Toward the modeling of mucus draining from the human lung: role of the geometry of the airway tree

Mucociliary clearance and cough are the two main natural mucus draining methods in the bronchial tree. If they are affected by a pathology, they can become insufficient or even ineffective, then therapeutic draining of mucus plays a critical role to keep mucus levels in the lungs acceptable. The manipulations of physical therapists are known to be very efficient clinically but they are mostly empirical since the biophysical mechanisms involved in these manipulations have never been studied. We develop in this work a model of mucus clearance in idealized rigid human bronchial trees and focus our study on the interaction between (1) tree geometry, (2) mucus physical properties and (3) amplitude of flow rate in the tree. The mucus is considered as a Bingham fluid (gel-like) which is moved upward in the tree thanks to its viscous interaction with air flow. Our studies point out the important roles played both by the geometry and by the physical properties of mucus (yield stress and viscosity). More particularly, the yield stress has to be overcome to make mucus flow. Air flow rate and yield stress determine the maximal possible mucus thickness in each branch of the tree at equilibrium. This forms a specific distribution of mucus in the tree whose characteristics are strongly related to the multi-scaled structure of the tree. The behavior of any mucus distribution is then dependent on this distribution. Finally, our results indicate that increasing air flow rates ought to be more efficient to drain mucus out of the bronchial tree while minimizing patient discomfort.     

SOME PHASES OF POSTOPERATIVE ATELECTASIS—The Role of Ciliary Action in Absorption of Air

The primary cause of postoperative atelectasis is the production of excessive quantities of mucus which occlude one or more air passages. This mucus is moved upward in the bronchial tree in successive masses, which carry with them bubbles of air, at the same time as the gases of the air are being absorbed by the venous blood. When all of the air has been removed, negative pressure develops which is equal to the effective ciliary power acting upon the contained masses of mucus. This negative pressure is, in all probability, maintained solely by ciliary action.     

SOME PHASES OF POSTOPERATIVE ATELECTASIS-The Role of Ciliary Action in Absorption of Air

The primary cause of postoperative atelectasis is the production of excessive quantities of mucus which occlude one or more air passages. This mucus is moved upward in the bronchial tree in successive masses, which carry with them bubbles of air, at the same time as the gases of the air are being absorbed by the venous blood. When all of the air has been removed, negative pressure develops which is equal to the effective ciliary power acting upon the contained masses of mucus. This negative pressure is, in all probability, maintained solely by ciliary action.     

Effects of bronchial secretions of patients with bronchial asthma and chronic bronchitis on the motility of ciliated cells of the ciliated epithelium

It has been shown that bronchial mucus from patients with bronchial asthma and chronic bronchitis can produce a ciliostatic effect when incubated with strips of frog palate mucosa. This effect can more often be found during clinical exacerbation and is supposed to be reversible. The ciliostatically active samples of bronchial mucus taken from asthmatic patients markedly inhibit reactivation glycerol models of frog's ciliated epithelium. It can therefore be suggested that the found activity acts directly on tubulin-dynein complex of the cilia. In contrast to these findings, the ciliostatically active samples of bronchial of glycerol models and therefore possibly act through some other mechanism.     

Further evidence for a flexible and highly expanded spheroidal model for mucus glycoproteins in solution.

The flexible and greatly expanded roughly spherical model for mucus glycoproteins proposed earlier, on the basis of hydrodynamic and n.m.r. data, is supported by new hydrodynamic results on a bronchial glycoprotein from a cystic-fibrosis patient. Furthermore, images from electron microscopy of this molecule and a lower-molecular-weight mucus glycoprotein (which closely resembles a glycopolypeptide) appear to be at least consistent with this model.     

Tachykinin receptors and the airways.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.     

Mucopolysaccharides in bronchial secretion of children.

The diseases of respiratory system are nowadays one of the important medical, economical and social problems of modern civilization. A common symptom of these diseases regardless of their etiology, character, klinical manifestation and morphological findings is hypersecretion of bronchial sputum. Tracheobronchial mucus is produced by mucous bronchial glands and goblet cells. This mucus represents at physiological condition about 5 micrometer thick cover on mucous of nasopharynx, larynx, trachea, bronchi up to terminal bronchioles. Protective function of mucus cover resposes in mechanical and humoral barrier and also in removing the inhaled particles and their transport to the upper part of the respiratory system. In spite of the fact, that bronchial secretion has an important role in protective mechanism and in thermal and water exchange of respiratory tract, this secretion is not sufficiently scrutinized mainly of children yet.     

