Studies on Airway Surface Liquid in Connection with Cystic Fibrosis

Transcription

1 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 393 Studies on Airway Surface Liquid in Connection with Cystic Fibrosis INNA KOZLOVA ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2008 ISSN ISBN urn:nbn:se:uu:diva-9358

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3 List of papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I II III IV V VI Kozlova I, Nilsson H, Phillipson M, Riederer B, Seidler U, College WH, Roomans GM. X-ray microanalysis of airway surface liquid in the mouse. Am J Physiol Lung Cell Mol Physiol 2005; 288: L874-L878. Kozlova I, Hühn MH, Flodström-Tullberg M, Wilke M, Scholte BJ, Roomans GM. Anaesthesia and the elemental content of mouse nasal fluid. Submitted, Kozlova I, Nilsson H, Henriksnäs J, Roomans GM. X-ray microanalysis of apical fluid in cystic fibrosis airway epithelial cell lines. Cell Physiol Biochem 2006; 17: Kozlova I, Vanthanouvong V, Almgren B, Högman M, Roomans GM. Elemental composition of airway surface liquid in the pig determined by X-ray microanalysis. Am J Respir Cell Mol Biol 2005; 32: Vanthanouvong V, Kozlova I, Johannesson M, Nääs E, Nordvall SL, Dragomir A, Roomans GM. Composition of nasal airway surface liquid in cystic fibrosis and other airway diseases determined by X-ray microanalysis. Microsc Res Techn 2006; 69: Kozlova I, Hjelte L, Roomans GM. Effect of mist tent on the ion content of nasal fluid in patients with cystic fibrosis. Submitted, Reprints were made with permission from the publishers.

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5 Contents Introduction Cystic Fibrosis The CFTR gene and CFTR protein Pathogenesis of cystic fibrosis lung disease The airways and airway epithelium Ion and water transport in the respiratory epithelium Airway surface liquid Survival and pharmacological treatment of CF Animal models Mouse models of CF Pig models of CF...25 Aims...27 Materials and methods Animals and anesthesia Mice (Papers I, II) Pigs (Paper IV) Cell lines (Paper III) Airway epithelial cell lines Liquid-liquid and air-liquid interface cell cultures Transepithelial electrical resistance (TEER) measurements ph measurements Frozen-hydrated samples (Papers I, III, IV) Pig tracheae and bronchi X-ray microanalysis Standards Mouse tracheae Liquid-liquid interface cell cultures Ion-exchange beads (Papers I, II, III, IV) Pig tracheae X-ray microanalysis Mouse tracheae Nasal fluid collection in mice Liquid-liquid interface cultures Plasma and serum samples (Papers I, IV)...36

6 3.5.1 Pig plasma and serum Mouse plasma and serum Morphological studies (Papers I, IV) Humans (Papers V, VI) Study design of paper V Study design of paper VI Defenition of chronic Pseudomonas aeruginosa colonization/infection Nasal fluid collection from humans X-ray microanalysis Standard calibration curve Statistical analysis...40 Results Paper I Paper II Paper III Paper IV Paper V Paper VI...45 Discussion Ionic composition of ASL in mice, pigs, air-liquid & liquid-liquid interface cell cultures (Papers I, II, III, IV) Ionic composition of nasal ASL in healthy and diseased humans (Papers V, VI)...52 Conclusions and future perspectives...57 Sammanfattning på svenska...59 Acknowledgements...61 References...63

7 Abbreviations F508 16HBE14o - ANOVA ASL ATP camp CF CFBE41o - CFTR EMEM ENaC PBS PCD PCL SEM Sephadex TEER TEM XRMA Deletion of phenylalanine in position 508 in the CFTR structure Human bronchial epithelial cell line (wild type CFTR) that maintains cell polarity and is transformed with an origin of replication defective simian virus (SV40) containing plasmid psvori -. The number of the clone is 16 and the number of passages is 14 originally Analysis of variance Airway surface liquid Adenosine triphosphate Cyclic adenosine monophosphate Cystic fibrosis Cystic fibrosis ( F508/ F508 homozygous) bronchial epithelial cell line that maintains polarity and is transformed with an origin of replication defective simian virus (SV40) containing plasmid psvori -. The number of passages is 41 originally Cystic fibrosis transmembrane conductance regulator Eagle s minimal essential medium Epithelial sodium channel Phosphate buffered saline Primary ciliary dyskinesia Periciliary layer Scanning electron microscopy Trade name for a cross-linked dextran gel with ionexchange capacity Transepithelial electrical resistance Transmission electron microscope X-ray microanalysis

