Inverse regulation of secretion and inflammation in human airway gland serous cells by neuropeptides upregulated in allergy and asthma

ABSTRACTAirway submucosal gland serous cells are sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and are important for fluid secretion in conducting airways from the nose down to small bronchi. We tested if serous cells from human nasal turbinate glands secrete bicarbonate (HCO3−), important for mucus polymerization, during stimulation with the cAMP-elevating agonist vasoactive intestinal peptide (VIP) and if this requires CFTR. Isoalted serous cells stimulated with VIP exhibited a ~20% cAMP-dependent decrease in cell volume and a ~0.15 unit decrease in intracellular pH (pHi), reflecting activation of Cl−and HCO3−secretion, respectively. Pharmacology, ion substitution, and studies using cells from CF patients suggest serous cell HCO3−secretion is mediated by conductive efflux directly through CFTR. Interestingly, we found that neuropeptide Y (NPY) reduced VIP-evoked secretion by blunting cAMP increases and reducing CFTR activation through Gi-coupled NPY1R. Culture of primary gland serous cells in a model that maintained a serous phenotype confirmed the activating and inhibiting effects of VIP and NPY, respectively, on fluid and HCO3−secretion. Moreover, VIP enhanced secretion of antimicrobial peptides and antimicrobial efficacy of gland secretions while NPY reduced antimicrobial secretions. In contrast, NPY enhanced the release of cytokines during inflammatory stimuli while VIP reduced cytokine release through a mechanism requiring CFTR conductance. As levels of VIP and NPY are up-regulated in disease like allergy, asthma, and chronic rhinosinusitis, the balance of these two peptides in the airway may control airway mucus rheology and inflammatory responses through gland serous cells.

Epinephrine stimulation of anion secretion in the Calu-3 serous cell model

Calu-3 is a well-differentiated human bronchial cell line with the characteristics of the serous cells of airway submucosal glands. The submucosal glands play a major role in mucociliary clearance because they secrete electrolytes that facilitate airway hydration. Given the significance of both long- and short-term β-adrenergic receptor agonists in the treatment of respiratory diseases, it is important to determine the role of these receptors and their ligands in normal physiological function. The present studies were designed to characterize the effect of epinephrine, the naturally occurring β-adrenergic receptor agonist, on electrolyte transport of the airway serous cells. Interestingly, epinephrine stimulated two anion secretory channels, the cystic fibrosis transmembrane conductance regulator and a Ca2+-activated Cl− channel, with the characteristics of transmembrane protein 16A, thereby potentially altering mucociliary clearance via multiple channels. Consistent with the dual channel activation, epinephrine treatment resulted in increases in both intracellular cAMP and Ca2+. Furthermore, the present results extend previous reports indicating that the two anion channels are functionally linked.

Chloride secretion by cultures of pig tracheal gland cells

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl− and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca2+ potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Mechanisms of Ca2+-stimulated fluid secretion by porcine bronchial submucosal gland serous acinar cells

The serous acini of airway submucosal glands are important for fluid secretion in the lung. Serous cells are also sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. However, the mechanisms of serous cell fluid secretion remain poorly defined. In this study, serous acinar cells were isolated from porcine bronchi and studied using optical techniques previously used to examine fluid secretion in rat parotid and murine nasal acinar cells. When stimulated with the cholinergic agonist carbachol, porcine serous cells shrank by ∼20% (observed via DIC microscopy) after a profound elevation of intracellular [Ca2+] ([Ca2+]i; measured by simultaneous fura 2 fluorescence imaging). Upon removal of agonist and relaxation of [Ca2+]i to resting levels, cells swelled back to resting volume. Similar results were observed during stimulation with histamine and ATP, and elevation of [Ca2+]i was found to be necessary and sufficient to activate shrinkage. Cell volume changes were associated with changes in [Cl−]i (measured using SPQ fluorescence), suggesting that shrinkage and swelling are caused by loss and gain of intracellular solute content, respectively, likely reflecting changes in the secretory state of the cells. Shrinkage was inhibited by niflumic acid but not by GlyH-101, suggesting Ca2+-activated secretion is mediated by alternative non-CFTR Cl− channels, possibly including Ano1 (TMEM16A), expressed on the apical membrane of porcine serous cells. Optimal cell swelling/solute uptake required activity of the Na+K+2Cl− cotransporter and Na+/H+ exchanger, both of which are expressed on the basolateral membrane of serous acini and likely contribute to sustaining transepithelial secretion.

Flagellin-stimulated Cl− secretion and innate immune responses in airway epithelia: role for p38

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10−7 M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl− secretion ( ICl) beginning after 3–10 min, reaching a plateau after 20–45 min at Δ ICl = 15–50 μA/cm2. Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated ICl in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1β (produced by both epithelia and neutrophils during infections) stimulated ICl similar to flagellin. Flagellin-, IL-1β-, ATP-, and forskolin-stimulated ICl were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1β altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-κB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1β-stimulated ICl by 33–50% but had smaller effects on IL-8 and NF-κB. It is concluded that: 1) flagellin and IL-1β activated p38, NF-κB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating ICl and IL-8 but not NF-κB; and 3) p38 was more important in flagellin- than IL-1β-stimulated responses. During P. aeruginosa infections, flagellin and IL-1β are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-κB, and IL-8 would persist.

The sld mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression

NFS/N- sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187–191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in “atypical” exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. “Atypical” cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

Bicarbonate and Chloride Secretion in Calu-3 Human Airway Epithelial Cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO−3 by a Cl −-independent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO−3 secretion and caused the secretion of Cl − by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO−3 secretion to Cl − secretion because the uptake of HCO−3 across the basolateral membrane is mediated by a 4,4 ′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive Na+:HCO−3 cotransporter. Since the stoichiometry reported for Na +:HCO−3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO−3 entry and favor the secretion of Cl −. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO−3 and Cl − secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO−3 and a Cl − channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO−3 secretion in the human airways warrants greater attention.