Role of mechanical stress in regulating airway surface hydration and mucus clearance rates

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Abstract

Effective clearance of mucus is a critical innate airway defense mechanism, and under appropriate conditions, can be stimulated to enhance clearance of inhaled pathogens. It has become increasingly clear that extracellular nucleotides (ATP and UTP) and nucleosides (adenosine) are important regulators of mucus clearance in the airways as a result of their ability to stimulate fluid secretion, mucus hydration, and cilia beat frequency (CBF). One ubiquitous mechanism to stimulate ATP release is through external mechanical stress. This article addresses the role of physiologically relevant mechanical forces in the lung and their effects on regulating mucociliary clearance (MCC). The effects of mechanical forces on the stimulating ATP release, fluid secretion, CBF, and MCC are discussed. Also discussed is evidence suggesting that airway hydration and stimulation of MCC by stress-mediated ATP release may play a role in several therapeutic strategies directed at improving mucus clearance in patients with obstructive lung diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).

Introduction

Mucus clearance is a term that refers to a coordinated integration of ion transport, water flow, mucin secretion, cilia action, and cough, resulting in the continuous flow of fluid and mucus on airway surfaces. Functionally, mucus clearance is an innate process involved in guarding the lung against inhaled bacteria, viruses, and other noxious particles. In addition to supporting mechanical clearance, the mucus lining the airways possess anti-bacterial agents such as lysozyme and lactoferrin (Boyton and Openshaw, 2002), migratory cells such as macrophages, and signaling molecules such as cytokines, to suppress microbial proliferation during the clearance process (Knowles and Boucher, 2002). Key to removing all of the trapped debris is the maintenance is the rate of cilia-mediated mucus clearance, or mucociliary clearance (MCC). While it is well known that this process is undoubtedly dependent on the rate of ciliary beating (Satir and Sleigh, 1990), it is clear that the rate of clearance is also strongly influenced by the mucus hydration state, and hence the viscoelastic properties of the mucus (Puchelle et al., 1995, Winters and Yeates, 1997, Tarran et al., 2001). In general, the more hydrated the mucus, the more efficiently it is cleared from the lungs.

The airway surface liquid (ASL) layer lining the airway surfaces is crucial for mediating mucociliary clearance rates (Boucher, 2002). More importantly, the clearance of mucus from airway surfaces requires the coordinated interaction of two separate layers that together comprise the ASL. As shown in Fig. 1, the first layer is the mucus layer which contains mucins secreted from goblet cells and glands (Rubin, 2002). The mucus layer is designed to bind and entrap virtually all of the particles deposited on the airway surface during normal breathing. In this system, the viscoelastic properties of the overlying mucus layer facilitate conversion of energy from beating cilia into vectorial mucus transport, facilitated by cough during stimulating irritation. As shown in Fig. 1., the mucus layer represents an unrestrained, tangled gel generated by the high-molecular-weight secreted, gel-forming, mucins (muc-5ac, muc-5b) that occupy this region (Thornton and Sheehan, 2004). The viscoelastic properties, and hence transportability, of this layer are determined by both the composition of the mucin macromolecules and, importantly, by the “hydration” state of this layer (Voynow, 2002, Boucher, 2003). As discussed below, the hydration state of the airways reflects the balance between the activities Na+ absorption and Cl secretion mediated by the epithelial sodium channel (ENaC) and the, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, respectively (Tarran et al., 2005). While the mucus layer is a viscoelastic gel with the major structural components being large mucin glycoproteins, normal mucus represents only about 1% mucins, with the balance being salt (1%), other proteins (1%), and water (97%) (Matsui et al., 2006).