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9 Introduction 1.1 Cystic Fibrosis Cystic fibrosis (CF), the most common fatal autosomal recessive disease among the white population, is characterized by dysfunctional chloride ion transport across epithelial cells causing the formation of a thick viscous secretion which plugs ducts in the lungs, intestine, pancreas, liver, and reproductive organs. A large number of symptoms, including sinus infections, poor growth, diarrhea and infertility, are the result of the effect of CF on the other parts of the body. Most of the CF patients have complications in the respiratory system, because of bacterial colonization, damage to the epithelium and exposure of matrix proteins that increase bacterial adherence. The cruel series of infection, inflammation and diminished mucociliary clearance eventually results in chronic obstructive lung disease and irreversible respiratory deficiency. CF occurs in approximately one in 2,500 live births. Approximately one in 20 whites carries a mutant CF gene allele [1]. Only about one in 17,000 black infants are afflicted, and CF is very rare in Asian populations [2]. Historical reference from the Middle Ages described children whose brows tasted salty and who died prematurely, but it was not until 1936 when Fanconi et al. were probably first to refer to the disease as cystic fibrosis with bronchiectasis and recognized it as a separate illness from celiac disease [3]. Andersen in 1938 described the clinical signs of pancreatic fibrosis and pulmonary disease in detail and used the term cystic fibrosis of the pancreas [4]. This disease was characterized by malabsorption of fat and protein, diarrhea, poor growth, and pulmonary infection. Pancreatic damage and lack of pancreatic enzyme secretion accounted for nutritional failure, which was assumed to lead to susceptibility to lung infection, often the final event. The thick, sticky mucus blocking the ducts of the airways as the main reason of the progressive lung disease was described by Farber who also gave rise to the alternative name mucoviscidosis [5]. The life expectancy was approximately 6 months and death often occurred from the lung infection. A significant discovery was made in 1948 by Paul di Sant Agnese, who detected that infants with CF had abnormal sweat with excess of sodium and chloride [6]. Elevated sweat chloride concentration offered a convenient 9

10 diagnostic test. The pilocarpine iontophoresis technique of Gibson and Cooke [7] allowed such testing in practice. Practically every patient with a clinical diagnosis of CF has a significantly high sweat chloride concentration, and hardly any other diseases produce elevated sweat electrolytes, but these diseases are clinically quite different from CF. Even milder patients without pancreatic insufficiency could be identified with the pilocarpine iontophoresis technique. The sweat test promptly became and remains the most common and reliable single parameter for differential diagnosis of CF, probably excluding genetic analysis. Paul Quinton in 1983 used sweat ducts to identify chloride transport as the basic physiologic defect in CF [8]. In parallel, Boucher and coworkers [9] and Knowles and colleagues [10] discovered increased sodium absorption across airway epithelia in CF patients. These results implied that abnormal absorption of Na + was dependent on the ENaC and was a regular characteristic of CF not only in sweat glands, but also in the airways. Only in 1989, the CF gene was discovered and its identity was examined using cells that originated from the sweat duct [11, 12]. This discovery demonstrated that the basic defect was in a camp-regulated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the CF gene, and this gave opportunities for new diagnostic tests, for research, and a hope for using gene therapy. At present, considerable progress in basic and clinical research has resulted in therapeutic improvements. Most patients are diagnosed before the age of two years. The life expectancy for patients with CF has improved significantly from 6 months to more then 30 years in the USA [3] and years in Sweden [13], with a treatment that does not depend on specific knowledge of the basic defect. Researchers around the world are still working on correcting the basic defect in CF and trying to find out new approaches to therapy which could help to extend and improve life of CF patients. For the future, several approaches have been proposed: (1) to treat the CF-related ion transport defects pharmacologically, decreasing sodium absorption and increasing chloride secretion; (2) to use gene therapy, inserting a normal copy of the CF gene into the appropriate cells. A possible future development is the use of stem cells for therapy. 10

11 1.2 The CFTR gene and CFTR protein The cystic fibrosis transmembrane conductance regulator gene is found in region q31.2 on the long (q) arm of human chromosome 7, is base pairs (bp) long and contains 27 axons (Figure 1). The CFTR gene encodes the CFTR protein which is 1480 amino acids long and has a molecular weight of 168,173 Da. The CFTR gene was identified in 1989 and RNA transcripts of CF gene were found in epithelial cells from various sources but not in brain or lymphoblast cell lines [11]. Figure 1. Gene location. Figure 2. Schematic drawing of the CFTR protein [11] The CFTR protein is anchored to the cell membrane of cells in the sweat glands, lung, pancreas, liver, digestive and reproductive tracts. The CFTR protein spans this membrane and acts as a channel connecting the cytoplasm to the surrounding fluid. This channel is primarily responsible for controlling the movement of chloride ions across the cell membrane. Sodium is the most common ion in the extracellular space and the combination of sodium and chloride creates a salt (NaCl), which is lost in large amounts in the sweat of individuals with CF. Structurally, the CFTR protein belongs to the ABC (ATP-binding cassette) class of ion channels or transporters (Figure 2). The protein possesses five domains: two membrane spanning domains each of which comprises 6 alpha helices and forms the chloride channel, two nucleotide-binding domains that bind and hydrolyze ATP (adenosine triphosphate), and a regulatory (R) domain. A regulatory binding site on the protein allows 11

12 activation by phosphorylation, mainly by a camp-dependent protein kinase [14]. Cystic fibrosis occurs when there is a mutation in both alleles of the CFTR gene. Based on their effect on protein synthesis and function, the numerous CFTR mutations are classified as follows [15] (Figure 3): Class I mutations that produce no protein due to a stop mutation or fatal errors in the CFTR mrna synthesis; Class II mutations in which the native CFTR fails to reach the apical membrane because of defective processing (e.g., F508 CFTR); Class III mutations that produce a protein that reaches the plasma membrane but fails to respond to camp; Class IV mutations that produce a camp-responsive channel with reduced conductance; Class V mutations that cause a decrease in the amount of CFTR proteins produced and that cause a small defect in maturation but normal or increased chloride transport activity (e.g., A455E, P574H); Class VI mutations that harbor nucleotide alterations that affect the regulatory properties of CFTR protein. Figure 3. CFTR mutations causing CF [15] 12

