In the present study, we found that IL-13 increased the cellular content and secretion of MUC5AC and decreased mucociliary transport. Interestingly, the cytokine caused small decreases in both G’ and G”, which were statistically significant. However, the changes were less than 2-fold, and the biological significance of this effect is probably minimal. None of the three mucoactive agents were cytotoxic at the concentrations tested, but they differed substantially in their properties. Only NAC potently scavenged free radicals, while only GGE had a concentration-dependent capacity to decrease the cellular content and secretion of MUC5AC. In contrast, all three drugs affected the rheology of the secretions.
Mucolytics generally decrease mucus viscosity by reducing the dicysteine bridges that contribute to the rigidity of the mucins
]. In the present study, NAC had relatively small effects on the cellular content or secretion of MUC5AC. However, it significantly decreased the viscosity and elasticity parameters of the secretions. Interestingly, NAC also appeared to reduce the total quantity of endogenous debris and, in particular, the fraction present as part of the mucus sheets. This aspect of the regulation of mucociliary function may merit additional study. Furthermore, 100 μM NAC decreased MTR for the particles associated with mucus sheets to below those of the IL-13-only cultures at all time points. It is possible this resulted from strong effects on viscosity that disrupted effective coupling between the mucus and the beating cilia.
Amb is used clinically to improve cough and discomfort from excessive mucus accumulation. Its mode of action is not fully understood, but may include antioxidant, anti-inflammatory or anaesthetic modes of action
]. It also may increase surfactant production
] and alkaline phosphatase secretion (which is associated with surfactant, but also reduces the effects of lipopolysaccharide)
], and alter ion transport
]. In our studies, Amb suppressed MUC5AC secretion and decreased MUC5AC cellular content slightly, but the effect was not concentration-dependent. Although this agent also altered the rheological parameters, the effect was substantially smaller than that of either NAC or GGE.
Several hypotheses for the mechanisms of action for GGE have been proposed. One suggested mechanism, the neurogenic hypothesis, involves stimulation of receptors in the stomach, resulting in vagal stimulation of respiratory tract fluid secretion, specifically cholinergic parasympathetic reflexes activating submucosal gland secretions
]. There is also evidence for direct effects on mucus adhesiveness and surface tension
], and on cough sensitivity
]. Significant effects on mucociliary clearance have also been demonstrated in chronic bronchitis patients but not in healthy controls
]. In contrast, no direct effects on mucociliary clearance measured as saccharine transport time in vivo
or ciliary beat frequency in nasal epithelial cell samples isolated from GGE-treated healthy volunteers were observed, although sampling artefacts in the isolation of the cells might have affected the results
]. In the present study, in contrast to the other agents tested, GGE produced substantial concentration-dependent decreases in both cellular MUC5AC content and secretion in the IL-13-stimulated cultures, similar to our previously reported results in unstimulated cells
]. The effects on MUC5AC content and secretion were paralleled by increases in MTR and significant decreases in both elasticity and viscosity parameters.
Added value in our study is provided by the correlations among MUC5AC content and secretion, MTR and rheology, which support the concept that the drugs affect selected parameters of the mucociliary transport system. In addition, the use of endogenous materials to track the MTR is an advantage. This method avoids potential dilution of the secretions and alterations in rheological properties that result when a suspension of exogenous particles is added to the surfaces of the cultures. This study also has some limitations. While the differentiated primary human cell cultures are more realistic models of the airway than cell lines or conventionally cultured primary cells, there is no actual clearance of the secretions, and the cultures must be washed at intervals. For this reason, the mucus levels vary from very little immediately after washing, to potentially large, depending on when the last washes occurred. We attempted to standardise these procedures, but in order to ensure sufficient mucus for the rheology measurements, the cultures for the MTR and rheology measurements included mucus produced and secreted during the 24 hr preceding drug treatment, while the cultures used to assess MUC5AC content and secretions were washed immediately prior to drug treatment. We did not attempt to measure ciliary beat frequency or periciliary fluid levels. In addition, the use of human lung cells precludes evaluation of effects mediated by other organs, such as the neurogenic mechanism postulated for the early effects of GGE
], which cannot be modelled in lung cell cultures. Metabolism by hepatic enzymes is also not included in the present model, and thus the time course for in vivo
responses may differ from those observed in vitro
. Finally, the contributions of mucins other than MUC5AC were not addressed in this study. These cultures exhibit modest numbers of MUC5AC-producing goblet cells
], which are increased by IL-13 treatment
], but not fully differentiated submucosal glands, the major source of MUC5B
]. However, MUC5B has been shown to be the major mucin in these cultures
]. It is, therefore, possible that the changes in mucus properties are due to the presence of MUC5B in the secretions. Future studies will address this possibility directly.