Glycated Albumin Triggers an Inflammatory Response in the Human Airway Epithelium and Causes an Increase in Ciliary Beat Frequency.

The role of inflammation in airway epithelial cells and its regulation are important in several respiratory diseases. When disease is present, the barrier between the pulmonary circulation and the airway epithelium is damaged, allowing serum proteins to enter the airways. We identified that human glycated albumin (GA) is a molecule in human serum that triggers an inflammatory response in human airway epithelial cultures. We observed that single-donor human serum induced IL-8 secretion from primary human airway epithelial cells and from a cystic fibrosis airway cell line (CF1-16) in a dose-dependent manner. IL-8 secretion from airway epithelial cells was time dependent and rapidly increased in the first 4 h of incubation. Stimulation with GA promoted epithelial cells to secrete IL-8, and this increase was blocked by the anti-GA antibody. The IL-8 secretion induced by serum GA was 10-50-fold more potent than TNFα or LPS stimulation. GA also has a functional effect on airway epithelial cells in vitro, increasing ciliary beat frequency. Our results demonstrate that the serum molecule GA is pro-inflammatory and triggers host defense responses including increases in IL-8 secretion and ciliary beat frequency in the human airway epithelium. Although the binding site of GA has not yet been described, it is possible that GA could bind to the receptor for advanced glycated end products (RAGE), known to be expressed in the airway epithelium; however, further experiments are needed to identify the mechanism involved. We highlight a possible role for GA in airway inflammation.

Nitric oxide activation by progesterone suppresses ATP-induced ciliary activity in oviductal ciliated cells.

Ciliary beat frequency (CBF) regulates the oviductal transport of oocytes and embryos, which are important components of the reproductive process. Local release of ATP transiently increases CBF by increasing [Ca2+]i. Ovarian hormones also regulate ciliary activity and oviductal transport. Progesterone (P4) induces nitric oxide (NO) production and high P4 concentrations induce ciliary dysfunction. However, the mechanism by which P4 affects CBF has not been elucidated. To evaluate the role of P4 in NO production and its effect on ATP-induced increases in CBF, we measured CBF, NO concentrations and [Ca2+]i in cultures of oviductal ciliated cells treated with P4 or NO signalling-related molecules. ATP induced a [Ca2+]i peak, followed by an increase in NO concentrations that were temporally correlated with the decreased phase of the transiently increased CBF. Furthermore, P4 increased the expression of nitric oxide synthases (iNOS and nNOS) and reduced the ATP-induced increase in CBF via a mechanism that involves the NO signalling pathway. These results have improved our knowledge about intracellular messengers controlling CBF and showed that NO attenuates oviduct cell functions. Furthermore, we showed that P4 regulates neurotransmitter (ATP) actions on CBF via the NO pathway, which could explain pathologies where oviductal transport is altered and fertility decreased.

An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

KEY POINTS: Extracellular ATP, in association with [Ca2+ ]i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. ABSTRACT: Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+ ]i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+ ]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml-1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1-100 µm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+ ]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium.

In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNFα) that can alter mucus viscosity; however airway ciliated cells have an autoregulatory mechanism to prevent the collapse of CBF in response to increase in mucus viscosity, mechanism that is associated with an increment in intracellular Ca+2 level ([Ca2+]i). We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca2+]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca2+]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways.

Acute lung injury induced by whole gastric fluid: hepatic acute phase response contributes to increase lung antiprotease protection.

BACKGROUND: Gastric contents aspiration in humans is a risk factor for severe respiratory failure with elevated mortality. Although aspiration-induced local lung inflammation has been studied in animal models, little is known about extrapulmonary effects of aspiration. We investigated whether a single orotracheal instillation of whole gastric fluid elicits a liver acute phase response and if this response contributes to enrich the alveolar spaces with proteins having antiprotease activity. METHODS: In anesthetized Sprague-Dawley rats receiving whole gastric fluid, we studied at different times after instillation (4 h -7 days): changes in blood cytokines and acute phase proteins (fibrinogen and the antiproteases alpha1-antitrypsin and alpha2-macroglobulin) as well as liver mRNA expression of the two antiproteases. The impact of the systemic changes on lung antiprotease defense was evaluated by measuring levels and bioactivity of antiproteases in broncho-alveolar lavage fluid (BALF). Markers of alveolar-capillary barrier derangement were also studied. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. RESULTS: Severe peribronchiolar injury involving edema, intra-alveolar proteinaceous debris, hemorrhage and PMNn cell infiltration was seen in the first 24 h and later resolved. Despite a large increase in several lung cytokines, only IL-6 was found elevated in blood, preceding increased liver expression and blood concentration of both antiproteases. These changes, with an acute phase response profile, were significantly larger for alpha2-macroglobulin (40-fold increment in expression with 12-fold elevation in blood protein concentration) than for alpha1-antitrypsin (2-3 fold increment in expression with 0.5-fold elevation in blood protein concentration). Both the increment in capillary-alveolar antiprotease concentration gradient due to increased antiprotease liver synthesis and a timely-associated derangement of the alveolar-capillary barrier induced by aspiration, contributed a 58-fold and a 190-fold increase in BALF alpha1-antitrypsin and alpha2-macroglobulin levels respectively (p < 0.001). CONCLUSIONS: Gastric contents-induced acute lung injury elicits a liver acute phase response characterized by increased mRNA expression of antiproteases and elevation of blood antiprotease concentrations. Hepatic changes act in concert with derangement of the alveolar capillary barrier to enrich alveolar spaces with antiproteases. These findings may have significant implications decreasing protease burden, limiting injury in this and other models of acute lung injury and likely, in recurrent aspiration.

Epithelial cell culture from human adenoids: a functional study model for ciliated and secretory cells.

BACKGROUND: Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. MATERIALS AND METHODS: In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca(2+) levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. RESULTS: Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. CONCLUSION: Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms.

Molecular modulation of airway epithelial ciliary response to sneezing.

Our purpose was to evaluate the effect of the mechanical force of a sneeze on sinonasal cilia function and determine the molecular mechanism responsible for eliciting the ciliary response to a sneeze. A novel model was developed to deliver a stimulation simulating a sneeze (55 mmHg for 50 ms) at 26°C to the apical surface of mouse and human nasal epithelial cells. Ciliary beating was visualized, and changes in ciliary beat frequency (CBF) were determined. To interrogate the molecular cascades driving sneeze-induced changes of CBF, pharmacologic manipulation of intra- and extracellular calcium, purinergic, PKA, and nitric oxide (NO) signaling were performed. CBF rapidly increases by ≥150% in response to a sneeze, which is dependent on the release of adenosine triphosphate (ATP), calcium influx, and PKA activation. Furthermore, apical release of ATP is independent of calcium influx, but calcium influx and subsequent increase in CBF are dependent on the ATP release. Lastly, we observed a blunted ciliary response in surgical specimens derived from patients with chronic rhinosinusitis compared to control patients. Apical ATP release with subsequent calcium mobilization and PKA activation are involved in sinonasal ciliary response to sneezing, which is blunted in patients with upper-airway disease.