The second layer, underlying the mucus layer, is the periciliary layer (PCL) (Fig. 1). This layer was previously thought (Tarran et al., 2001) to simply be a thin (∼7 μm), low-viscosity aqueous layer that acts as a lubricant layer for cilia beating and the movement of the mucus layer over the epithelial surface. However, more recent hypotheses have suggested that the PCL is also a gel layer; in this case, a “grafted polyanionic gel” (reviewed in Randell, 2006). Here, the cell surface of the human airways contains large membrane-bound glycoproteins as well as tethered mucins (muc-1, muc-4, and muc-16). Not only does such a structure provide an efficient lubricating layer for cilia to beat, but also serves as a barrier to restrict access of particles from accessing the cell surface directly due to the close approximation of these long-branched carbohydrate molecules (Randell and Boucher, 2006). As with the overlying mucus layer, the hydration state of the PCL-gel layer reflects the balance of Na+ and Cl ion transport activities (Tarran et al., 2005).

The rate of mucus clearance from the airways is dynamic and can, under appropriate conditions, be stimulated to increase clearance. This has been demonstrated in a number of studies measuring the basal rates of mucus clearance in healthy subjects, as well as the changes that occur in disease and in response to inhalation of exogenous toxicants and drugs. For example, it has been shown that the rate of mucus clearance can be increased by ∼3 fold over basal levels in response to inhalation of exogenous agonists (Wanner et al., 1996). Despite a large body of pharmacologic data, it has only been recent that studies have been undertaken to understand how the mucociliary clearance apparatus is endogenously regulated at the microscopic level, i.e., how the hydration status of the airways, ciliary beat frequency (CBF), and mucin secretion rates are coordinated to effect efficient clearance under both basal and stimulated conditions. In part, based on studies designed to explore the pathogenesis of obstructive lung diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), much attention has focused on understanding how these mechanisms that provide “innate” airways defense and how they are modulated in airways diseases characterized by chronic bacterial infection.

Key to this work are the findings from a number of studies which demonstrate that endogenous factors are responsible for accelerating mucus clearance in reaction to airway stresses exposure as part of a “lung defense” response. As will be discussed throughout this review, a fundamental finding of this work was that the release of endogenous 5′ nucleotide triphosphates, such as ATP, along with its metabolite adenosine, interact with airway epithelial purinergic receptors, to serve as major autocrine regulators of mucus clearance rates, and hence innate airways defense under basal, as well as, in response to inhaled toxins/irritants. It has also become apparent that the oscillatory motion generated during breathing is an important aspect of airway as well as lung physiology. While it has long been known that normal tidal breathing interspersed with sighing is important for maintaining alveolar integrity (Dietl et al., 2001), recent studies provide a mechanism that links the oscillatory motion of pulmonary ventilation to mucus clearance (Tarran 2005; Button, 2007), together suggesting that the mechanical stresses generated by tidal breathing are necessary for maintaining proper airways health.

Section snippets

Airway ion transport and ASL regulation

Airway epithelia have low transepithelial electrical resistances (Boucher, 1994) and are highly water permeable (Farinas et al., 1997, Matsui et al., 2000, Crews et al., 2001). These features suggest that large, osmotic gradients cannot be maintained across airway epithelia. This property initially appears disadvantageous during vectoral ion/water transport since extra energy is expended to move ions/water against a continual backflux. However, leaky epithelia, such as the airways, are capable

Nucleotides and ion transport

Despite the importance of understanding the regulation of ASL hydration on lung health, key aspects of its physiology remain unexplored. While much is known about the processes that mediate transepithelial ion transport, including ion channels, pumps, and co-transporters, significantly less is known about the “signals” that coordinate the activity of this complex system to reciprocally modulate both Na+ absorption and Cl secretion, to provide the proper balance of the ASL hydration. Airway

Stress-stimulated ATP release

As discussed throughout this issue, the lung is a unique organ in that it is subjected to complex physical forces during breathing, coughing and vascular perfusion, which all likely contribute to the regulation of lung function (Schumacker, 2002). Of import to this review, the cells lining the airway surfaces are subjected to oscillatory stresses during tidal respiration. It has long been appreciated that oscillatory motion of the lungs represents a key feature of pulmonary health, as

