Our study demonstrated an association between goblet cell metaplasia and increased rates of nucleotide release in a model relevant to in vivo airway epithelia, primary HBE cultures. Our data further demonstrated that nucleotide release occurs coordinately with mucin secretion and that goblet cells can be important contributors to nucleotide and nucleoside concentrations in ASL. Nucleotide release coordinated with mucin secretion enables goblet cells to signal neighboring ciliated cells via purinoceptors to increase ion and liquid secretion and, hence, properly hydrate newly released mucins.
Goblet cell metaplasia was induced in primary HBE cultures via two independent approaches, RSV infection and IL-13 treatment. In both models, ATP release and mucin secretion rates directly correlated with goblet cell number ( and ). We also observed that nonstimulated (i.e., “basal”) mucin secretion and ATP release rates were similarly reduced by an intracellular Ca2+
chelator, BAPTA-AM ( and ). These data suggest that mucin secretion and ATP release from resting goblet cells are not completely “constitutive” (i.e., independent of regulation by second messengers) but likely consisted of “constitutive” and “basally Ca2+
–regulated” components. We speculate that both processes may reflect basally regulated rates of granule release as proposed by Davis and Dickey (20
ATP release and mucin secretion were similarly inhibited by reagents that inhibit secretory pathways (e.g., nocodazole, brefeldin A, and N-ethylmaleimide) in goblet-cell metaplastic cultures (). Conversely, agonists that increased mucin exocytosis and secretion (i.e., UTP and ionomycin) () also increased nucleotide release in control (non–goblet-cell metaplastic) cultures and to a greater extent in goblet-cell metaplastic cultures (). The increase in nucleotide release in response to stimuli was in proportion to the goblet cell numbers and the increase in mucin secretion. Because the use of a pharmacological agent could elicit off-target effects, it is important to confirm the results with several different agents or methods.
Our recent studies indicate that a nucleotide–sugar UDP-glucose is secreted concomitantly with mucins from goblet-like cell lines (6
). Thus, although the focus of this study was on the release of adenine nucleotides that enhance MCC functions, we investigated a correlation between goblet cell metaplasia and release of a nucleotide sugar that is known to be secreted via a vesicular-mediated mechanism (27
). Goblet-cell metaplastic cultures released UDP-glucose at greater rates than nonmetaplastic control cultures, strengthening the notion that increased vesicular-mediated release produced paralleled increases in mucin and nucleotide sugar concentrations in ASL (). UDP-glucose may modulate airway luminal innate immune responses, given that its receptor, P2Y14
, is expressed in neutrophils, lymphocytes, and macrophages (31
) and that UDP-glucose has been reported to promote secretion of inflammatory mediators from lung epithelial cell lines (34
The simplest hypothesis to account for the association between nucleotide release and mucin secretion is that nucleotides and mucins are contained in common granules and are released when vesicles fuse with the apical membrane. Recent studies have supported this hypothesis by demonstrating that subapical granules of goblet-like cell lines contain mucins and a spectrum of adenyl nucleotides (5
) and are apically secreted via Ca2+
–regulated exocytosis, producing simultaneous mucin secretion and nucleotide release (6
). The increased concentrations of a range of adenyl purine species on airway surfaces with goblet cell metaplasia () could reflect selective release of ATP from mucin granules with extensive ecto-ATP metabolism. Conversely, predictions from mathematical models (18
) and data from isolated mucin granules from the Calu-3 lung epithelial cell line (5
) suggest that granules exocytotically release a spectrum of adenyl nucleotides (ATP, ADP, and AMP).
An alternative hypothesis is that ATP release channels could be expressed in the mucin granule membrane and inserted into the plasma membrane during granule exocytosis. However, carbenoxolone, an inhibitor of the primary candidate for ATP release channels in airway epithelia (i.e., connexin and pannexin hemichannels) (29
), failed to affect basal or stimulated ATP release from control or goblet-cell metaplastic cultures (). Thus, it is unlikely that ATP release from airway epithelial goblet cells is predominantly mediated by vesicular insertion of connexin and pannexin ATP-releasing hemichannels.
Primary HBE cultures, reflecting in vivo airway morphology, are dominated by ciliated cells (~90%) under normal conditions. Data that ATP release rates (in the absence of shear stress) correlated with the number of mucus-producing cells ( and ) illustrate that mucus cells are a major contributor to ATP release under such conditions. Data that HBE cultures with 10 and 70% goblet cells released 0.3 and 1.2 pmol/cm2/min of ATP, respectively (), suggest that, if the correlation between the percentage of goblet cells and ATP release rates is linear, cultures with 0 and 100% goblet cells would release 0.15 and 1.65 pmol/cm2/min of ATP, respectively. Thus, it is estimated that goblet cells contributed to ATP release approximately 11 times more than ciliated cells under resting conditions.
On the other hand, airway cells in vivo
are constantly exposed to shear stress (e.g., from breathing and coughing). Mechanisms of ATP release and contributions of ciliated versus goblet cells to ATP release under shear stress are likely to be different from those in the absence of shear stress. A previous study demonstrated that shear stress caused an approximately 30-fold increase in ATP release as compared with resting conditions (i.e., to a rate of 9 pmol/cm2
/min) in ciliated cell–dominant (~90%) primary human airway cultures (19
). These data suggest that ciliated cells are a significant contributor to ATP release under shear stress.
Our data may have relevance to chronic airway diseases. For example, increased ATP release and adenosine accumulation on the surfaces of goblet-cell metaplastic airway epithelia may be particularly pertinent to asthma pathogenesis. Increased airway adenosine concentrations are a hallmark of asthma (35
), and increased airway ATP concentrations have been speculated to play a key role in asthma pathogenesis by activating dendritic cell functions (37
). However, the source for extracellular airway ATP and adenosine in asthma has been unclear. Our studies, using IL-13–treated cultures as a model of asthma-induced goblet cell metaplasia, suggest that metaplastic goblet cells could represent an important source of ASL nucleotides under stimulated conditions ().
Our data may also be relevant to viral airways infection. Common acute viral infections (e.g., RSV) produce respiratory symptoms consistent with mucus hyperproduction and inflammation that may persist for many weeks after initial infection (38
). Data from models that span this time frame (e.g., virus-infected mice and primary HBE cultures) suggest that this late postviral phase is associated with goblet cell metaplasia (40
). However, the cellular origins of increased mucus production and inflammatory signals that produce the postviral syndrome have not been unambiguously identified. The current study demonstrated that RSV induces goblet cell metaplasia in HBE cultures, which not only provides a source of increased mucin secretion but also increases ASL nucleotides and nucleosides that may participate in inflammatory signaling ( and ) (42
In summary, nucleotide release rates increase in parallel to mucin secretion rates in RSV- and IL-13–induced goblet-cell metaplastic HBE. The increase in goblet-cell nucleotide release proportional to increased mucin secretion may provide a compensatory mechanism to hydrate newly secreted mucus and promote airway surface clearance. In parallel, increased release into the extracellular environment of adenine nucleotides and nucleosides may promote airway luminal inflammatory responses via purinoceptors by stimulating secretion of inflammatory mediators from airway epithelia (42
), secretion of airway and alveolar fluids (19
), mucin gene transcription (43
), neutrophil migration (47
), and regulating endothelial barrier function (49
). Accordingly, goblet cell–mediated increases in ATP, adenosine, and UDP-glucose concentrations in airway lumens may coordinately activate innate host defense mucociliary clearance and cellular inflammatory pathways. These compensatory mechanisms are parts of the complex pathophysiologic scheme pertinent to many chronically diseased airways characterized by goblet cell metaplasia.