13 There are over 1300 different mutations that can produce CF and there are several mechanisms by which these mutations cause errors in the CFTR protein [16]. The most common mutation, F508 is a deletion ( ) of three nucleotides that results in the loss of the amino acid phenylalanine (F) at the 508 th position of the protein [11] (Figure 4). This mutation is present in 70% of the chromosomes of CF patients worldwide and 90% of CF patients in the United States [17]. The F508 mutation, for instance, creates a protein that does not fold normally. 95% of the F508-CFTR is retained in the endoplasmic reticulum, from where it is degraded by the ubiquitin-proteasome system [18]. Once the CFTR protein reaches the cell membrane, its turnover is relatively fast and the wild-type (wt) CFTR has a half-life of ~72 hours, while the half-life of F508-CFTR is ~4 hours [19]. The fully mature CFTR is a kDa protein and is recycled by endocytosis or degraded by the lysosomal proteases. Figure 4. The F508 deletion [11] 1.3 Pathogenesis of cystic fibrosis lung disease The airways and airway epithelium The airway is lined by a number of different cell types: columnar ciliated cells, non-ciliated columnar cells, goblet (mucus-secreting) cells, basal and Clara cells. Basal airway cells rest on a basement membrane and are instrumental in attaching the columnar cells to the basement membrane and underlying connective tissue [20]. The most abundant type of cell is the ciliated (fluid-secreting) columnar cells. Each cell has about 300 cilia on its apical surface [21] with an average length of 6 m [22]. In CF patients the number of microvilli per ciliated cell is decreased by 40% [23]. Epithelial cells have a unique structure and function [24]. The fundamental feature that characterizes an epithelial cell is its polarity. The apical membrane of the airway 13

14 epithelial cells has cilia. The cilia transport the mucus, which traps inhaled particles, towards the pharynx where it can be transferred to the digestive tract. Mucus is produced by goblet cells and by the submucosal glands. The columnar cells contribute to the mucus by secretion of fluid containing ions and water which forms a fluid layer around the cilia to facilitate their movement. The lateral membrane may have gap junctions and is adjacent to a lateral intercellular space. These two membranes are separated by a tight junction that encircles the cells and serves as a boundary between apical and basolateral membranes. Tight junctions have a selective permeability to ions and other solutes. Thus there are two pathways that ions and solutes may follow in crossing the epithelium: the cellular pathway (through the cells) and the paracellular pathway (between the cells). The ciliated cell is the only cell type expressing CFTR in nasal, bronchial, and proximal bronchiolar surface epithelia. But CFTR was not or sparsely present in the submucosal gland cells in tissue specimens from normal subjects [25]. The apical regions of ciliated cells of the airway epithelia of CF patients ( F508 mutation) were completely devoid of CFTR immunostaining signal, a finding confirmed by the absence of mature CFTR protein in biochemical studies of the same samples [26] Ion and water transport in the respiratory epithelium It is generally assumed that the ion transport processes in the airway epithelium are similar in the upper and lower airways [27]. During the last decades evidence has accumulated that a defective Cl - transport across epithelial cells plays a fundamental role in the etiology of CF. An understanding of ion transport abnormalities may ultimately lead us to a more rational development of a specific therapy. In the airway epithelia ciliated cells perform two main ion fluxes: Cl - secretion and Na + absorption (Figure 5) [28]. Figure 5. Ion and water transport in airways 14

15 Ion transport depends on the maintenance of an electrochemical gradient across the membrane. A Na-K-ATPase in the basolateral membrane pumps Na + out of the cell and K + into the cell, and as a result the intracellular concentration of Na + is low (20mM), and the concentration of K + is high (150 mm) [29]. With the help of this gradient a Na + /K + /2Cl - cotransporter may accumulate Cl - within the cell against its electrical gradient. Amassed Cl - leaves the cell through chloride channels in the apical membrane. Secretion of chloride is electrically coupled to efflux of K + through a basal K + conductance channel [30, 31]. In normal airway epithelium the paracellular water transport may be coupled to the secretion of chloride ions via a calciumdependent chloride channel (CaCC) and a camp-dependent chloride channel (CFTR) [32], which are located in the apical membrane of epithelial cells. It is now widely accepted that the CFTR Cl - channel is the predominant Cl - channel in the apical membrane of epithelial cells and that a genetic defect in the activity of this channel is the underlying cause of cystic fibrosis. It has been shown that CFTR also functions as a regulator of other ion channels, principally the epithelial Na + channel (Figure 6) [33, 34]. Figure 6. Multifunctional CFTR [32] Sodium is absorbed across the epithelium in a two-step process passive entry into the cell down an electrochemical gradient via a conductive pathway in the apical membrane and extrusion from the cell across the basolateral membrane by the activity of Na-K-ATPase pump [35]. Sodium channels are ubiquitous in nature and are classified either as neuronal voltagegated sodium channels, which are selectively blocked with neurotoxins or as epithelial sodium channels (ENaC), which are selectively blocked by amiloride [36]. Both the electrical and the chemical gradients favor sodium entry. 15

