PHYSIOLOGICAL REVIEWS   Vol. 79 No. 1 January 1999, pp. S215-S255
Copyright ©1999 by the American Physiological Society

Role of CFTR in Airway Disease

JOSEPH M. PILEWSKI AND RAYMOND A. FRIZZELL

Departments of Medicine and of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania

I. INTRODUCTION
II. GENETICS AND PHYSIOLOGY OF CFTR GENE MUTATIONS
    A. Cellular Genotype-Phenotype Comparisons: Classes of Mutations
    B. Correlation of Genotype With In Vivo Function
    C. Relation of CFTR Mutations to Disease Severity
    D. Importance of CFTR in Organ Physiology and Development of Different Organs
III. CLINICAL COURSE OF CYSTIC FIBROSIS: TURNING POINTS IN PATHOGENESIS
    A. Earliest Pathological Manifestations of Pulmonary Disease
    B. Airway Infection and Inflammation Lead to Bronchiectasis
    C. Role of Inflammation in the Progression of Airway Pathology
IV. DETERMINANTS OF AIRWAY SURFACE LIQUID
    A. Basic Principles
    B. Transport Functions of Proximal Airways
    C. Other Transport Functions of CFTR
    D. Airway Fluid Transport and Water Permeability
    E. Composition and Thickness of Airway Surface Liquid
    F. Lessons From Other Genetic Diseases
V. MUCOCILIARY CLEARANCE
    A. Factors Contributing to Normal Clearance
    B. Mucociliary Clearance and Sputum Properties in CF
    C. Effect of Salt Concentration on Mucus Transport
    D. Comparison With Dyskinetic Cilia Syndromes
VI. AIRWAY INFECTION
    A. Organisms and Their Mechanisms
    B. How the Airway Environment in CF Permits Infection
VII. INFLAMMATORY MECHANISMS
    A. Immune Processes: Defects in Opsonization
    B. Defects in Anti-inflammatory Cytokines: Interleukin-10
    C. Oxidant Environment and Glutathione Transport
    D. Defective Apoptosis Related to CF Mutations
    E. Proinflammatory Effects of Bacterial DNA
VIII. SUMMARY
REFERENCES

	ABSTRACT

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Pilewski, Joseph M., and Raymond A. Frizzell. Role of CFTR in Airway Disease. Physiol. Rev. 79, Suppl.: S215-S255, 1999.  Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), which accounts for the cAMP-regulated chloride conductance of airway epithelial cells. Lung disease is the chief cause of morbidity and mortality in CF patients. This review focuses on mechanisms whereby the deletion or impairment of CFTR chloride channel function produces lung disease. It examines the major themes of the channel hypothesis of CF, which involve impaired regulation of airway surface fluid volume or composition. Available evidence indicates that the effect of CFTR deletion alters physiological functions of both surface and submucosal gland epithelia. At the airway surface, deletion of CFTR causes hyperabsorption of sodium chloride and a reduction in the periciliary salt and water content, which impairs mucociliary clearance. In submucosal glands, loss of CFTR-mediated salt and water secretion compromises the clearance of mucins and a variety of defense substances onto the airway surface. Impaired mucociliary clearance, together with CFTR-related changes in the airway surface microenvironment, leads to a progressive cycle of infection, inflammation, and declining lung function. Here, we provide the details of this pathophysiological cascade in the hope that its understanding will promote the development of new therapies for CF.

	I. INTRODUCTION

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In patients with cystic fibrosis (CF), pulmonary disease is the major cause of morbidity and mortality. With the development of treatments for intestinal obstruction and pancreatic insufficiency, patients typically survive beyond infancy, and at some point, virtually all patients develop chronic bacterial infection, abnormal airway secretions, and airway inflammation. This typically results in progressive bronchiectasis, respiratory failure, and death. However, the question of how CF transmembrane conductance regulator (CFTR) mutations cause lung disease continues to be one of the most perplexing and poorly understood chapters in the story of CF and airway epithelial cell pathophysiology. In short, we know that the CFTR is a regulated anion channel that accounts for the cAMP-regulated Cl conductance of the apical membranes of airway epithelial cells, and we know that CFTR mutations either eliminate or markedly impair this conductance pathway. But the question of how the loss of CFTR causes CF remains incompletely understood. Several years ago, there were educated guesses regarding the possible links from CFTR to lung disease; today, there are well-reasoned and testable hypotheses. Hopefully, the next several years will provide an understanding that provides for more effective treatments.

Why focus on CFTR's function as an ion channel to explain CF pathology? First, in exocrine tissues and intestine, the pathophysiology of CF appears to be explained well by CFTR's channel function. Intestinal obstruction in CF is due to a drying out of the luminal contents that is caused by insufficient net fluid secretory activity. The elevated salt in CF sweat and salivary secretions is explained by a lack of anion channel function (218). Second, if one surveys different organs or across species, the pathophysiology of CF generally varies with the capacity of different epithelia to express alternate ion channel pathways. This appears to at least partly explain the relative absence of lung pathology in CF mice, and it is the lead theory for why different strains of mice show different degrees of intestinal impairment when the CF gene is knocked out (see review by Grubb and Boucher, this supplement). These findings suggest that other anion channels can compensate for the loss of CFTR. Finally, most mutations that interfere only with the magnitude of the CFTR Cl conductance, as opposed to its processing and targeting to the plasma membrane, produce CF, but usually in a milder form (254, see sect. IIC). Thus the simplifying principle is that lung disease is manifest in some way because of the absence of a cAMP-regulated anion channel in airway cells, and this leads either to physical or compositional impairment in the properties of the airway surface fluid that make the mucous gel more difficult to clear and easier for bacteria to colonize.

In this review, we first summarize the physiologically relevant molecular genetics and clinical turning points in the pathogenesis of CF lung disease. We then attempt to relate the cellular functions that have been ascribed to CFTR to the properties of the ASL, its role in mucociliary clearance, and to aberrant inflammatory mechanisms that may explain how mutations in CFTR cause pulmonary disease.

