distinguishes CF from all other obstructive lung diseases is the complete loss of a special kind of secretion: the HCO₃^–-rich fluid secretion from gland serous cells, which we propose leads to premature gelling of abnormally concentrated CF mucus within the thin and multiply branched tubules of submucosal glands. (Note that the concept of a deficient periciliary liquid layer, if important in the airways, should be even more critical in the small-diameter ducts of the glands.) We propose that idiosyncratic features of airway glands may cause a small proportion of them to develop tethered mucus plugs that persist for days, weeks, or months within the airways. These long-resident plugs continue to bind bacteria, but it has been shown, at least for nasal mucus, that microbes cocultured with mucus start to proliferate after a suppression period lasting as little as 24 hours (8).

The persistence of pathogens in the airway mucus is detected by macrophages patrolling the periciliary liquid layer, which release an array of cytokines to trigger an adaptive immune response (47). This includes an influx of inflammatory cells, especially neutrophils, into the submucosa and the airway lumen. The adaptive inflammatory immune response is essential, but we hypothesize that its success, and especially its resolution, also depend critically on the patency of mucus clearance. If clearance is impaired, spent inflammatory cells and their toxic products accumulate, host tissues are damaged, and more inflammatory cells are recruited. This destructive cycle is progressive.

In addition to stasis, CF airway mucus may be inherently less hostile to certain organisms than normal mucus (48). Definitive experiments to test this possibility are difficult because of access problems, but not only that. The ability of normal mucus to suppress pathogen growth almost certainly depends on synergistic actions among its complex components, which range from water and electrolytes to cells (49). Thus, the loss of CFTR-mediated electrolyte and fluid transport might impair the chemical shield of mucus in addition to its profound effects on clearance.

The multifactorial mucus clearance hypothesis of CF airway disease is consistent with the obstructive pathology characteristic of other CF organs that secrete mucus or macromolecules, including the sinuses, vas deferens, pancreas, and intestine, which become partially or completely filled with inspissated secretions, leading in some cases to complete blockage and degeneration (50–52). There is an emerging consensus that inadequate hydration of epithelial fluids, secondary to the loss of CFTR, underlies all of this pathology.

	SUBMUCOSAL GLANDS AND PCD AIRWAY DISEASE

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Microscopy of living submucosal glands immediately makes clear the importance of ciliary movement in mixing and expelling mucus from the glands (53). Some authors have proposed that mucus is squeezed from the glands by contractions of the myoepithelial cells, but it seems more plausible that the main function of the myoepithelial cells is to help provide the structural integrity needed to withstand the large hydrostatic pressure generated by ion and water transport into the gland lumen. During stimulation, hydrostatic pressure will ensure that mucus exits the gland, but when stimulation stops and the myoepithelial cells relax, mucociliary transport within the gland ducts may be important for clearing residual mucus and ensuring that it does not remain tethered to the glands. We propose that mucus tethering to gland ducts does occur in PCD and contributes to the characteristic airway infections of PCD. Because PCD gland mucus contains normal amounts of water, such tethering is predicted to be less severe than in CF, and more amenable to methods that stimulate clearance.

	UNANSWERED QUESTIONS AND ADDITIONAL MECHANISMS

TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 
Why Have Submucosal Glands in Airways?
Submucosal glands greatly amplify the ability of airways to produce mucus. Unlike the ciliated surface epithelium, which is a universal feature of mammalian airways, glands are absent from tracheas of rabbits, are rare in mice and hamsters, and are reduced in horses relative to other species (54–56). These species differences may be related to the efficiency of the nasal cavities in trapping particles and the extent to which animals are obligate nose breathers (J. H. Widdicombe, personal communication).

What Is the Role of VIP-stimulated Gland Secretion?
In spite of an extensive literature (13), our present understanding of the normal role of VIP and non-VIP pathways in controlling gland secretions is inadequate to predict the consequences of the absolute defect we discovered in VIP- and forskolin-mediated fluid secretion by CF glands. We need to know in much more detail how the nervous system controls the airway submucosal glands. In the absence of such evidence, we can only speculate that VIP pathways (or more generally all pathways capable of mediating serous cell-dominated secretion) may be involved in airway health via mechanisms very different from those emphasized in this review. One example is the modulation of inflammation (57).