Stochastic Modeling Predictions for the Clearance of Insoluble Particles from the Tracheobronchial Tree of the Human Lung

Bronchial clearance of deposited particles was simulated using a stochastic model of the tracheobronchial tree. The clearance model introduced in this study considers (1) a continuous decrease of the mucus thickness from the trachea to the terminal bronchioles according to a linear or an exponential function, (2) the possibility of mucus discontinuities, which are mainly found in intermediate and distal airways of the tracheobronchial compartment, (3) mucus production in proximal airways, (4) a slow bronchial clearance phase due to the capture of a defined particle fraction f _s in the periciliary sol phase, and (5) an eventual delay of the mucociliary transport at carinal ridges of airway bifurcations. Based on the concept of mucus volume conservation in single bifurcations, a reduction of the thickness of the mucus blanket from proximal to distal airways causes a significant increase of the mucus velocities in small ciliated airways compared to other stochastic modeling predictions assuming a constant thickness of the mucus layer throughout the conducting airways. This effect is further enhanced by the consideration of mucus discontinuities. In contrast, the ability of bronchial airways to produce a certain volume of mucus has a decreasing effect on the mucus velocities. In all generated clearance velocity models, mucociliary clearance is completely terminated within 24 h after exposure, consistent with the experimental evidence. Implementation of a slow bronchial clearance phase predicts a long-term retention fraction, which is fully cleared from the lung after several weeks. For 1-μm MMAD particles, 24-h retention varies between 0.42 and 0.52, in line with the suggestions of the ICRP. Mucus delay at carinal ridges only affects short-term clearance by increasing the retained particle fraction at a given time, while long-term retention is not influenced.     

Localization of catecholamines, serotonin and histamine in the cells and mucus of bronchial washings from apparently healthy subjects

By means of luminescent-histochemical methods localization of catecholamines, serotonin, histamine has been studied in cells and mucus of bronchial washings of 30 practically healthy persons. Cytofluorometry is performed in mucus, pulmonary epithelium, alveolar macrophages, lymphocytes and neutrophils of the bronchial washings. In the alveolar macrophages a high level of the compounds studied is detected.     

Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease

Excessive production of airway mucus is a cardinal feature of bronchial asthma and chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality in these diseases. IL-13, a Th2-type cytokine, is a central mediator in the pathogenesis of bronchial asthma, including mucus overproduction. Using a genome-wide search for genes induced in airway epithelial cells in response to IL-13, we identified pendrin encoded by the SLC26A4 (PDS) gene as a molecule responsible for airway mucus production. In both asthma and COPD mouse models, pendrin was up-regulated at the apical side of airway epithelial cells in association with mucus overproduction. Pendrin induced expression of MUC5AC, a major product of mucus in asthma and COPD, in airway epithelial cells. Finally, the enforced expression of pendrin in airway epithelial cells in vivo, using a Sendai virus vector, rapidly induced mucus overproduction in the lumens of the lungs together with neutrophilic infiltration in mice. These findings collectively suggest that pendrin can induce mucus production in airway epithelial cells and may be a therapeutic target candidate for bronchial asthma and COPD.     

Elasto-thixotropic properties of bronchial mucus and polymer analogs. I. Experimental results

The non-newtonian viscous and elasto-thixotropic properties of native and lyophilized pathological bronchial mucus and of polymer solutions (3% and 6% PIB in decalin) used as mucus analogs were analyzed using a cone-plate Carri-Med rheometer and a Couette viscoelastometer that we have specifically developed for measuring the rheological properties of bronchial mucus in clinical practice. The master curves obtained for apparent viscosity under steady conditions as a function of shear rates (gamma: 2.6 X 10(-3) to 6.9 X 10(1) sec-1) were fairly similar, whatever the apparatus used. Under transient conditions, at low shear rate (gamma less than 1.4 sec-1), PIB and mucus exhibited a typical viscoelastic behavior: the shear stress increased slightly up to a steady-state value. At higher gamma, a transitory overshoot of sigma characteristic of the elastothixotropic systems appeared. Such a behavior can be interpreted as resulting from structural changes such as formation and rupture of the three-dimensional network present in bronchial mucus as in polymer solutions.     

The role of mucus sol phase in clearance by simulated cough

Using a simulated cough machine, we analyzed the effect of adding tensio-active liquids as sol phase simulant on the clearance of gel mucus simulant by cough. Polysaccharides crosslinked with sodium tetraborate were used at different concentration as gel mucus simulant. A drop of gel mucus simulant was deposited either directly on the model trachea or on a sol phase layer simulant (2% sodium dodecyl sulfate in water). The clearance of the mucus simulants was quantified by observing the movement of marker particles in the gel layer. The viscoelastic properties of gel mucus simulants were determined by using a viscoelastometer (SEFAM). The adhesive properties were analyzed by means of the platinum ring technique. The wettability of the mucus simulants was quantified by the automatic measurement of the contact angle of the drop of gel on the model trachea. We found that the addition of a sol phase significantly decreased by about 50% the adhesivity and wettability of the gel mucus simulants. This decrease was associated with a marked enhancement of cough clearance, whatever the viscoelastic properties of the gel mucus simulants. These results suggest that the sol phase is essential in bronchial respiratory mucus clearance by the cough mechanism.     