	II. GENETICS AND PHYSIOLOGY OF CFTR GENE MUTATIONS

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To date, over 700 mutations in the CFTR gene have been associated with a CF disease phenotype (S. FitzSimmons, CF Foundation, personal communication). From a physiological perspective, the grouping of mutations into five classes based on the primary mechanism responsible for reduced CFTR Cl channel function has provided a useful framework for considering genotype-phenotype relationships. As proposed by Welsh and Smith (313) and summarized in Figure 1, class 1 mutations, such as G542X and R553X, are those in which stop codons or frameshift mutations lead to premature termination of mRNA translation, and thus essentially no protein production. In class 2 mutations, CFTR protein fails to mature properly in the biosynthetic pathway, with degradation of translated protein before it can progress past the endoplasmic reticulum (see review by Kopito, this supplement). The F508 mutation, the prototypic class 2 and the most common CF mutation, results in a temperature-sensitive defect in protein processing; at 37°C, little or no mature protein is detectable at the plasma membrane (51), but at 27°C, some F508 CFTR traffics to the cell membrane where it forms partially functional Cl channels (50, 72, 171). Most laboratories agree that F508 CFTR exhibits a reduced open probability, however (72). Thus both class 1 and 2 mutations prevent sufficient CFTR expression at the cell membrane. As would be expected, clinical studies have confirmed that these mutations are associated with typical multiorgan disease, including male infertility, pancreatic insufficiency, and progressive obstructive pulmonary disease.

View larger version (118K): [in this window] [in a new window]   FIG. 1.   Classes of cystic fibrosis (CF) gene mutations. CF mutations can be divided into 5 classes that define the mechanism for defective chloride conductance. In normal epithelia (top panel), the CF gene is transcribed into mRNA, which is translated in the endoplasmic reticulum (ER) to cystic fibrosis transmembrane conductance regulator (CFTR) protein. After translation, nascent CFTR is glycosylated in the Golgi apparatus before insertion in the cell membrane. In class 1 CF mutations, there is failure of CFTR translation, typically due to stop mutations such as G542X. In class 2 mutations, which include the most common CF mutation, F508, CFTR fails to mature and is degraded by proteases in the ER. Class 3 mutations are fully processed and inserted in the membrane, but mature protein is refractory to activation, as for example, the G551D mutation fails to conduct chloride in response to stimulation with protein kinase A. In class 4 mutations, the mature protein is activated normally, but the chloride conductance of channel is diminished. Finally, class 5 mutations are splice site mutations that result in decreased abundance of full-length mRNAs, hence a decrease in the quantity of fully functional CFTR at cell membrane. [Modified from Tsui, L.-C., and P. Durie. Hosp. Pract. 32: 115, 1997. Original illustration by Seward Hung.]

In contrast, class 3 and 4 mutations allow protein production and transit to the apical surface, but they result in channels that are insensitive to activation or display altered Cl conductance. Class 3 mutations, such as G551D, are regulatory mutations in which single amino acid substitutions or deletions result in a properly processed protein that is virtually insensitive to channel activation. For example, reduced ATP binding to G551D CFTR results in a severely diminished macroscopic Cl conductance (173) that would intuitively be associated with a severe disease phenotype. Clinical studies have confirmed this prediction, because there is no evidence that either pancreatic or pulmonary disease severity in these patients differs significantly from patients with class 1 or 2 mutations (102). Class 4 mutations, such as R117H, R347P, and R334W, respond to activation by cAMP agonists but exhibit reduced Cl channel conductance or channel open probability (254). As such, these mutations would be expected to result in mild disease manifestations, and several reports have confirmed that this is the case for pancreatic disease (11, 65, 90, 112, 159).

More recent studies have suggested that splice site mutations, which affect the efficiency of normal mRNA splicing and thereby alter the abundance of normally processed and functional CFTR at the cell membrane, should be considered a fifth class of CF mutations. The prototype of this class is the 3849+10 kb C to T mutation, in which a nucleotide substitution at a splice site reduces but does not preclude correct mRNA splicing. Consequently, this mutation yields at least some mRNA capable of producing functional protein that would be expected to be associated with mild disease. Clinical correlation with the limited number of patients who have this mutation has documented a mild phenotype for pancreatic disease, and surprisingly, this is true for the male genital tract as well (109, 268). Congenital bilateral absence of vas deferens (CBAVD) is a phenotype that is normally very sensitive to mutation of CFTR (see sect. IID).

Several approaches have been used to demonstrate ion transport differences in vivo among the classes of CFTR gene mutations, and in general, these have provided evidence for residual Cl secretory capacity in some class 4 mutations. One approach has been to measure transepithelial potential difference across the nasal epithelium to assess amiloride-inhibitable Na transport and cAMP-mediated Cl secretion (see Ref. 149 for review of technique). In patients with gene mutations in which CFTR traffics to the membrane (G551D, A455E, R117H), there was significantly more residual Cl secretion than in patients with mutations that do not permit protein synthesis or trafficking (F508, W1282X, Q493X) (111). Moreover, there was a positive correlation between the amount of residual Cl secretion and the forced expiratory volume, an indicator of airway function. As predicted by the class of mutation paradigm, no Cl secretory response was observed in patients homozygous for F508 or in those with a F508 mutation and a truncation mutation (G542X or R553X). Thus there is suggestive evidence for a correlation between gene mutations and ion transport function; however, the sensitivity of the nasal potential difference assay for residual Cl secretory activity, and the correlation with disease severity, remains to be determined.

A second in vivo approach to correlate genotype with physiology has been to examine the relationship between genotype and carbachol-induced Cl secretion in rectal biopsies. Compared with patients with class 1 or 2 mutations, patients with an A455E mutation had higher residual Cl secretion. This was associated with a later age of CF diagnosis, a lower incidence of pancreatic insufficiency, and a higher achieved age (304). Collectively, the in vivo studies of nasal and rectal epithelial function support the in vitro observation that at least some class 4 mutations permit residual Cl secretion that reduces disease severity. Of note is that residual Cl secretion was also observed in a subset of homozygous F508 patients. This suggests that other factors, such as the expression of other Cl channels, or the trafficking of some mutant F508 CFTR to the membrane, or perhaps genetic polymorphisms, contribute to the variations in disease severity.

Recently, several polymorphisms within the CFTR gene have been identified and found to influence the penetrance of some CFTR mutations. Chu and Cutting (55) identified variations in the length of the polypyrimidine tract in the intron 8 splice acceptor site (the Tn locus) and found that three length variants were associated with varying efficiencies of exon 9 splicing. The 5 thymidine (5T) variant was associated with inefficient splicing and frequent transcripts lacking exon 9, which is critical for formation of a functional CFTR protein (271). In a subsequent study of the relationship between polypyrimidine variants and the phenotype of patients with the R117H mutation, the 5T variant was most clearly associated with the typical CF phenotype, whereas the 7T variant was observed both in patients with pancreatic sufficient CF and in patients with CBAVD alone (137). Thus it appears that the 5T variant often leads to lower levels of partially functional R117H-CFTR, and hence clinical CF. The splice efficiency of the 7T variant is not consistent; thus the combination of R117H and 7T may or may not result in clinical disease.