16 In cystic fibrosis airway epithelium there is excessive Na + (and volume) absorption and an inability to secrete Cl - (and volume) across the epithelium. These abnormalities would predispose airway secretion to dehydration, which results in defective mucociliary clearance and lung defense. Consequently, drugs that tend to reverse these abnormalities may have therapeutic potential. Both apical and basolateral cell membranes are normally relatively impermeable for Cl - [31]. However, several substances such as adenosine and epinephrine are able to induce Cl - secretion in CF [28]. Signals that regulate ion transport in many epithelia come from the basolateral surface of the cells. In the airways it seems convenient to have some regulation also on the apical surface to be able to respond to the demands on mucociliary clearance that inhaled particles may cause. Pharmacologic blocking of the epithelial Na + channel, which is rate-limiting for volume absorption from the airway surface, constitutes a novel therapeutic target. Studies of mucus clearance both in animal models and human subjects demonstrate that blocking of the epithelial Na + channel is associated with an acceleration of mucus clearance, suggesting that epithelial Na + channel blocking may indeed constitute a rational form of therapy for chronic bronchitis and for cystic fibrosis [37]. Intracellular ph (ph i ) is an important intrinsic factor controlling a multitude of cellular functions [38], and it has the potential to act as rapid signal for coordination of intracellular processes. Cellular mechanisms that contribute to regulation of the free intracellular activity of protons include metabolic production of protons and bicarbonate, an intracellular buffer system, and specialized transport mechanisms for these ions, e.g. Na + /H + and Cl - /HCO 3 - exchangers, proton ATPases, and conductive pathways for H + and HCO 3 -. The CF gene defect affects several ion transport mechanisms in the airway epithelial cell membrane, including apical Na + channels and Cl - channels. CFTR is able to transport both chloride and bicarbonate [39, 40], as evidenced, for example, by CFTR-dependent alkaline pancreatic fluid secretion [41, 42]. It has thus been postulated that defective CFTR-facilitated bicarbonate transport in the airways might produce an abnormally acidic ASL, which could promote airway infection in CF by reducing mucociliary clearance and antimicrobial action, and increasing the viscosity of ph-sensitive mucous glycoproteins [43, 44]. There is evidence that ASL and/or gland fluid ph might be regulated by cell membrane ion transporters such as CFTR, anion and cation exchangers, and H + pumps. In primary cell cultures of the airway surface epithelium, Coakley et al. [45] reported a relatively acidic ASL in CF cell cultures, with camp-induced alkalization in normal but not CF cells. They found evidence for ouabain-sensitive H + -K + -ATPase activity that could acidify ASL ph; however, its activity was similar in normal and CF cells. Krouse et al. [46] also reported ouabain-sensitive H + -K

17 ATPase activity in Calu-3 cells, a model of submucosal gland cells that secrete bicarbonate in response to forskolin. Fischer et al. [47] reported greater H + secretion in human tracheal epithelial cells than in Calu-3 cells, and inhibition of H + secretion by mucosal ZnCl 2. In contrast, Inglis et al. [48] reported that luminal acidification in pig distal airways could be inhibited by bafilomycin A1, an inhibitor of the vacuolar-type H + ATPase. Thus, although the evidence is somewhat conflicting, it appears that multiple H + /HCO - 3 transporters, including CFTR, are involved in establishing ph in primary gland fluid secretions and in maintaining/regulating airway surface fluid ph. Airway epithelial ion transport processes play a major role in the regulation of the volume and composition of the airway surface liquid (ASL) by generating osmotic gradients that provide a driving force for transepithelial fluid movement. CF-associated epithelial ion and fluid transport abnormalities include upregulated amiloride-sensitive Na + absorption and impaired Cl - secretion [49, 50] that lead to ASL depletion, which promotes mucus adhesion and chronic bacterial colonization with Pseudomonas aeruginosa [44, 51]. The chronic bacterial infection gives rise to an inflammatory reaction in which neutrophils dominate. Gradually, the lung tissue becomes fibrotic and lung function deteriorates. Treatment of the pulmonary disease of CF is symptomatic, and includes physiotherapy to remove the obstructive mucus, aggressive treatment with antibiotics, and, more recently, treatment with antibodies against Pseudomonas aeruginosa [52]. In the end stage of the disease, lung or heart-lung transplantation may be an option. Of note, fluid transport across the apical membrane of the respiratory epithelial cells is not only determined by chloride transport via CFTR but also by chloride transport by other chloride channels, and also by Na + -uptake via the ENaC channel. This has led to two alternative strategies in pharmacological treatment of CF, namely (1) attempts to activate other chloride channels (to provide compensatory chloride efflux), and (2) attempts to inhibit Na + - uptake via ENaC Airway surface liquid The airway surface liquid (ASL) is a thin layer of fluid covering the airways. The ASL possesses a sol layer (periciliary liquid layer (PCL)) that keeps mucus at an optimal distance from the underlying epithelium and through which cilia beat freely, and an overlying mucus layer that entraps particles to be transported by coordinated ciliary movement (Figure 7) [53, 54]. 17

18 Figure 7. Schematic model of the ASL [53] The ASL secreted by airway epithelium and submucosal glands is the first line of defense of the airway epithelium and ensures the mucociliary transport of inhaled particles. ASL contains glycoproteins such as mucins, and other proteins including defensins, lactoferrin, lysozyme, lipids, peptides, ions, and water. The ionic composition of the ASL plays a crucial role in airway host defense by controlling the ciliary activity, mucin release [55], and antimicrobial activity [55] in the airways. Studies of patients with cystic fibrosis have strongly suggested that the volume and/or composition of the fluid that lines the airway surface are important components of lung defense [51, 56, 57, 58, 59]. Abnormal composition and physical properties of the ASL and glandular secretions are proposed to promote chronic bacterial colonization of the airways by impairing mucociliary clearance (Figure 8). Figure 8. Normal and CF ASL [53] 18

