Mucus clearance as a primary innate defense mechanism for mammalian airways (original) (raw)
Primary ciliary dyskinesia: the importance of cough clearance. Patients with primary ciliary dyskinesia (PCD) exhibit recurrent middle ear infections, chronic airway infection, predominantly lower-lobe bronchiectasis, male sterility, and sometimes situs inversus. Patients with PCD exhibit a milder airway disease than do those with CF and typically live at least to middle age. This syndrome reflects defective ciliary function, with abnormalities in beat, stroke, or coordination, associated with genetically determined abnormalities of the structural/functional components of the ciliary shaft. The first of the genes associated with PCD has recently been cloned; it encodes an intermediate chain dynein (IC78) that appears important in assembling and coordinating the activities of several proteins, including the heavy chain dyneins of the ciliary shaft (36, 37).
The syndrome of PCD is informative with respect to many aspects of mucus clearance in lung defense. For example, measurements of mucociliary clearance, using external radionuclide tracers, have revealed virtually no basal, cilium-dependent mucus clearance in patients with PCD (38–40). However, studies of mucus clearance with inhaled radionuclide tracers show that cough-dependent mucus clearance is well preserved in PCD patients (39, 40). Thus, PCD patients may have a nearly normal rate of mucus clearance over time that is mediated solely by repetitive coughing. It appears likely that the retention of coughing as a backup mechanism for mucus clearance in PCD explains the milder pulmonary phenotype of PCD relative to CF.
The presence of effective cough-dependent mucus clearance in PCD patients suggests that the volume of liquid on airway surfaces is normal or increased. Indeed, the few reports quantifying ion transport in airway tissues freshly excised from PCD patients or in nasal epithelia studied in vivo have revealed no abnormalities that might adversely affect the volume of ASL (41). Thus, the apparently normal ASL volume in PCD patients may both preserve the lubricant function of the PCL and provide sufficient hydration and height of mucus for effective cough-dependent clearance. Interestingly, agents such as UTP that increase the volume of liquid on airway surfaces can acutely increase the efficiency of cough clearance in PCD patients (39). Long-term studies, however, will be required to test the clinical benefit of such therapeutic strategies in these patients.
Cystic fibrosis: the importance of the periciliary liquid and mucus hypoxia. Although controversy still exists, it now seems likely that CF lung disease reflects chronic depletion of PCL volume rather than high salt concentrations in the CF ASL. This assertion rests upon evidence that ASL is isotonic both in the normal state (in humans and other large mammals) and in CF (5, 6, 42–44). Further, it is congruent with the findings that, under physiologic conditions, the rates of net epithelial Na+ transport and isotonic volume absorption are higher in CF than in normal airway epithelia (6, 14, 45, 46). Consistent with accelerated volume absorption are reports of an increased number of Na+-K+-ATPase binding sites and activity, increased amiloride-sensitive and ouabain-sensitive O2 consumption in CF airway epithelia, and the early pathology of the non-infected CF lung (33, 41, 47–49). Finally, recent data have directly connected reduced ASL volume, but not ion composition, with morphologic evidence of spontaneous airway disease in CF mice (50).
What is new in the area of CF pathogenesis is the detailed understanding of the relationships between volume depletion on airway surfaces and mechanisms for mucus transport. Volume depletion on airway surfaces could be associated with selective removal of liquid from the mucus layer, or could reflect depletion of both the mucus layer and the PCL. In vitro studies have revealed that volume depletion normally occurs sequentially, first from the mucus layer, and then from the PCL. Specifically, early volume depletion occurs from the mucus layer, until about 50% of volume is removed, after which the volume is removed from the PCL (51). Thus, the mucus layer can normally act as a reservoir for liquid, but in CF, where volume regulation on airway surfaces is severely perturbed, the capacity of the mucus layer to buffer the PCL volume may be exceeded.
Another important insight from these studies has been identification of the relative importance of depletion of the PCL in the pathogenesis of CF. Depletion of the PCL predicts adverse interactions between the mucus layer and the airway epithelial cell surface (Figure 4). Indeed, in vitro data indicate that depletion of the PCL allows the gel-forming mucins in the mucus layer, such as MUC5AC and MUC5B, to come in contact with the tethered mucins MUC1 and MUC4 on the cell surface (Figure 4). Similar findings have been made in analyses of frozen sections of freshly excised CF airways. Thus, it appears likely that the annealing that occurs between the carbohydrate side chains of the mobile mucins and cell surface mucins, which effectively glues mucins in mucus to cell surfaces (the “Velcro effect”), will abolish cough clearance. Hence, excessive ASL volume absorption constitutes a double hit on mucus clearance mechanisms. The loss of volume depletes the PCL, which removes the liquid in which cilia can extend and beat, thus inhibiting mucociliary clearance. It simultaneously compromises the PCL’s lubricant function, allowing the mucus layer to adhere to cell surfaces and inhibiting cough clearance. This dual effect may account for the severe phenotype of CF lung disease, relative to PCD. Interestingly, the importance of tethered mucins in the pathogenesis of organ obstruction has been better documented in the gut, where the meconium ileus syndrome associated with the CF mouse intestine has been ameliorated by genetic ablation of the MUC1 mucin, which serves as the cell surface adhesive ligand for meconium in the intestine (52).