The presence of other polymorphic loci has been proposed to account for the partial penetrance of polymorphic Tn loci and may also contribute to the observed phenotypic variability within CFTR mutations. Evidence for partial penetrance of the 5T allele was recently derived from an analysis of the 5T allele in a large ethnically similar population. An increased frequency of the 5T allele was found in patients with CF or atypical CF who did not have CFTR mutations on the chromosome carrying the 5T allele. Moreover, within families, the same 5T allele was associated with a wide range of clinical presentations, from healthy fertile male to CBAVD to clinical CF, supporting the notion that the 5T allele is a splice variant with partial penetrance (134). Two other polymorphisms were recently demonstrated to contribute to the variable penetrance of the 5T allele. Cuppens et al. (62) examined the effects of polymorphisms at the (TG)m and M470V loci and found that a higher number of TG repeats on the 5T allele was associated with disease, whereas a low number was observed in healthy CF fathers. Moreover, the number of TG repeats influenced the exon 9 splice acceptor efficiency, and CFTR Cl channel activity varied with the polymorphism at the 470 locus. These data provide strong evidence that polymorphisms contribute to the partial penetrance of the 5T allele and suggest that such polymorphisms may contribute to heterogeneity in both CFTR Cl channel conductance and disease phenotype among individuals with the same CFTR mutation.

Heterogeneity in CF phenotype has raised the logical question of whether the clinical variability could be explained on the basis of genotypic differences, with the hypothesis that mutations having residual CFTR function would be associated with milder phenotypes. Several approaches have been used to correlate genotype with phenotype, including the in vivo ion transport studies discussed above and a number of epidemiological studies. The large case control analysis from the multinational CF genotype-phenotype consortium clearly demonstrated that certain mutations are associated with pancreatic sufficiency; however, a correlation between genotype and severity of pulmonary disease could not be identified. Comparison of F508 homozygous patients with a limited spectrum of compound heterozygotes revealed that the class 4 R117H mutation was associated with pancreatic sufficiency, later age at CF diagnosis, and lower sweat Cl concentrations (101). Other studies have demonstrated that another class 4 mutation (A455E) and a class 5 mutation (3849+10 kb C to T) are also associated with pancreatic sufficiency (109). These studies demonstrate that at least some class 4 or 5 mutations are associated with mild pancreatic disease and thereby support the notion that for pancreatic function, a class 4 or 5 mutation provides enough residual CFTR Cl channel function to result in a mild phenotype.

Case control studies that have attempted to correlate genotype with pulmonary disease severity have disclosed that A455E (a class 4 mutant) is associated with mild disease (88); however, studies of groups of patients with other uncommon mutations, including siblings, have revealed wide variation in pulmonary disease severity. Patients with an A455E mutation had a higher likelihood of pancreatic sufficiency, better pulmonary function, and a lower likelihood of airway colonization with Pseudomonas aeruginosa (88). Interestingly, two studies have demonstrated that heterologous expression of A455E CFTR resulted in a diminished but significant halide permeability compared with wild-type CFTR (253, 308). In contrast, nasal potential difference studies to determine the presence of a cAMP-mediated Cl secretory response in nasal epithelium of A455E patients revealed a modest response in only one of five patients (308). Thus there appears to be a correlation between in vitro Cl secretion and disease severity for some class 4 mutations. The failure to discern a nasal potential difference response could reflect a low expression level of CFTR in surface epithelium or indicate that the nasal potential difference assay lacks sensitivity for detection of residual Cl secretion.

The larger study by the CF genotype-phenotype consortium (101) failed to identify differences in pulmonary disease severity between F508 homozygous patients and those who were compound heterozygotes for F508 and several other mutations, including G542X, R553X, W1282X, N1303K, 621 + 1G to T, 1717-1G to A, and R117H. The wide intragenotype variation in pulmonary disease severity has been proposed to reflect environmental factors, the presence of polymorphisms or modifier genes (see above), or differences in therapy and/or patient compliance. Together with the limited number of patients having a class 4 or 5 mutation, the variability in pulmonary disease makes identification of mild pulmonary mutations more difficult. A recent longitudinal analysis of pulmonary function in CF patients revealed that F508 homozygous patients had a higher rate of pulmonary function decline than patients who were heterozygous for F508 or had two non-F508 mutations (60). Similar analyses of larger cohorts of patients having "mild" mutations, or collection of survival data from large registries, may eventually prove more fruitful than case control studies for determining the influence of genotype on pulmonary function.

Considered collectively, the studies of CFTR channel function in vitro and correlation with in vivo ion transport and disease phenotype suggest that the amount of residual Cl channel function of a given mutation influences disease severity in the pancreas and, in some cases, the lung. At least some class 4 mutations permit sufficient CFTR function to yield a less severe phenotype (159); however, other factors must be implicated to account for the wide variation in disease severity within a group of patients having the same mutation. Moreover, further studies are necessary to determine whether residual CFTR Cl channel function causes a less severe disease phenotype or whether such function is merely a marker of nonchannel CFTR function that may be more important in pathophysiology (see below).

Recent studies have suggested that variations in the organ system manifestations of CF reflect differences in the level of CFTR function necessary for normal organ function. These variations are particularly intriguing for male genital tract disease. Over 95% of males with multiple organ manifestations of CF suffer from infertility due to bilateral absence of the vas deferens. At the other end of the clinical spectrum are patients with CBAVD but no recognized pulmonary, pancreatic, or sinus disease. Close evaluation has revealed that more CBAVD patients are heterozygous at the CFTR locus (50 vs. 4% in the general population). In addition, several patients have two CFTR mutations, one of which is R117H (70). Other studies have found a high incidence of the 5T exon 9 splice variant on the normal allele in the heterozygous CBAVD patients (52), suggesting that a reduction of functional CFTR to ~10% of wild-type levels may result in genital tract disease. These observations suggest that CBAVD is a form of CF disease, and they further demonstrate that mild mutations, such as R117H, may provide sufficient residual Cl secretory capacity to prevent the development of typical CF pulmonary and pancreatic disease.