19 Several hypotheses have been proposed to explain how mutations in the CFTR contribute to airway disease in CF. Based on measurements of the elemental composition of ASL obtained from CF patients and healthy subjects, together with in vitro and in vivo models using bronchial epithelial cells, one hypothesis proposes that mutations in CFTR cause abnormalities in electrolyte transport across airway epithelia leading to an elevation in the NaCl concentration in the ASL [56, 58, 60, 61]. The "high salt hypothesis" is based on meticulous experiments in which airway epithelial cells from normal and CF individuals were grown on filters. Bacteria were then placed into the fluid covering the apical surfaces of the cultured cells. The remarkable finding was that bacteria flourished in the cultures of CF airway cells, but were killed in the cultures of normal cells. Bacteria placed in the media beneath normal cells flourished, suggesting that normal cultured airway cells produce apical factors that kill bacteria. A further remarkable discovery was made when the salt content of the apical fluid was manipulated. Merely adding pure water to the fluid from CF cells rendered it bactericidal while adding NaCl to fluid from normal cells allowed bacteria to grow. Thus it could be concluded that both normal and CF airway epithelial cells release antibacterial substances into the surface liquid, but in CF cultures the antibiotics are rendered ineffective by a higher salt concentration. Other studies, however, have failed to detect differences in NaCl concentrations in ASL between CF patients and healthy persons [62, 63, 64, 65]. These studies have led to a second hypothesis in which mutations in CFTR promote increased absorption of isotonic ASL leading to dehydration and volume reduction of the ASL and impaired mucociliary clearance, leading to obstruction and infection [66]. The "low volume hypothesis" is not based on CFTR s function as a channel. Instead, it is based on CFTR s ability to inhibit the activity of the epithelial sodium channel (ENaC). According to this hypothesis, both normal and CF ASL have plasma-like levels of salt, but in CF, CFTR s inhibition of ENaC is eliminated, resulting in increased Na + uptake. Increased Na + uptake drives increased absorption of Cl - and water. Both hypotheses support the view that the ASL is abnormal in CF and that this abnormality arises as a consequence of the loss of CFTR activity. Both hypotheses have also focused attention on the mechanism by which abnormalities in ASL composition affect the function of airway epithelial cells. Figure 9 [67] summarizes the principal hypothesis-related abnormalities in ASL and glandular physiology in CF to chronic bacterial infection and progressive deterioration in lung function. The "low ph hypothesis" postulates that the ASL is abnormally acidic in CF, inhibiting mucociliary clearance mechanisms [43]. Defective CFTR-dependent bicarbonate transport in CF is proposed to acidify the ASL. 19

20 Figure 9. Hypotheses linking defective CFTR function to airway disease in CF [67]. The "low oxygenation hypothesis" postulates that the oxygen content of the ASL is low in CF airway epithelial cells and possibly slows oxygen diffusion in the ASL, resulting in enhanced Pseudomonas aeruginosa growth and biofilm formation and impaired clearance [68]. The "defective gland function hypothesis" postulates that the primary defect in CF is reduced fluid secretion by airway submucosal glands and possibly altered secretion of mucous glycoproteins [69, 70, 71]. One motivation for this hypothesis is that the much greater expression of CFTR in CF airway glands would impair salt and water secretion, resulting in reduced secretion fluid volume, increased protein concentration, and increased viscosity. Many studies have concentrated on the composition of the ASL, but it has been difficult to determine the exact composition of the ASL. It might seem odd that there could be a disagreement about something as simple as the composition of airway surface liquid in human airways. However, there is no consensus on this point because the volume of fluid to be sampled is tiny and rapidly altered when disturbed. Airway surface liquid has been estimated to be anywhere from μm in depth depending upon species and method. The sol layer is usually about the same depth as the cilia, or approximately 10 μm. If a typical thickness is taken as 30 μm, then a square cm area would contain only 3 microliters, and the entire surface of the conducting airways, an area about 2 m 2, would be bathed in 60 milliliters or 4 tablespoons of fluid [72]. Published data on the composition of the ASL are divergent and show a range from very hypotonic to hypertonic. Tracheal and/or bronchial ASL has been collected both from humans and from other species, mainly from mouse [67]. Published data for the ionic composition of human ASL 20

21 are fairly closely together and range from mm for Na + and mm for Cl - [60, 62, 65, 73]. For human xenographs in immunodeficient mice, however, lower values (64 mm Na +, 65 mm Cl - ) have been reported [74]. For the mouse the variation is very large: 6 mm Na + and 1 mm Cl - was reported by Baconnais and coworkers [75], 87 mm Na + and 57 mm Cl - by Cowley and colleagues [76], and values in the isotonic range (~ mm Cl - ) by several groups [77, 78, 79]. For monkey and rabbit, values for Cl - of 112 and 114 mm have been found [80]. High values were found for dogs, where Na + concentrations ranged from mm and the Cl - concentration was 134 mm [81], and in ferret where Na + was 167 mm and Cl mm [82]. Clearly hypotonic values were found for the rat, where Na + was 41 mm and Cl - 45 mm [83]. A number of different techniques for sampling and analysis have been used, and it would appear that, apart from possible species differences, one source of variation between the results is the sampling technique. In addition to the studies on mice mentioned above, Zahm and colleagues [84] give data in mmol/kg dry weight, which is not comparable with the absolute data in mm given in the other studies. Furthermore, studies have been performed on cell cultures of airway epithelial cells, but also these studies did not result in agreement being obtained. According to Matsui and coworkers [64], values for Na and Cl were nearly isotonic, whereas Zabner and colleagues [58] found values for Na and Cl around 50 mm, and McCray and colleagues [85] found a value for Cl of 18 mm. Widdicombe and coworkers [86] used X-ray microanalysis to measure the composition of ASL on top of cultured airway epithelial cells, but this study was purely qualitative. There is still little information about ASL composition in lower airways, where airway disease in CF has been postulated to begin. Also, there are few data about the possibility that ASL ionic composition might be altered in response to various agonists and factors released by inflammatory cells/bacteria or other components of CF airway fluid. CF patients are born with apparently normal lungs, but this is followed by the acquisition of chronic, unrelenting bacterial infections of the airways (bronchi) in the first few years of life. Thus, in the simplest view, CF lung disease reflects the failure of the innate defense mechanisms of the lung against inhaled bacterial organisms [54]. Viral infections occur in all infants with cystic fibrosis and later in life, most CF patients become infected with Pseudomonas aeruginosa, which once established persists usually for life. More than 80% of adult CF patients are chronically infected [87] and develop lung infection and airway inflammation that lead to respiratory failure, which is the most common cause of morbidity and mortality [88]. Clearance of mucus from the diseased airway is of primary importance because it minimizes the bacterial contamination, reduces infections and improves the quality of life of the patients. Effective clearance of mucus is a critical innate airway defense 21