Mechanical stress mechanotransduction and ATP release

While it is known that external mechanical stress represents a ubiquitous mechanism to stimulate ATP release, the underlying sensors and response mechanisms whereby mechanical shear stresses are recognized and transduced into cellular effects by airway epithelia remain poorly understood. However, force transduction in endothelia has been extensively studied, and it is possible to speculate about the nature of shear stress sensing in airways based on this research. For example, it is widely

Techniques to produce oscillatory mechanical stress in airway epithelia

While it has been shown that ATP release represents a common response to a wide array physical stimulation, a key issue has been to measure the effect of stresses in the physiologically relevant range, generated by stresses similar to those exerted on airway epithelial cells during normal breathing. To this end, our laboratory has designed two approaches to deliver mechanical stress to airway epithelial surfaces in a quantitative/controllable manner, mimicking forces exerted on the airway

Human airway cultures under “thin-film” conditions

The culture of primary airway epithelial cells at air–liquid interface on porous culture substrates has become the gold-standard technique for study human and animal airway epithelia. These cultures recapitulate the mixed mucus-secretory and ciliated surface phenotype expressed in vivo (Matsui et al., 1998), and has been extraordinarily useful in elucidating many links between ion transport regulation and ASL volume homeostasis (Matsui et al., 1998, Tarran et al., 2001, Button et al., 2007).

Regulation of ASL volume and mucus clearance by stress-mediated ATP release

The effect of physiologically relevant levels of mechanical stress on stimulating ATP release by human airway epithelial cell cultures has been investigated during both oscillatory shear (Tarran et al., 2005) and compressive stress (Button et al., 2007). In these studies, well-differentiated human airway epithelial cultures under physiological air–liquid interface conditions, were subjected to a range of oscillatory shear and compressive stresses for 30 min prior to quantitation of apical [ATP],

Differential effects of oscillatory versus non-oscillatory stress

In addition to oscillatory forces associated with tidal breathing, airway epithelia can be subjected to continuous, non-oscillatory, transmural pressures (Tschumperlin and Drazen, 2006). One example is seen in the folding of the epithelial lining of the airway wall is a well-recognized process (Lambert et al., 1994) where bending of the epithelium and its substrate can give rise to long-term local shear deformations and pressure gradients (Wiggs et al., 1997). For example, during an asthmatic

Therapeutic implications of oscillatory motion in the airways

In addition to the in vitro data, above, there are in vivo data that suggest that stress-stimulated released nucleotides regulate components of the mucociliary system, e.g., ion transport, and mucociliary clearance. For example, studies of chest wall oscillatory ventilation in dogs revealed that mucus clearance doubled when chest-wall pressure oscillations were administered (King et al., 1983, Rubin et al., 1989). However, the authors could provide no simple explanation as to why vibration of

Systems integration approach to understanding mucus clearance

It has become clear that to fully understand how this complex system regulates ASL hydration and mucus clearance, mathematical models of these processes are necessary. Therefore, a diverse group of scientists has been assembled to combine experimental analysis with mathematical modeling to investigate how physical phenomena and the putative feedback mechanisms that regulate mucus height, viscoelasticity and cilia beat frequency interrelate to regulate mucus clearance. This group, called the UNC

Summary

Mucus clearance is an essential innate immune protective mechanism in the airways. Recent advances have greatly increased our understanding of the structure and function of the mucus clearance apparatus, its homeostatic regulation in normal airways, and how both genetic and acquired diseases disrupt its function to ultimately degrade lung function. Further, recent studies culminated in the observations that physiologically relevant mechanical stresses can stimulate mucus clearance via increases

Acknowledgments

The authors thank their many colleagues at the UNC CF Center and the UNC Virtual Lung Group for sharing thoughts and data cited in this review. Work cited here was supported, in part, by grants from the Cystic Fibrosis Foundation (BUTTON04I0 and BUTTON06G0) and the National Institutes of Health (DK065988, EB002025, and HL077546). We also would like to thank Lisa Brown for providing graphics and editorial assistance.

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