The correlation between the level of functional CFTR and phenotype has been further extended to estimate the reduction of functional protein necessary in the lung and pancreas to cause disease [proposed by Davis et al. (64) and summarized in Table 1]. Individuals carrying one mutant allele (heterozygotes or disease carriers) are expected to express 50% of normal CFTR, and they have no disease phenotype. This suggests that a 50% reduction in CFTR expression is developmentally and physiologically insignificant. Patients with A455E plus a severe CFTR mutation are estimated to have ~4% of normal CFTR function, since ~8% of A455E CFTR reaches the cell surface (253). Most of these patients have high sweat Cl concentrations but milder CF pulmonary disease and pancreatic sufficiency. This suggests that lung and sweat duct dysfunction occurs when the level of functional CFTR is less than ~5%. With the severe mutations (class 1, 2, or 3), the level of functional CFTR is generally <1%. Patients with two of these mutations typically present with pancreatic insufficiency in addition to severe pulmonary disease, suggesting that pancreatic disease occurs with <1% functional CFTR. On the basis of this analysis, the rank order of organ susceptibility to CFTR mutations from the most-to-least sensitive is the vas deferens, the sweat duct, the lung, and the pancreas. Exceptions to this generalization, such as the observation of fertility and significant but delayed onset pulmonary disease in male patients with the splicing mutation 3849+10 kb C to T (268), suggest that other mechanisms, such as alternative splicing in different organs (281) or organ-specific modifier genes, contribute to this already difficult effort of rigorously defining the correlation between genotype and phenotype.

	III. CLINICAL COURSE OF CYSTIC FIBROSIS: TURNING POINTS IN PATHOGENESIS

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As discussed briefly in section I, obstructive pulmonary disease is the major cause of morbidity and mortality in CF. Before turning to a discussion of the pathogenesis of CF airway disease, we review the clinical turning points to place the subsequent discussion of ASL within a broader context.

Clinical and pathological studies have suggested a number of turning points in the generally progressive course of CF (summarized in Table 2). It is noteworthy that although the CF lung is macroscopically normal at birth, subtle abnormalities in mucus secretion appear very early and may represent the first turning point in pathogenesis. Pathological descriptions of mucus inspissation in submucosal glands as early as the second trimester of development imply that abnormal mucus secretion occurs in CF in the absence of airway infection (203). Histopathological analysis of the conducting airways from six of seven fetuses with CF revealed dilatation of the tracheal submucosal glands with accumulation of inspissated mucus. Similar changes have been observed in CF newborns dying of meconium ileus (76, 77); however, a separate group was unable to confirm consistent submucosal gland pathology in newborns (54). Moreover, the specificity of gland dilatation to CF has been called into question by other investigators who found similar pathology in submucosal glands in newborns dying of other airway diseases (201). These uncertainties may relate to differences between the late fetal and postnatal lung. Among the pathological studies, however, the studies of CF fetuses and newborns imply a defect in mucus secretion that precedes infection.

The link between CFTR and the histological findings during lung development is unclear, because CF mutations do not appear to alter morphogenesis. The CFTR is expressed in conducting airway during lung development. With the use of sensitive assays (reverse transcription PCR), CFTR mRNA was detected in the lung of 18-wk human fetuses (108). Subsequent studies in human and rabbit fetuses confirmed expression of CFTR mRNA in the pseudoglandular stage of lung development and demonstrated expression of CFTR protein from the pseudoglandular stage through birth (184, 186). In situ hybridization revealed CF gene expression in both large bronchi and small airway epithelium throughout lung development, with decreasing expression in distal epithelium in the later developmental stages (283). Interestingly, although CFTR is abundantly expressed in the serous cells of submucosal glands in postnatal lung (74, 121), no CF gene expression was observed in fetal submucosal glands (283). Moreover, despite differences in the volume of lung secretions and transepithelial potential difference between CF and non-CF second trimester fetal lung explants maintained in short-term organ culture, there were no gross morphological differences in second trimester fetal lungs (184). Collectively, these data suggest that CFTR does not play an important role during lung development, presumably because of alternative secretory pathways, such as the ClC-2 Cl channel (197). The importance and mechanisms of secretory pathways in lung development is beyond the scope of this review; the reader is referred to a number of recent reviews in this area (28, 269).

Although it has been suggested recently that there is a lack of causation between airway infection and inflammation in the CF airway (see sects. VI and VII), bacterial infection and airway inflammation appear to be the second and third turning points in the pathogenesis of CF airway disease. The airways of CF patients are preferentially colonized by specific bacterial pathogens, often in the first year of life. Studies of CF patients from the clinically well newborn to the severely affected adult have implicated airway inflammation as critical to the pathophysiology of CF lung disease, with most clinical studies suggesting that bacterial infection drives the airway inflammation. With the advent of newborn screening for CF, patients were identified before the onset of overt pulmonary disease, allowing for serial assessments of bacterial colonization and an early evaluation of lower airway inflammation. These studies have demonstrated an evolution of bacterial pathogens: Staphylococcus aureus (SA) and Hemophilus influenzae (HI) appear to inhabit the CF airway early, often before the onset of clinical symptoms, while airway infection with Pseudomonas aeruginosa (PA) almost universally follows these other pathogens (1). The age at first positive culture for SA was significantly earlier (mean of 12.4 mo) than for PA (mean of 20.8 mo), and patients infected with PA typically had SA and HI isolated from the airway before infection with PA. Other studies have confirmed this sequence of bacterial infection (133). Moreover, infants in whom PA could be isolated were more likely to have chronic cough and had a higher frequency of hospitalizations for respiratory disease than patients without PA in lower airway cultures (1). This suggests that the presence of PA has pathophysiological significance (see below). Persistent bacterial isolation from the CF airway has been considered colonization, which implies a harmless interaction between host and organism. However, the above clinical studies and the observation that inflammation and protease excess persists through periods of clinical stability (see below) support the contention that bacterial persistence represents a harmful stimulus to the airway by driving inflammation. In brief, the CF airway may most accurately be perceived as chronically infected with bacterial pathogens rather than colonized (44).

The implication of these clinical observations is that PA, particularly the mucoid strain, plays a major role in the pathogenesis of CF airway disease and that acquisition of mucoid PA be considered a fourth turning point in pathogenesis. An alternative explanation has been that PA colonization is not itself pathogenic but merely reflects the severity of airway dysfunction. One attempt to resolve this issue was to examine the relationship between colonization with PA and the decline in pulmonary function. In a longitudinal study of bacterial isolates and clinical course, Kerem et al. (132) found that persistent isolation of PA from the sputum or throat was associated with 10% lower lung function relative to patients in whom PA was not isolated. This suggested that airway infection with PA is pathogenic and not merely a marker of airway pathology.