22 mechanism and under appropriate conditions can be stimulated to enhance clearance of inhaled pathogens. It has become clear that extracellular nucleotides (ATP and UTP) and nucleosides (adenosine) are important regulators of mucus clearance in the airways as a result of their ability to stimulate fluid secretion, mucus hydration, and ciliary beat frequency (CBF) [89]. The mist tent therapy was a recommended treatment for CF patients in the 1960s and 1970s and aimed at increasing the hydration and to change the properties of mucus in order to make it easier for the patients to remove it from airways by coughing. It was assumed that water particles present in mist were deposited in the lower respiratory tract of the patients. The efficacy of the mist tent therapy was supported by clinical trials and by ventilatory function studies in patients with CF [90, 91]. Other studies re-examined the clinical effects of mist tent therapy in CF patients using clinical observations, and pulmonary function tests, and concluded that the nocturnal mist tent therapy had no beneficial effect for CF patients. The severity of the pulmonary disease and the type of nebulizer used had no apparent effect on the result [92, 93, 94]. The efficiency of the method was doubted and its use was largely discontinued. However, at a few CF centers mist tent therapy was regularly recommended particularly for small children with CF [95]. Potential ways to increase antimicrobial activity in the CF lung include increasing the secretion of antibacterial proteins and promoting the secretion of antibacterial factors that may not be present under basal conditions. Because bacterial killing activity is dependent on the protein concentration of antibacterial factors [96], this approach could be of benefit to patients with CF, irrespective of the issues about differences in the salt content of ASL. Although there are a large number of identified antibacterial proteins and peptides in ASL, and in other organs as well, a complete elucidation of these factors has not been achieved. Defensins, one of the most intensively studied classes of antimicrobial peptides, have been identified in a wide variety of animals, including birds, rodents, and humans [97]. Defensins are small cationic peptides containing arginine-rich amino acids with three disulfide bonds, which can be divided into - and - defensin subfamilies in human subjects [97]. The main function of defensins is believed to be to kill bacteria and fungi either on the surfaces of the epithelial cells or within phagolysosomes of phagocytes. 22

23 1.4 Survival and pharmacological treatment of CF In the 1950s when CF was first described, the children were dying of pancreatic insufficiency and failure to thrive. As the pancreatic enzyme replacement therapy solved the problem with the pancreas and children started living longer, the full picture of the respiratory problems became evident [98]. Nowadays the mortality in CF is associated with chronic obstructive pulmonary disease and respiratory failure being the primary cause of death among 90% of CF patients [99]. Double lung or heart-lung transplantation evolved to a standard treatment modality for patients with cystic fibrosis suffering from end-stage lung disease. Overall 3-year survival is about 60% in lungtransplant patients with cystic fibrosis [100] although patients infected with Burkholderia cepacia complex were reported to have a poor prognosis [101]. A time point at which a decision about transplantation needs to be made that most centers would agree upon is a forced expiratory volume in 1s (FEV 1 ) < 30% of the expected value in a patient receiving maximum medical treatment [102]. In another transplantation modality, living lobar lung transplantation, the time point of the surgery is also very critical but the decision is based on defining a point when a potential recipient is too ill to justify placing two healthy donors at risk of donor lobectomy [103]. Progress in the supportive care of CF patients had more than doubled the median age of survival in the past decades from 14 years in 1969 to over 40 in 2008 [104]. Country-specific differences in survival exist, which might be associated with availability and access to specialized care, treatment strategies and socioeconomic status [105, 106]. The most consistent aspect of therapy for cystic fibrosis is limiting and treating the lung damage caused by the thick mucus and the infections with the goal of maintaining the quality of life. Antibiotics have clinical benefits in patients with cystic fibrosis [107]. Intravenous, inhaled, and oral antibiotics are used to treat chronic and acute infections. Mechanical devices and inhaled medications are used to dilute and clear the abnormally viscous mucus. Many mechanisms have been proposed, including the effects on neutrophil function [108], IL-8 production [109], sputum rheology [110], goblet cell hypersecretion [111], the alginate biofilm produced by Pseudomonas aeruginosa [112], and direct antipseudomonal activity [113]. However, the precise mechanisms are still uncertain [114]. Other aspects of CF therapy involve treatment of diabetes with insulin, pancreatic disease with enzyme replacement, and infertility with advanced reproductive techniques. Since cystic fibrosis is a monogenic disease, gene therapy may become a possible treat- 23