Lymphocytes and bronchial hyperresponsiveness

Non-specific bronchial hyperresponsiveness can be defined as an increased responsiveness of the respiratory airways to physical, chemical and pharmacological stimuli. It is a characteristic feature of asthma. Knowledge of the mechanisms contributing to bronchial hyperresponsiveness can provide an insight into the pathogenesis of asthma and could lead to an improved therapy. Several abnormalities have been postulated to underlie the hyperresponsiveness, such as a beta-adrenoceptor dysfunction, hyperreactivity of airway smooth muscle, epithelial dysfunction or damage, increased reflex bronchoconstriction, mucus plugging and mucosal oedema. It is possible that more than one of these abnormalities or other, as yet unknown, mechanisms are involved. In contrast to the role of lymphocytes in the regulation of IgE antibody production, the role of these cells in bronchial hyperresponsiveness has received little attention. We review evidence indicating that lymphocytes are involved in the development of non-specific bronchial hyperresponsiveness in some animal models and in patients with asthma.     

Mucociliary interactions and mucus dynamics in ciliated human bronchial epithelial cell cultures

The airway epithelial surface liquid is generally considered to be composed of two layers, a periciliary layer and a continuous thick mucus layer moving in bulk. This view may not be appropriate for all areas of the lung. Our hypothesis, that mucus may form a discontinuous layer with dynamic attachments to the surface, is investigated using a culture system. We used live-cell confocal microscopy to investigate thin mucus layers and fluorescent beads and exogenous MUC5B to visualize mucus dynamics on ciliated human bronchial cultures. A continuous mucus layer was not observed. In sparsely ciliated cultures, mucus attached to ciliated cells; however, in highly ciliated cultures, mucus formed strands several hundred micrometers long. As with increases in ciliation, increases in bead concentration caused the appearance of mucus strands. We confirmed the involvement of mucins in the binding of mucus to cilia by adding labeled purified MUC5B to the cultures. These data suggest that mucins may have an intrinsic ability to form attachments to cilia. The significance of these findings is that aberrant modulation of such an intrinsic property may explain the initiation of highly adherent mucus in cystic fibrosis lung disease.     

Chemical and physical properties of human bronchial mucus glycoproteins.

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.     

A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma

Mucus hypersecretion is a crucial feature of pulmonary diseases such as asthma, chronic bronchitis and cystic fibrosis. Despite much research, there is still no effective therapy for this condition. Recently, we showed that the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is required for mucus secretion by human bronchial epithelial cells in culture. Having synthesized a peptide corresponding to the N-terminal domain of MARCKS, we now show that the intratracheal instillation of this peptide blocks mucus hypersecretion in a mouse model of asthma. A missense peptide with the same amino acid composition has no effect. Based on quantitative histochemical analysis of the mouse airways, the peptide seems to act by blocking mucus release from goblet cells, possibly by inhibiting the attachment of MARCKS to membranes of intracellular mucin granules. These results support a pivotal role for MARCKS protein, specifically its N-terminal region, in modulating this secretory process in mammalian airways. Intratracheal administration of this MARCKS-related peptide could therapeutically reduce mucus secretion in the airways of human patients with asthma, chronic bronchitis and cystic fibrosis.     

Complex structure of human bronchial mucus glycoprotein

Human bronchial mucus glycoproteins or mucins were isolated from the sputum of two patients by a method avoiding reducing agents and involving water extraction and gel filtration on Sepharose CL-2B in 6 M guanidinium chloride. The chemical analysis indicated approximately 25-40% lipid. The amino acid and carbohydrate analysis differ quantitatively from that of mucins purified after prior reduction of mucus. These fractions also have a higher proportion of aspartic and glutamic acids than that of the mucins from reduced sputum. These mucins are still contaminated by small amounts of peptides but do not seem to contain disulfide-attached cross-linking protein. Human bronchial mucins have a strong tendency to form aggregates except in 6 M guanidinium chloride. Electron microscopy performed with various procedures indicates the presence of both micelles and flexible threads measuring 200-1000 nm. Delipidation removes most of the micellar forms. Thereafter mucins appear mainly as polydisperse flexible extended threads and also as aggregates. These features of bronchial mucins do not fit with the generally accepted idea of mucin subunits linked by disulfide bridges (unless they are linked end to end) and alternatively favour a model where mucin molecules behave like filaments that could easily aggregate according to the solvent system (mucin concentration, absence of dissociating conditions).     

Production of tissue-engineered three-dimensional human bronchial models

We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.