Studies of the clinical course of CF suggest that bacterial infection of the airway leads to an inflammatory response. Persistence of infection and inflammation through periods of clinical stability (156) ultimately leads to bronchiectasis, that is, to abnormal and generally irreversible dilatation of the airways. More recent studies using samples from the lower airway of infants with CF have confirmed early airway infection with SA and supported the notion that bacterial infection begets airway inflammation. As expected, the majority of infants who had greater than 10⁵ cfu bacteria/ml of lower airway fluid had increased numbers of neutrophils and higher total cell counts. The few infants with increased inflammatory cells without bacterial or viral isolates were thought to have an alternative etiology, such as aspiration lung disease (12, 13). However, a second study raised the question of whether inflammation precedes significant airway infection. Seven of 19 CF infants who had negative cultures for bacterial pathogens or common respiratory viruses had evidence of airway inflammation [increased leukocytes and concentration of the potent chemoattractant interleukin (IL)-8] (136). Although plausible explanations for this finding are that the sensitivity of bronchoscopic sampling for detection of viruses and bacteria is suboptimal, or that inflammatory cells persist during the resolution phase of a subclinical infection, this observation raised the intriguing hypothesis that airway inflammation may precede bacterial infection in the CF airway. A recent study by Armstrong et al. (13), however, identified a larger cohort of infants lacking a detectable airway pathogen. In this larger population, several markers of inflammation [bronchoalveolar lavage (BAL) cell count, concentrations of IL-8, and elastolytic activity] were no different from a control population of infants with stridor, a condition characterized by upper airway obstruction (13). Moreover, serial BAL samples from the same individuals suggested a close correlation between inflammatory markers and the presence of bacterial pathogens. Thus the findings of Khan et al. (136) that suggest a discordance between airway inflammation and infection remain to be confirmed.

Studies describing the sequence of pathological changes in the lung indicate that the submucosal glands are involved uniformly and that airway disease involves both proximal and distal airway segments. Morphological changes in the airway wall and lumen occur shortly after hyperplasia and obstruction of tracheal and bronchial submucosal glands (22, 267). Inflammatory infiltrates in the airway submucosa and plugging of bronchi and bronchioli with mucus and inflammatory cells were observed in the majority of patients who died in the first 4 mo. However, the hallmark pathological change in CF, bronchiectasis, was unusual at this age (22). In a subsequent morphological study, changes in the more distal airways varied depending on the age at death, with airway dilatation being prominent in younger patients (267). More recent studies have suggested that over time, chronic infection and inflammation in the proximal airway lead to a destruction of bronchial cartilage (200) that contributes to both expiratory airflow limitation and the progression of bronchiectasis. Thus pathological and clinical studies support the pathophysiological sequence summarized in Figure 2 and the hypothesis that airway inflammation due to infection is necessary for the development of bronchiectasis.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Typical clinical course of airway disease in CF. On left is an approximate time line for the highlights in development of airway disease in the typical patient with CF. Although there is considerable variation in the timing of each event, with some patients not presenting with lung disease until adulthood, the linear sequence is generally observed.

	IV. DETERMINANTS OF AIRWAY SURFACE LIQUID

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View larger version (29K): [in this window] [in a new window]   FIG. 3.   Schematized morphology of proximal and distal airways. Elements of mucociliary defense and clearance mechanism are depicted. Shown is the conventional view that separate surface gel and periciliary sol layers comprise the airway surface fluid. See text for further discussion.

View larger version (75K): [in this window] [in a new window]   FIG. 4.   Surface scanning electron micrograph of human bronchial epithelium in primary culture at an air-liquid interface. Presence of ciliated and nonciliated cells mirrors the morphology of proximal airway in vivo. The gel layer that forms over the polarized, differentiated epithelium has been removed by washing before processing. (Courtesy of Drs. D. Devor, S. C. Watkins, and J. M. Pilewski.)

View larger version (35K): [in this window] [in a new window]   FIG. 5.   Proximal airway schematic showing sites of salt and water transport and mucin secretion that lead to formation of airway surface fluid. Relative level of CFTR expression is indicated by shading.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Cellular models for NaCl absorption (surface epithelium) and secretion (serous cells of submucosal gland). CFTR is apical Cl conductance shown in both cell types. The transepithelial electrical potential difference (Vt) is lumen negative across both absorptive and secretory cells with physiological solutions at both surfaces. Vt arises from differences in the apical and basolateral membrane voltages as shown; average values are given for open-circuit conditions.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   Interactions of CFTR with other cellular events. Steps in CFTR biosynthesis dictate its presence in intracellular compartments where its activity can contribute to their acidification. Phosphorylation-dependent regulation of CFTR leads to its insertion and retrieval at plasma membrane. Time constants for these CFTR trafficking reactions are several minutes, consistent with time course of stimulation of transepithelial Cl current. Time constants of biosynthesis and degradation are ~10 h. Activity of CFTR Cl channels in intracellular compartments may lead to alteration in processing of glycoproteins or in functional expression of other Cl channels [e.g., outwardly rectifying Cl channels (ORCC)] or amiloride-sensitive Na channel (ENaC). PPase, protein phosphatase. See text and reviews by Bradbury and Schwiebert et al. for discussion.

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The composition and volume of the ASL are critical in determining how changes in cellular ion transport properties associated with the loss of CFTR lead to pulmonary disease. There are basically two views of the mechanism by which altered salt transport in CF can lead to a breakdown of lung defenses. One view relates primarily to the volume of the airway lining fluid. It proposes that an increase in the rate of liquid absorption (and/or a decrease in liquid secretion) changes the volume of the ASL, decreases mucociliary clearance, and allows bacteria and mucus to accumulate (32, 33). The contrasting view is based on differences in composition of the ASL. It holds that changes in ASL salt composition alter the ability of airway defense mechanisms to kill bacteria and that this leads to bacterial colonization and airway obstruction (261). Reports of elevated ASL salt concentration in CF (see below), together with the known salt dependence of airway defense molecules, suggest that these mechanisms fail because CF airway epithelia do not have the capacity to lower surface salt concentration, as do normal airway epithelia. Thus the volume and composition of the ASL becomes a key issue in resolving the mechanism of airway defense and how it is compromised in CF.

It is almost embarrassing that we do not know the ASL salt composition with certainty. Despite its importance in helping to resolve the issues raised above, reports of ASL composition are few. This is due to difficulties in sampling the very thin liquid layer at the airway surface and to likely changes in its volume and composition produced by the sampling process. Most approaches have utilized an ASL collection technique based on the application of filter paper planchetes to the airway surface. The results from several reports suggested that the Cl concentration of ASL in CF patients was higher than that found in normal subjects (92, 127). In earlier reports (see below), salt concentration in CF was slightly hypertonic to plasma and exceeded values determined from non-CF airways, which were hypotonic. As indicated by Quinton (219), there is no known fluid transport process in flat simple epithelia that would allow for the formation of a primary secretion that is hypotonic to plasma, as implied from the measurements in normal subjects; rather, it has been suspected that evaporative water loss occurring during the measurement or during sample processing may produce falsely high values of NaCl concentration (127).