24 ment. It is conceivable that drugs used by some of the patients, especially CF and allergy patients, could affect the salt content of the nasal fluid. However, the effect of drugs on the ion content of the nasal fluid has not been investigated, and it is therefore impossible to attribute particular changes to drugs used. This will require a separate, systematic investigation. 1.5 Animal models Mouse models of CF The cloning of the CFTR gene made it possible to generate mice, in which gene function was deleted either by homologous recombination, or by introduction of specific mutations in the murine CFTR gene [115, 116, 117]. This strategy was the first attempt to generate an animal model that reproduces the basic ion transport defects of human CF airways, such as deficient camp-dependent Cl - secretion and increased Na + absorption, to study their role in the in vivo pathogenesis of chronic airway disease. In several studies it was shown that CF mice exhibit characteristic defects in intestinal ion transport producing a severe CF-like gastrointestinal phenotype similar to meconium ileus in CF infants [115, 116]. Surprisingly, even when CF mice were rescued from neonatal mortality due to intestinal blockage by treatment with an osmotic laxative, lack of functional CFTR did not cause CF-like airway disease [117]. In contrast to CF patients, in whom chronic obstructive lung disease remains the major cause of morbidity and mortality, CF mice do not exhibit airway mucus plugging, goblet cell metaplasia or chronic inflammation and bacterial infection of the lower airways. Subsequently, detailed functional analyses of epithelial ion transport in different regions of the airways demonstrated that CF mice exhibit Na + hyperabsorption and deficient camp-dependant Cl - secretion, and an associated phenotype of ASL depletion and goblet cell metaplasia in the nasal epithelium [79, 117, 118]. Some studies demonstrated that a deficient CFTR-mediated Cl - secretion is compensated by CFTR-independent Ca 2+ -activated Cl - channels (CaCC) in the lower airways of CF mice [119, 120]. More recently, it became possible to measure mucociliary transport in small animals by a technique that determines the clearance of a fluorescent dye deposited into the lower airways with microdialysis [121]. Consistent with normal ion transport properties and lack of pathology in lower airways, it was demonstrated that in vivo mucus clearance rates are normal in CFTR-deficient mice [121]. In a normal airway, an appropriate balance between Na + absorption (mediated by ENaC in the apical membrane) and anion secretion (mediated by apical CFTR and alternative anion channels) determines the volume of fluid on the 24

25 airway surface. This transport balance regulates the height of the periciliary liquid (PCL) in which the cilia beat to permit effective mucus clearance (Figure 10) [122]. Mall and coworkers created a -ENaC overexpressing mouse to increase sodium absorption, this reduced periciliary liquid depth, impeding ciliary movement and mucus clearance. Normally, CFTR regulates ENaC activity, a process that does not occur in cystic fibrosis because CFTR is absent or defective. In -ENaC transgenic mice, and perhaps in cystic fibrosis airways, failure of mucus clearance promotes accumulation of airway secretory products, including glycosaminoglycans (GAGs) and a pool of chemokines (e.g., interleukin-8), neutrophil proteases and growth factors that promote airway inflammation and mucus cell hyperplasia [123]. Figure 10. Modeling cystic fibrosis [122] Pig models of CF Despite nearly 20 years of research and more than 5000 PubMed cited papers [124], knowledge of the underlying CFTR defect has not been effectively translated into improved clinical therapies. One reason has been poor understanding of how the CFTR defect produces CF lung disease. Transgenic mouse models of CF manifest subtle to no CF lung disease [125]. The long-awaited CF pig holds great promise as a model of CF (Figure 11) [126]. 25

26 Recent success in applying somatic cell nuclear transfer technology to generate heterozygous CFTR null and F508-CFTR knock-in pigs [126] assures that homozygous CF pigs are on their way. Pigs have already been used extensively to study a wide variety of lung functions and pathologies, such as surfactant biology, asthma, acute lung injury, and gene transfer [124]. Of relevance to CF, information already exists in pigs on airway salt transport, nasal potential differences, submucosal gland function, and mucociliary clearance mechanisms [127, 128, 129, 130]. Will CF pigs provide new insight into the pathogenesis of CF lung disease? Research into the link between CFTR dysfunction and CF lung disease has had adverse history. Figure 11. Photo of the first CFTR +/ - piglet taken at 1 day of age [126] The two major expected uses of CF pigs include research on the pathogenesis of CF lung disease and the testing of CF therapies. Hopefully, pigs will develop the major manifestations of human lung disease. Rogers and coworkers [131] showed many similarities between human and pig lungs: similar anatomy, histology, electrolyte transport, submucosal gland function, and immune and inflammatory responses. If CF pigs do not develop lung disease spontaneously, then exposure to Pseudomonas aeruginosa might initiate the process. However, a study reported by Ostedgaard and coworkers indicated partial cellular processing and significant plasma membrane targeting of porcine F508-CFTR [132], raising concerns about the phenotypic consequences of the F508 pig model. Another study by Liu and coauthors confirmed the species difference in cellular processing of F508-CFTR reported by Ostedgaard et al., but their results indicated rather mild processing defect of F508-pCFTR and suggested that the transgenic F508-CFTR pig model may not develop the desired CF phenotypes [133]. It was estimated that plasma membrane targeting of 10% F508-CFTR protein could avoid the CF phenotype in human subjects [134]. 26