PCL is required for effective mucociliary and cough clearance. (a) Schema of microanatomy of normal ASL. Note mixing of bacteria in “turbulent” mucus. (b) Schema depicting hypothetical volume depletion of ASL covering CF airway epithelial surfaces. Note that the volume depletion is reflected in both the generation of a more concentrated mucus layer and the depletion of the PCL. PCL depletion allows interactions to occur between the tethered mucins of the glycocalyx and the mucus layer. Note motile bacteria penetrating into thickened, stationary mucus. (c) Evidence for ASL volume depletion in CF airway epithelia. ASL height was measured immediately, 12 hours, and 24 hours after deposition of PBS containing Texas red dextran on the epithelial surface of the cell (pseudocolored green). Left: Representative confocal microscopy images. Right: Mean data for normal (circles) and CF (squares) ASL heights. *CF ASL is significantly shallower than normal (P < 0.05; n = 6 per group). (d) Mucus (bead) rotational velocity 24 hours after administration of PBS containing fluorescent markers. At t = 0 hours, both normal and CF cultures exhibited rotational velocities of about 45 μm/s. (*P < 0.05, CF vs. normal; n = 6 per group). (e) Low-power electron micrograph of perfluorocarbon/osmium–fixed CF airway culture 24 hours after volume addition and with rotational mucus transport abolished. Note close apposition of mucus layer and the glycocalyx covering flattened cilia and the cell surface. (f) Light micrograph of freshly excised CF bronchus stained with Alcin blue periodic acid-Schiffs for mucus. As in the in vitro model, note close apposition (annealing) between secreted mucins and the cell surface (indicated by white arrow). NL, normal.
Recently, a link between mucus stasis and a predilection for Pseudomonas aeruginosa infection in CF has been revealed (33). In brief, despite the failure of mucus clearance, goblet cells continue to secrete, generating thick mucus plaques and plugs on airway surfaces. CF airway epithelia exhibit high rates of cellular O2 consumption to fuel raised Na+ transport, which creates hypoxic zones in adherent mucus plaques near the cell surface. P. aeruginosa inhaled and deposited on the surfaces of mucus plaques “swim” into the mucus plaques and adapt to the hypoxic zones with alginate production and biofilm formation, setting the stage for chronic infection.
The initiating event in CF, ASL volume hyperabsorption, reflects two CF-specific defects in airway epithelial ion transport. Recent in vitro studies suggest that active Na+ absorption by normal airway epithelia mediates ASL volume absorption under basal conditions and that normal airway epithelia can slow Na+ absorption and induce Cl– secretion, when ASL volumes are depleted (51). In CF, there appear to be defects in both the Na+-absorptive and Cl–-secretory mechanisms for regulating ASL volume. Specifically, CF airway epithelia have an accelerated basal rate of Na+ (and volume) absorption that reflects the absence of the tonic inhibitory effect of CFTR on the epithelial Na+ channel (ENaC) activity (53, 54). However, CF airway epithelia are also missing the capacity to add liquid back to airway surfaces when ASL volumes are depleted, due to the absence of CFTR functioning as a Cl– channel (55, 56). Thus, therapies directed both at slowing the abnormally raised volume absorption, e.g., Na+ channel blockers, and at initiating Cl– channel activity, e.g., UTP-dependent activation of Ca2+-regulated Cl– channels, provide routes for the treatment of the primary defect of the CF airway epithelium (20, 57).
Pseudohypoaldosteronism: a surprise with respect to mucus clearance. Pseudohypoaldosteronism (PHA) is a clinical syndrome that reflects, at the genetic level, loss-of-function mutations in ENaC subunits (58). Characterization of the pulmonary phenotype of this syndrome became pertinent because of the contrasting predictions of the compositional (chemical shield) and volume (mucus clearance) hypotheses with respect to the effects of the loss of Na+ channel function on ASL composition versus volume. Specifically, the compositional theory predicted that PHA patients would have raised ASL NaCl concentrations and inactivation of defensins, with resultant chronic airway infections. In contrast, the volume hypothesis predicted that PHA patients would have an excess of isotonic volume on airway surfaces, perhaps with a syndrome of airway obstruction.
Studies of PHA subjects showed that Na+ transport is absent, at least as measured by the transepithelial potential difference technique (59). Analyses of both the upper (nasal) and lower (bronchial) airways revealed that the ASL is isotonic in PHA patients, as it is in healthy subjects, but that the volume of their ASL is greatly increased. These patients suffer intermittent airway obstruction and infection as young children, but after the age of 6 years, their lung function is normal, and they are free of chronic bacterial airway infections or bronchiectasis, consistent with normal lung defense. Studies of mucus clearance in these subjects with the external radiolabeled tracer technique (59) show that basal mucus clearance occurs at an astonishing rate, equaling or exceeding rates in normal subjects after acute exposures to β-agonists or purinergic agents. No comprehensive explanation for this observation is available, but, given the reservoir function of the mucus layer, extra liquid added to this mucus layer could account for a modest increase in mucus clearance rates. Indeed, recent in vitro studies have confirmed that liquid added to the ASL preferentially partitions into and swells the mucus layer, accelerating mucus transport (51). Whereas liquid addition probably improves mucus viscoelasticity, it likely also changes the characteristics of the PCL, perhaps decreasing its viscosity, which could also accelerate mucus transport.
Therefore, the mucus transport data from the PHA subjects have highlighted two principles. First, as long as mucus clearance is maintained, chronic airway infections do not occur, despite any postulated adverse effects of isotonic (high salt) ASL on antimicrobial activity. Second, expansion of ASL volume triggers a series of events in the ASL compartment that accelerates the rate of mucus transport.