A recent study by Knowles et al. (150) highlights the problems associated with these measurements. First, how does one define the composition of a 5- to 10-µm-thick periciliary liquid layer without perturbing it? A liquid layer 10 µm thick would contain only ~20% of the liquid volume collected during sampling. Is the reminder of the sampled liquid derived from axial or radial flow? The sampling process, as well as any cellular damage which may occur, will draw liquid across or along the epithelium. Thus the measurement is likely to assess a combination of the steady-state composition of the ASL, plus aspects of the salt and water permeability properties of the underlying epithelium. The serum protein levels and K concentrations in the collected liquid are low, and this is used as an argument against cell damage. However, another possible complication arises from the mechanical stimulation of epithelial reflexes during sampling, which may evoke transport responses from the surface or gland epithelia (see below). This is a good example of Heisenberg's principle, where the system's properties are likely perturbed in the measurement itself. These perturbations may amplify or even generate differences in the liquid at the surface of CF and non-CF airways.

The nasal mucosa provides an accessible site for assessing the salt concentrations of the ASL, and liquid can be sampled as it accumulates during nasal occlusion. Using this approach, Knowles et al. (150) found a time-dependent increase in liquid volume, accompanied by a reduction in salt concentration. After equilibration, Na concentration (109 mM) was lower than the plasma value, the Cl concentration was similar to that of plasma, and K was higher (Na plus K was similar to plasma). They found no difference in the ionic composition of liquid obtained from CF and normal subjects. Importantly, the measured salt concentrations exceed values that are optimal for the activity of salt-dependent defensins in the respiratory tract. These studies were extended to the lower airway, using a filter paper sampling technique. Here, ion concentrations were significantly lower than in the nasal liquid collection studies. The sum of Na plus K suggested that the solution on bronchial surfaces is hyposmotic to plasma. Again, no CF-related differences in Na, Cl, or K concentrations of ASL were detected.

Similar findings have been reported recently by Hull et al. (114). They measured ASL salt concentrations in the lower trachea in infants undergoing bronchoscopy also using a porous membrane sampling technique. They correlated their findings with the extent of airway inflammation. No difference in ASL Na concentration was detected between CF and non-CF infants without pulmonary inflammation; however, ASL Cl concentration was significantly lower in CF (a ~30% reduction). In CF subjects with inflammation, ASL Cl concentrations were higher and not different from non-CF values. The role of inflammation in this effect is unclear; nevertheless, these results do not support the concept that an increase in ASL salt concentrations in CF is the basis of impaired airway defense against pulmonary pathogens.

One important consideration in the study of Knowles et al. (150) was the possibility that gland secretions contribute to the volume and composition of liquid on airway surfaces. They explored the possibility that the filter paper sampling technique, the common method for the lower airway, elicits liquid secretion from submucosal glands. They attempted to separate the contributions of surface and gland epithelia by collecting accumulated liquid from the nose. Reflex stimulation of nasal glands was evoked by having subjects chew chili peppers, and this resulted in a similar increase in the volume of fluid collected in both CF and normal subjects. They interpreted this to indicate that non-CFTR-dependent secretory events underlie this component of fluid secretion. Stimulation of nasal fluid secretion diluted the concentrations of the measured ions; the Na plus K decreased ~30-40 mM with a corresponding decrease in estimated osmolarity. This finding raises the possibility that secretion from the submucosal glands of a hyposmotic solution can modify ASL composition. As discussed in section IVB2, these findings suggest that we should model the submucosal glands like other exocrine glands that elaborate an isosmotic primary secretion that is modified by salt transport in the ducts leading to the airway surface. The ducts would need to absorb NaCl without water to produce a hypotonic secretory product and are therefore predicted to have a low water permeability (absence of aquaporins) and a mechanism for Na reabsorption from dilute solutions. This concept is consistent with the expression of amiloride-sensitive Na channels at this site, as reported from the in situ hybridization studies of Burch et al. (41). Thus a hyposmotic ASL, obtained from filter paper sampling of the lower airways, could result from a reflex stimulation of gland secretion. Consistent with this idea, Knowles et al. (150) found that as the volume of fluid collected increased, its osmolarity fell. This could be a consequence of stimulating secretion of a hyposmotic fluid. Because Na plus K did not differ in CF and normal ASL, the secretion from the glands stimulated in this manner does not differ between normal and CF subjects. If this is true, then the Cl permeability properties of the duct, on which the reabsorption of NaCl to form a hypotonic secretory product depend, would have to be independent of CFTR and must therefore rely on some other Cl transport process. The submucosal gland properties would differ from those of sweat ducts in this respect but would be similar to salivary ducts, where a hypotonic secretory product is formed (339).

This discussion indicates that the composition of the ASL in lower airways remains relatively uncertain. The findings of Knowles et al. (150) suggest that its measurement may be compromised by stimulation of gland secretion and, due to the size of the sampled compartment, the measurement itself may perturb cellular transport processes. However, we should bear in mind that the formation of a hypotonic secretory product in submucosal gland ducts could lead to activation of defense molecules. Because the volume of liquid secreted by the glands can exceed the transport rates of surface cells by about sixfold (see above), the secretion of a hypotonic solution containing defense molecules could have significant protective effects for the glands themselves and for the surface microenvironment at the site of secretion. However, this effect would be transient, since a high water permeability of the surface epithelium is expected to equilibrate the tonicity of secreted liquid.

Finally, a recent study performed using well-differentiated proximal airway cells in primary culture implies that the axial transport of sol and gel layers may be linked (180). This study used fluorescent markers for gel and sol to track their individual axial transport rates. In contrast to expectation, both layers moved at the same rate. Removing the mucus layer reduced lateral liquid transport ~80%, inferring that movement of the sol fluid depends in some way on the transport of mucus by the cilia. These findings are consistent with prior measurements of mucociliary clearance, which suggest that the mucus itself plays a role in ciliary transport (91, 238, 318). As mentioned earlier, most prior studies have assumed that the sol layer is relatively static and that mucins ride atop this layer, propelled by the ciliary beat. However, a recent study suggests that periciliary liquid moves with mucus and that its movement depends on mucus clearance. As summarized by Matsui et al. (180), the amount of fluid transported axially by ciliary clearance can be estimated from the area of ciliated epithelium (~2,400 cm², Ref. 181), the ciliary length (~5 µm), and a mucociliary transport rate of 0.5 cm/min (333) to be ~860 ml/day. This is approximately the volume of fluid thought to reside in the distal airways (see sect. IVA2). It is consistent with the concept that there is a continual migration of liquid from distal to proximal regions, and because of regional differences in ASL volume, this liquid must be absorbed by the surface cell salt and water transport processes discussed earlier. This implies that water absorption accompanies salt absorption, which would require a high epithelial water permeability and would not favor maintenance of a low ASL salt concentration. These findings argue for changes in mucociliary clearance that stem from reduced ASL volume in CF; that is, enhanced salt and water absorption at the airway surface impairs the ciliary clearance mechanism.