27 Aims The aims of this study were to: To determine the composition of the ASL in mice, the apical fluid of normal (16HBE14o - ) and CF (CFBE41o - ) bronchial epithelial cell lines, and pigs, using two different methods: X-ray microanalysis of frozenhydrated samples and X-ray microanalysis of dextran beads equilibrated with the ASL or apical fluid, respectively. To find similarities and differences in structure and physiology of mouse, pig, and human airways. To carry out an ultrastructural study of the airway epithelium at various osmotic values. To investigate CFTR-mediated HCO 3 - conductance and the role of HCO 3 - in regulating ASL ph. To study the effects of agonists and antagonists of ion transport on changes in ion concentrations in mouse nasal fluid. To investigate the influence of repeated anaesthesia on the ion concentration in the mouse nasal fluid. To study the effects of antagonists on ion concentrations in the apical fluid of normal and CF bronchial epithelial cell lines. To determine the elemental composition of nasal fluid in healthy subjects, CF patients, CF heterozygotes, patients with rhinitis, or with primary ciliary dyskinesia (PCD). To investigate whether CF patients become colonized with Pseudomonads during mist tent treatment, and what effect a night spent in a mist tent has on the ion content of nasal ASL in CF patients and healthy controls. 27

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29 Materials and methods 3.1 Animals and anesthesia All animals were housed in an environmentally controlled vivarium and allowed free access to a standard pellet diet and water. All experimental procedures were conducted in accordance with guidelines of the Swedish National Board for Laboratory Animals and had previously been approved by the Swedish Laboratory Animal Ethical Committee in Uppsala Mice (Papers I, II) For the physiological experiments, ~50 NMRI mice (B&K, Sollentuna, Sweden) of both sexes, ~1 month old, were used. In addition, five NMRI mice of ~15-18 months were used as controls for comparison with CFTR(-/-) mice of similar age. The CFTR tm1cam (-/-) mice (female, ages months) were raised in Hannover (Germany), transported by air to Uppsala (Sweden), and kept for 1 week before the start of the experiment on a crude fiberdeficient diet with addition of a laxating salt solution (Oralav; B. Braun, Melsungen, Germany). These mice were anesthetized by spontaneous inhalation of isoflurane (Forene; Abbott Scandinavia, Kista, Sweden). The inhalation gas was administered continuously through a breathing mask (Simtec Engineering, Switzerland) and contained a mixture of 40% oxygen, 60% nitrogen, and 2.2% isoflurane. Before harvesting of the trachea, the mice were terminated by spinal translocation. The NMRI mice were anesthetized with pentobarbital. In some experiments, the anesthetized animals were injected intraperitoneally with pilocarpine (50 mg/kg body weight) or isoproterinol (10 mg/kg body weight), and the trachea was removed 15 minutes after injection. In experiments where nasal fluid was collected, the animals received an intraperitoneal injection with pilocarpine, isoproterenol (dose as above), or phenylephrine (10 mg/kg body weight). C57BL/6J mice (F12 backcross) heterozygous for the F508 mutation (Cftr Tm1EUR ) [135] were bred as heterozygotes and the offspring was genotyped by PCR analysis. Homozygous F508del mice (Cftr Tm1EUR ) and wild-type control mice were 29

30 temporally anesthetized with a solution of 2,2,2 tribromoethanol in 2- methylbutan-2-ol (Avertin). The mice were anesthetized on two subsequent days and nasal fluid was collected after anesthesia. After the first sampling, the animals were allowed to recover and it was noted that their behavior was normal, and no negative symptoms of the anesthesia were observed. After the last sampling, the animals were sacrificed, and nasal fluid was collected post-mortem Pigs (Paper IV) Ten healthy pigs of mixed breed (Hamshire, Yokeshire, and Swedish landrace) with a body weight of kg were used in this study. Before transport to the laboratory, premedication with 40 mg azaperon (Stresnil; Janssen Pharmaceutical, Beerse, Belgium) was given by intramuscular injection. Anesthesia was induced with 0.5 mg atropine (Atropine; NM Pharma AB, Stockholm, Sweden) in a mixture of 100 mg tiletamin and 100 mg zolazepam (Zoletile forte vet; Virbac Laboratories; Carros, France) diluted in 5 ml medetomidin 1 mg ml -1 (Domitor, Orion Corporation, Farmos, Finland); 1 ml per 20 kg body weight intramuscularly. The animals were placed in supine position on a heating pad and intubated with a cuffed endotracheal tube with 6.0 mm inner diameter and ventilated mechanically in a volume- or pressure-controlled mode with 3 cm H 2 O of positive end expiratory pressure (PEEP) (Siemens Servo 900C; Siemens-Elema AB, Solna, Sweden). The I:E ratio was 1:2. The tidal volume was 14 ml/kg plus compensation for dead space volume and the frequency was adjusted to an end tidal CO 2 of 5.5 kpa. A bolus injection of 0.2 mg fentanyl (Fentanyl; Antigen Pharmaceuticals, Roscrea, Ireland) was given intravenously after the intubation. Anesthesia was maintained by infusion of 5 ml kg/h of 4 g ketamin (Ketamin Veterinaria, Zürich, Switzerland), 1 mg fentanyl in 1,000 ml Rehydrex with glucose (Pharmacia and Upjohn, Stockholm, Sweden). The animals had no signs of respiratory infections or inflammation on visual inspection. The animals were also used for another study and sacrificed at the end of the experiment. 3.2 Cell lines (Paper III) Airway epithelial cell lines 16 HBE14o - (16HBE) and CFBE41o - (CFBE) (homozygous for the F508 mutation) cells were kind gifts of Dr D. C. Gruenert, San Francisco [136]. 30