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

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).

Fig. 1. Representation of the two layers that compose the airway surface layer (ASL)

The overlying mucus layer, composed of gel-forming mucins (muc-5b and muc-5ac, shown here as entangled strands) secreted by glands and superficial goblet cells, is a highly viscoelastic material that traps inhaled particles for clearance. The lower, or periciliary layer (PCL), is a grafted-gel structure composed of cell surface tethered mucins (muc-1 and muc-4) and glycolipids within the domain around the cilia and microvilli.

Fig. 2. Regulation of airway ion transport processes by extracellular nucleotides and nucleosides

Autocrine/paracrine actions of nucleotides in the airways epithelial cells. ATP release and metabolism to adenosine (ADO) promotes activation of CaCC and CFTR channels and inhibition of ENaC via activation of P2Y₂ receptors (ATP) and A_2b receptors.

Fig. 3. Approaches used to deliver mechanical stress to airway epithelial surfaces in a quantitative/controllable manner

(A) Shear stress device. To generate shear stress, 4 cultures were rotated in a stop-go fashion for various times inside a highly humidified incubator. The device controlled via a stepper motor connected to a microprocessor based controller. Shear stress, over the range from .005 to 10 dynes·cm⁻² could be produced by varying the rate of acceleration of the stepper motor (Tarran et al., 2005). (B) System used to produce cyclic compressive stress (CCS) on human airway cultures. Here, a microprocessor controlled the pressure amplitude and timing of the transepithelial pressure to the apical surface of the cultured human airway epithelial cells using high-speed solenoids (Button et al., 2007). Pressure could be delivered under oscillatory or static (non-oscillatory) conditions over a range of pressures (1 to 100 cmH₂O).

Fig. 4. Release of ATP during physiological stress

Relationship between magnitude of oscillatory shear stress (A) and compressive stress (B) vs. apical ATP concentration at steady-state (at 30 min). Lines above denote the physiological range of shear (Tarran et al., 2005) and compressive stress (Button et al., 2007) during tidal breathing (see text).

Fig. 5. Oscillatory stress stimulates ASL hydration and mucociliary transport rates

(A) Representative XZ confocal images of ASL at 0, 6, and 48 h after mucosal addition of 30 µl of “excess” fluid containing Texas-red dextran to the apical side of normal human airway epithelial cultures under control conditions and undergoing CCS (20 cmH₂O, 20 CPM). Bar=10µm. (B) Time-course of changes in ASL height after fluid absorption under control (●) or CCS (□, 20 cmH₂O, 20 CPM) conditions. Note: dotted line denotes the length of the outstretched cilia. (C) Mucociliary transport rates before (T=0) and during control (●) or CCS (□, 20 cmH₂O, 20 CPM) conditions. In these experiments, MCT rate was determined by the movement of 1 µm florescent beads added to cultures as previously described (Matsui et al., 1998).

Fig. 6. Differential effects of cyclic and constant stress on ATP release

(A) Example of pressure tracings from cultures exposed to atmospheric pressure (control), oscillatory compressive stress (CCS) @ 20 cmH₂O/ 20 CPM, and non-oscillatory compressive stress (SCS) @ 20 cmH₂O. (B) Comparison of ATP release rates in cultures undergoing CCS and SCS for 30 min. Rates were determined by measuring ASL [ATP] over 30 min in cultures pre-treated with a cocktail of ATPase inhibitors (Button et al., 2007).

Fig. 7. Model systems diagram of mucus clearance