1. Liddle's syndrome

In Liddle's hypertension, truncation of the COOH terminus of the - and -subunits of ENaC are associated with salt-sensitive hypertension due to a primary increase in Na channel activity (

	V. MUCOCILIARY CLEARANCE

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Although there is considerable controversy over the ionic concentrations of ASL in CF (see sect. IVE), recent evidence suggests that increases in the salt concentration of sputum increase ciliary transportability and mucociliary clearance. Using the mucus-depleted bovine trachea model, Wills et al. (318) examined the effects of salt concentration on the ciliary transportability of sputum obtained from CF and non-CF bronchiectasis patients. Notably, there was no difference in transportability between CF and non-CF sputum, nor were there differences in the response to changes in salt concentration. Incubation of sputum in either excess isotonic or hypertonic saline (200-600 mosM PBS) or solid NaCl (to increase NaCl concentration ~90 mM) significantly increased the ciliary transportability and decreased the viscosity and elasticity of both CF and non-CF sputum without affecting ciliary beat frequency. In contrast, increasing the hydration of sputum by incubation in hypotonic solutions reduced ciliary transportability. Based on these observations, and those of previous investigators (106), this group suggests that ciliary transportability depends on saline content and osmolality of sputum. In CF, excessive Na absorption could reduce the salt concentration of sputum, thereby increasing mucus viscoelasticity and decreasing ciliary transportability.

What data exist to support the suggestion that sputum from CF patients has lower salt concentrations than normal? Few studies have attempted to measure the salt concentration of sputum itself and those that have relied on sputum from patients with laryngectomies for "normal" sputum. Matthews et al. (182) reported significantly higher salt concentrations in laryngectomy samples compared with CF patients (165 vs. 101 mM for Na, 162 vs. 75 mM for Cl). More recent studies have also reported hypotonic Na and Cl concentrations in CF sputum (284). However, comparisons of CF to non-CF bronchiectasis sputum revealed small differences (319), raising the question of whether changes in salt concentration are due to chronic infection and inflammation rather than solely to CF mutations. Thus, as with the ASL, the effect of CFTR mutations on sputum salt concentration, and a role for altered NaCl concentration on mucociliary clearance, remains to be defined.

Comparison of the clinical course and physiological derangement in mucociliary clearance between CF and the dyskinetic cilia syndromes strongly suggests that defects in mucociliary clearance cannot solely account for the progressive obstructive lung disease in CF. Patients with dyskinetic cilia syndromes, in which structural deformities in the ciliary motor apparatus impair ciliary movement, have a longer life span and better preserved pulmonary function than patients with CF. This occurs despite the fact that mucociliary clearance is more severely impaired in dyskinetic cilia syndromes than in CF (153). Moreover, in patients with bronchiectasis due to diseases other than CF, PA colonization is less frequent and appears to occur much later in the course of the disease, when airway obstruction is severe (78). Moreover, patients with dyskinetic cilia have a near-normal life expectancy (3) compared with the current ~30-yr median survival for CF patients. These observations suggest that although mucociliary clearance is generally reduced in CF (see sect. VB), the increased susceptibility to bacterial infection is disproportionate to the severity of mucociliary clearance impairment. This implies that other mechanisms contribute to pathogenesis of the progressive airway obstruction in CF.

	VI. AIRWAY INFECTION

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The important studies of Smith and Welsh (261) used polarized airway cells cultured from normal subjects to show that these epithelia killed bacteria when they were placed on the apical surface. Cystic fibrosis epithelia did not. The bacterial challenge was 1,000 cfu or less. The killing generally occurred in ~10 h, and if the bacterial load exceeded ~1,000 cfu, the system was overwhelmed. The fluid washed from the apical surface of normal airway epithelia possessed bacteriocidal activity, but the fluid washed from CF cells did not kill bacteria in vitro. This suggested that some defense factor was present on the surface of normal but not CF monolayers, or that the factor might be secreted by both normal and CF epithelia, but that differences in the environment at the airway surface affected its potency.

The defensins are a group of low-molecular-weight cationic proteins secreted by epithelia and immune cells. Like other airway antimicrobials, their ability to kill bacteria is salt sensitive. Their activity is usually markedly diminished at NaCl concentrations that approximate plasma values (e.g., 100-140 mM NaCl). Accordingly, Smith et al. (261) added salt to the solution washed from the normal airway surface and found that this reduced its potency. Conversely, diluting the fluid at the apical surface of CF cells enhanced its bacterial killing activity. The important conclusion from these observations was that if the ASL had an abnormally high NaCl concentration in CF, this higher concentration of salt would inactivate defensins and generally reduce the ability of endogenous antimicrobials to kill bacteria. This was a property of the apical surface; no activity against bacteria was found in the solution at the basolateral side of epithelium.

In a subsequent series of experiments, Goldman et al. (93) basically replicated these findings using the repopulated mouse xenograft model. They implicated a particular human defensin, hBD-1, as the major bacteriocidal factor. This observation was based on the ability of antisense oligonucleotides, which targeted the synthesis of hBD-1, to eliminate the ability of normal epithelia to kill bacteria.

There are several questions that attend these important observations. First, is a single agent or a combination of agents responsible for the bacteriocidal activity of the ASL? Can a single factor such as hBD-1 explain the capacity of the airway surface to kill bacteria? A mixture of antimicrobials having different mechanisms of action is normally present. If these substances work together to control the microbial population, how would the effects of antisense suppression of hBD-1 alone be explained? It is possible that combinations of bacteriocidal factors have synergistic effects. For example, lysozyme also shows salt sensitivity and operates by digesting bacterial cell walls. Lysozyme together with an agent that disrupts bacterial ion gradients, like a -defensin, may therefore have profound effects on colonization. Thus the antisense experiments might be explained by the synergism between antimicrobials that have different mechanisms of action. It appears unlikely that a single substance like hBD-1 can account for antimicrobial activity of the airway, but its elimination may remove its synergistic effects with other defense substances. Second, we do not understand whether cultured cells fully replicate the in vivo condition. Submucosal glands are missing from both the epithelial monolayer cultures and from the repopulated xenografts studied by Smith et al. (261) and Goldman et al. (93). However, the glands are a major source of defense substances, and clogging of their ducts would physically eliminate a major component of the antimicrobials that are normally available at the airway surface. Finally, we still do not know whether salt concentrations are altered in the ASL from CF patients, as discussed above. Given the relatively low bacterial load where this mechanism is operative, this line of defense is likely breached early in the disease process so that mucociliary clearance and cell-mediated inflammatory mechanisms quickly come into play.

