Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.     

Endoplasmic reticulum stress in chronic obstructive lung diseases

Chronic airway inflammation characterizes several airway diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). The altered airway milieu that results from the pathogenic processes in these disorders affects the airway epithelia, leading to an up-regulation of their innate defense. In human airway epithelia, luminal inflammatory stimuli induce an adaptation characterized by an expansion of the endoplasmic reticulum (ER) and its Ca(2+) stores. This epithelial adaption mediates Ca(2+)-dependent "hyperinflammatory" responses, and recent studies have shown that activation of the unfolded protein response (UPR) by ER stress is involved in the process. The UPR is also known to be activated by cigarette smoke, the primary trigger for development of COPD. These studies illustrate the functional role of UPR pathways during airway inflammation and suggest that targeting the UPR may be a therapeutic strategy for obstructive airway diseases. This article reviews the link between airway epithelial inflammation and activation of the UPR, and discusses how UPR activation might be relevant for CF and COPD airways disease.     

Basolateral chloride current in human airway epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) > I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement.     

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.     

Gene Expression in Obliterative Bronchiolitis-Like Lesions in 2,3-Pentanedione-Exposed Rats

Obliterative bronchiolitis (OB) is an irreversible lung disease characterized by progressive fibrosis in the small airways with eventual occlusion of the airway lumens. OB is most commonly associated with lung transplant rejection; however, OB has also been diagnosed in workers exposed to artificial butter flavoring (ABF) vapors. Research has been limited by the lack of an adequate animal model of OB, and as a result the mechanism(s) is unclear and there are no effective treatments for this condition. Exposure of rats to the ABF component, 2,3-pentanedione (PD) results in airway lesions that are histopathologically similar to those in human OB. We used this animal model to evaluate changes in gene expression in the distal bronchi of rats with PD-induced OB. Male Wistar Han rats were exposed to 200 ppm PD or air 6 h/d, 5 d/wk for 2-wks. Bronchial tissues were laser microdissected from serial sections of frozen lung. In exposed lungs, both fibrotic and non-fibrotic airways were collected. Following RNA extraction and microarray analysis, differential gene expression was evaluated. In non-fibrotic bronchi of exposed rats, 4683 genes were significantly altered relative to air-exposed controls with notable down-regulation of many inflammatory cytokines and chemokines. In contrast, in fibrotic bronchi, 3807 genes were significantly altered with a majority of genes being up-regulated in affected pathways. Tgf-β2 and downstream genes implicated in fibrosis were significantly up-regulated in fibrotic lesions. Genes for collagens and extracellular matrix proteins were highly up-regulated. In addition, expression of genes for peptidases and peptidase inhibitors were significantly altered, indicative of the tissue remodeling that occurs during airway fibrosis. Our data provide new insights into the molecular mechanisms of OB. This new information is of potential significance with regard to future therapeutic targets for treatment.     

Regulation of airway tight junctions by proinflammatory cytokines

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.     

Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y(2)-R activation in CF airway epithelia is an expansion of the apical ER Ca(2+) stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca(2+)(i) signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.     

Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway.

Recombinant adenoviruses are being evaluated for gene therapy of cystic fibrosis lung disease with the goal of reconstituting the expression of the cystic fibrosis transmembrane conductance regulator in pulmonary epithelia by direct administration of the virus into the airway. The therapeutic potential of recombinant adenoviruses is limited in part by the relative inefficiency by which gene transfer occurs. This study uses a human bronchial xenograft model to study adenovirus infection in the human airway in an attempt to define the molecular events that limit gene transfer. Our studies of the human airway confirm previous observations of cell lines that have indicated a two-step process for adenovirus entry, which begins with the binding of the virus to the cell through the fiber protein and continues with internalization via interactions among cellular integrins and an RGD motif (Arg-Gly-Asp) in the penton base. Furthermore, the level of maturity of the epithelia in xenografts has a major impact on gene transfer. Undifferentiated epithelia express high levels of alpha v beta 5 integrins and are easily infected with recombinant adenoviruses; gene transfer is completely inhibited with excess fiber and partially inhibited with RGD peptide and alpha v beta 5 integrin antibody. Pseudostratified epithelia do not express alpha v beta 5 integrin in differentiated columnar cells and are relatively resistant to adenovirus-mediated gene transfer; what little gene transfer occurs is inhibited by fiber but not by RGD peptide or alpha v beta 5 integrin antibody. These studies suggest that the expression of integrins in human airway epithelia limits the efficiency of gene transfer with recombinant adenoviruses. However, low-level gene transfer can occur in fully mature epithelia through alpha v beta 5 integrin-independent pathways.     

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

BACKGROUND: In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. METHODS AND RESULTS: As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. CONCLUSIONS: This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.     

Pseudomonas aeruginosa flagellin and alginate elicit very distinct gene expression patterns in airway epithelial cells: implications for cystic fibrosis disease

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa phenotypes relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lung by mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelial cells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast, infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.     