Does CF Disease Start in Small Airways?
It is often stated that CF lung disease begins in small airways, and therefore gland dysfunction is discounted as a primary contributor to CF airway disease. However, we are unaware of definitive evidence that bacterial airway infections or mucus plugging in patients with CF generally begins in the smallest bronchioles and then spreads to the larger airways. Glands are found in cartilagenous airways as small as 1–2 mm, as they enter pulmonary lobules. High-resolution computed tomography of CF lungs clearly shows evidence of peripheral air trapping and bronchial wall thickening as the earliest signs of disease, but inspection of published figures suggests that the regions of earliest air trapping are usually large enough to be explained by restrictions in the smallest gland-containing bronchi (58). Moreover, the extreme heterogeneity of CF disease is difficult to explain by a mechanism whereby the airways "fill up" from the smallest to largest airways, but is rather more consistent with intermittent plugging of larger airways.

What Mechanisms Are Required for the Dispersion of Exocytosed Granules?
Preliminary observations in our laboratory of ongoing mucus formation in single glands support prior evidence (53) that exocytosed material may remain as condensed packets for a long time after secretion. We hypothesize that the process whereby these packets of material are dispersed within mucus may be defective in CF airway glands. This hypothesis is based on a comparison of airway gland secretion with secretion in the intestinal crypts. Paneth cells can be thought of as the serous cells of the intestinal crypts. In a landmark study, Clarke and colleagues have shown that in CF mice, Paneth cell granule contents are trapped as undispersed packets within crypt lumen (59, 60). The mechanism for this is unknown, but may be related to deficient HCO₃^– and water secretion by the CF mouse crypts. If a similar phenomenon occurs within airway submucosal glands, the effectiveness of antimicrobials would be greatly diminished, even though their concentrations, as assayed by typical methods, might be unchanged. We need to know whether this does, in fact, occur.

What Interventions Should Work?
The characterization of CF lung disease presented above leads us to propose an intervention strategy that departs significantly from present practices. The main lesson of innate defense is that it is constantly vigilant, attacking immediately when pathogens are least numerous and most susceptible. We suggest that the closest strategy to compensate for the compromised innate defenses in CF lungs is a daily regimen of clearance (exercise may be best, but this is highly individualistic) followed by inhaled antibiotics. (If clearance methods were effective, inhaled antibiotics might not be required.) Because we think that CF lung disease starts with localized airway obstruction and infection, pulmonary function tests are inadequate to give early warnings. A marked improvement is to combine them with high-resolution computed tomography scans (61); the greatly reduced radiation levels of modern scanners makes that feasible.

There are, of course, objections to these strategies, the most usual being a fear of developing antibiotic resistance, the high cost of treatments, and the time required. However, the most effective treatment strategies that have passed muster in controlled trials come close to this model (62), except that they commence only after patients have become chronically infected. As similar trials are run in ever younger patients, we think it likely that the field will eventually converge on the model suggested by studies of airway innate defenses.

Conclusions
Our working hypothesis is that secretion by CF submucosal glands lacks the electrolyte-driven fluid component normally supplied by serous cells. Macromolecular secretion by both serous and mucous cells, and electrolyte-driven fluid secretion by mucous cells, are hypothesized to be intact. Because the rheologic properties of mucus depend critically on the concentration of macromolecules during initial formation of the gel and are resistant to subsequent changes, we hypothesize that a deficiency in electrolyte-driven water transport deep within the gland tubules will increase the concentration of gland mucus, adversely affecting mucus clearance from the glands and dispersal of antimicrobials, and contributing to impaired mucociliary and cough clearance.

	ACKNOWLEDGMENTS

 
The authors thank transplant recipients and the Stanford Transplant Team for aid in obtaining posttransplant tissue specimens. They are grateful to M. E. Krouse and R. B. Moss for criticism of the manuscript. They are especially grateful to the families of organ donors, whose generosity allows both life-saving transplants and research into the root causes of lung diseases.

	FOOTNOTES

 
Supported by National Institutes of Health grants DK-51817 and HL-60288, and by the Cystic Fibrosis Foundation.

(Received in original form June 19, 2003; accepted in final form August 29, 2003)

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TOP ABSTRACT MUCUS CLEARANCE MUCUS PRODUCTION BY SUBMUCOSAL... METHODS FOR STUDYING GLAND... GENERAL FINDINGS SECRETION BY CYSTIC FIBROSIS... IONIC COMPOSITION OF PURE... WORKING MODEL FOR SUBMUCOSAL... CYSTIC FIBROSIS GLAND DEFECT:... CYSTIC FIBROSIS AIRWAY DISEASE:... SUBMUCOSAL GLANDS AND PCD... UNANSWERED QUESTIONS AND... REFERENCES

 

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