Neutrophil elastase induces mucus cell metaplasia in mouse lung

Goblet cell hyperplasia in the superficial airway epithelia is a signature pathological feature of chronic bronchitis and cystic fibrosis. In these chronic inflammatory airway diseases, neutrophil elastase (NE) is found in high concentrations in the epithelial lining fluid. NE has been reported to trigger mucin secretion and increase mucin gene expression in vitro. We hypothesized that chronic NE exposure to murine airways in vivo would induce goblet cell metaplasia. Human NE (50 microg) or PBS saline was aspirated intratracheally by male Balb/c (6 wk of age) mice on days 1, 4, and 7. On days 8, 11, and 14, lung tissues for histology and bronchoalveolar lavage (BAL) samples for cell counts and cytokine levels were obtained. NE induced Muc5ac mRNA and protein expression and goblet cell metaplasia on days 8, 11, and 14. These cellular changes were the result of proteolytic activity, since the addition of an elastase inhibitor, methoxysuccinyl Ala-Ala-Pro-Val chloromethylketone (AAPV-CMK), blocked NE-induced Muc5ac expression and goblet cell metaplasia. NE significantly increased keratinocyte-derived chemokine and IL-5 in BAL and increased lung tissue inflammation and BAL leukocyte counts. The addition of AAPV-CMK reduced these measures of inflammation to control levels. These experiments suggest that NE proteolytic activity initiates an inflammatory process leading to goblet cell metaplasia.     

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl(-) conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca(2+)-activated Cl(-) conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl(-)/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca(2+) signal in response to mucosal nucleotides that may contribute to the increased Cl(-)/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca(2+) signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.     

Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia

The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca(2+) signal in those cells undergoing a Ca(2+)-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca(2+) signal to activate Ca(2+)-dependent K(+) channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca(2+)-dependent K(+) channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca(2+) entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca(2+) entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca(2+) signal and the subsequent activation of Ca(2+)-dependent K(+) channels.     

Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.     

Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia

A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-mum periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to >or=7 microm are poorly understood. Using bumetanide as an inhibitor of Cl- secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated "blending" of both Cl- secretion and Na+ absorption must occur to effect ASL volume homeostasis. We then investigated how ASL volume status is regulated by the underlying epithelia. Cilia were not critical to this process as (a) ASL volume was normal in cultures from patients with primary ciliary dyskinesia with immotile cilia, and (b) in normal cultures that had not yet undergone ciliogenesis. However, we found that maneuvers that mimic deposition of excess ASL onto the proximal airways, which occurs during mucociliary clearance and after glandular secretion, acutely stimulated Na+ absorption, suggesting that volume regulation was sensitive to changes in concentrations of soluble mediators in the ASL rather than alterations in ciliary beating. To investigate this hypothesis further, we added potential "soluble mediators" to the ASL. ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl- secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume.     

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.     

FITC-induced murine pulmonary inflammation: CC10 up-regulation and concurrent Shh expression

We describe an immunohistochemical study of the acute and chronic effects of fluorescein isothiocyanate (FITC) on Sonic hedgehog (Shh) expression and Clara cell secretory protein (CC10) up-regulation in murine lung. FITC was dissolved in PBS and instilled non-surgically into adult mouse lungs via the trachea. During the acute phase (120h) of the FITC response, CC10 staining within Clara cells increased markedly but the protein did not leak into the tissue spaces or the airways, and no fibrosis was apparent. An immune response was evident, characterised by infiltrating T and B lymphocytes. There was no concomitant expression of Shh. During the chronic phase (6 months post-instillation), significant tissue degeneration was observed in the airways. There was moderate to severe fibrosis in the lung fields that stained positively for FITC and significant inflammatory cell infiltrate was observed. Shh was expressed, and CC10 showed multiple sites of diffuse staining consistent with release from Clara cells into alveolar air spaces. PBS controls showed no fibrosis after 6 months, but there was positive Shh staining below the airway epithelia and minimal extracellular CC10 staining. The results may throw some light on the role of CC10 in pulmonary inflammation. The relationship of Shh expression and CC10 leakage to lung damage and repair is discussed.     

Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR?(/)? pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR?(/)? epithelia showed markedly reduced Cl? and HCO?? transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR?(/)? pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl? conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl? and HCO?? in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.     

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes

Throughout the body, the epithelial Na⁺ channel (ENaC) plays a critical role in salt and liquid homeostasis. In cystic fibrosis airways, for instance, improper regulation of ENaC results in hyperabsorption of sodium that causes dehydration of airway surface liquid. This dysregulation then contributes to mucus stasis and chronic lung infections. ENaC is known to undergo proteolytic cleavage, which is required for its ability to conduct Na⁺ ions. We have previously shown that the short, palate lung and nasal epithelial clone (SPLUNC1) binds to and inhibits ENaC in both airway epithelia and in Xenopus laevis oocytes. In this study, we found that SPLUNC1 was more potent at inhibiting ENaC than either SPLUNC2 or long PLUNC1 (LPLUNC1), two other PLUNC family proteins that are also expressed in airway epithelia. Furthermore, we were able to shed light on the potential mechanism of SPLUNC1''s inhibition of ENaC. While SPLUNC1 did not inhibit proteolytic activity of trypsin, it significantly reduced ENaC currents by reducing the number of ENaCs in the plasma membrane. A better understanding of ENaC''s regulation by endogenous inhibitors may aid in the development of novel therapies designed to inhibit hyperactive ENaC in cystic fibrosis epithelia.Key words: mucociliary clearance, chronic airway disease, cystic fibrosis, protease, airway surface liquid, Na⁺ absorption