	VII. INFLAMMATORY MECHANISMS

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Mechanisms of airway inflammation in CF have been investigated extensively, but only recently has there been evidence of CFTR-related dysregulation in the inflammatory response. In general, it appears that the coordinated network of cytokines and cell adhesion molecules that regulate the inflammatory response to bacterial infections in other airway diseases are intact in CF. A detailed discussion of these mechanisms is beyond the scope of this article and is available in a number of recent reviews (68, 135, 156). The following discussion focuses on aspects of the inflammatory response that may be unique to CF.

Phagocytic function in CF neutrophils from peripheral blood is normal, and humoral antibody responses are intact (reviewed in Ref. 156). However, cleavage of opsonic receptors on neutrophils by elastase may impair phagocytosis in the CF airway lumen. Antibody attachment to bacterial surfaces normally activates the complement cascade, with enzymatic cleavage of complement component C3 generating C3b and iC3b. In turn, C3b and iC3b serve as ligands for complement receptors 1, 3, and 4, respectively, on neutrophils and thereby promotes phagocytosis of bacteria. In the airway of CF patients, proteolytic enzymes such as elastase appear to overwhelm the endogenous antiprotease activity. Bronchoalveolar lavage fluid from CF patients has been shown to cleave iC3b and complement receptor 1 (24), resulting in "opsonin-receptor mismatch" (288), and elastase disrupts other opsonin-receptor interactions by cleaving the neutrophil antibody receptor FcRIIIB (287) and IgG (80). Although the relative importance of opsonophagocytic defects in the CF airway remain to be defined, the localized impairment of some components of the complement and humoral defense systems appears to contribute to bacterial persistence.

As reviewed in section I, a major early feature of the histopathology in CF is neutrophilic bronchitis and bronchiolitis; however, the appropriateness of the inflammatory response relative to the bacterial burden remains unclear. The long-standing paradigm, summarized in Figure 2, is that bacterial infection of the airway initiates an inflammatory response that successfully contains the infection in the airway lumen but generally fails to eradicate the bacteria. Analyses of cytokines (IL-1) and chemoattractants (leukotriene B₄) in BAL from CF patients have revealed elevated levels compared with healthy controls and levels comparable to other diseases in which there is bacterial infection of the lung (324). An alternative paradigm suggested by the finding of inflammation without typical viral and bacterial pathogens (136) is that the CF gene defect itself somehow contributes to excessive airway inflammation, such that the neutrophilic inflammatory response exceeds that driven by bacterial infection. The data to support this notion are limited to the observation that there is decreased production of the anti-inflammatory cytokine IL-10 in CF (30). Cytokine analysis of BAL fluid and macrophages from CF patients and healthy control subjects demonstrated elevated concentrations of the proinflammatory cytokines tumor necrosis factor- (TNF-), IL-1, IL-8, and IL-6, but decreased concentrations of IL-10 (31). Normal macrophages stimulated with lipopolysaccharide expressed intracellular cytokines to a similar extent as macrophages from CF patients, suggesting that the cytokine production in the CF airway is in part driven by lipopolysaccharide (31). The same investigators determined that IL-10 is constitutively produced by bronchial epithelial cells from normal individuals but that freshly isolated CF epithelial cells fail to produce IL-10 (30). The mechanism for these observations remains unclear. The simplest interpretation is that the inflammatory milieu in the CF airway downregulates IL-10 production and that CFTR does not itself alter the ability of macrophages or epithelial cells to produce IL-10. Nevertheless, the notion that airway inflammation in CF may continue unabated because of a deficiency in the production of anti-inflammatory cytokines like IL-10 merits further investigation, including a comparison with other diseases characterized by chronic airway infection.

A recent study of oxidant formation in CF neutrophils suggests a CFTR-related abnormality that may contribute to the pathogenesis of CF airway disease. Neutrophil oxidant function is regulated by NADPH oxidase activation and myeloperoxidase activity. Activation of NADPH leads to the generation of superoxide anion and hydrogen peroxide. Myeloperoxidase in neutrophil granules utilizes hydrogen peroxide to generate hydrochlorous acid, which reacts with amines to produce chloramines, which have a long half-life relative to other oxidants. As excess oxidant activity in the CF airway could contribute significantly to airway injury (reviewed in Ref. 40), investigators compared the neutrophil oxidant activities of CF children without active infection to their parents and healthy controls. There were no differences in NADPH oxidase activity among the three groups; however, both myeloperoxidase-dependent oxidant activity and chloramine release were increased in CF patients and, to a lesser extent, the parents compared with controls (327). Treatment of CF neutrophils with amiloride or choline buffer reduced intracellular myeloperoxidase-dependent oxidant generation and extracellular myeloperoxidase release, suggesting that altered myeloperoxidase activity in CF neutrophils may be regulated in part by intracellular pH or ion concentrations (327). Because CF gene expression has been reported in neutrophils (338), and CFTR has been implicated in the regulation of pH in intracellular organelles (see discussion above), these data raise the possibility that CF gene mutations may contribute to exuberant myeloperoxidase activity, and hence excessive oxidant generation that may promote airway injury through lipid peroxidation. However, further studies are necessary to demonstrate that the observed differences are not due solely to activation of circulating neutrophils, as has been suggested by previous investigations (96, 225, 232).

In addition to possible neutrophil oxidant excess in CF, a recent study provided evidence that CFTR may be involved in regulating the level of glutathione, an important antioxidant, in ASL. Using membrane patches from cells transfected with CFTR and CFTR channel blockers, Linsdell and Hanrahan (172) determined that CFTR is permeable to both glutathione and oxidized glutathione from the intracellular solution. This suggests that CFTR may provide a conductive pathway for glutathione to reach the ASL and account for both the reportedly high levels of glutathione in bronchoalveolar fluid from normal lung (45) and the reduced levels reported in CF patients (236). Thus CFTR mutations may directly impair glutathione transport, thereby disabling an important oxidant defense mechanism and increasing susceptibility of the CF airway to oxidative injury.

In addition to the above potential mechanism of neutrophil dysfunction in CF, recent reports suggest that mutations in CFTR may lead to abnormal programmed cell death, or apoptosis, in epithelial cells and leukocytes, thereby contributing to DNA release and mucus viscosity. Acidification of intracellular compartments accompanies apoptosis, raising the possibility that defective intracellular acidification related to CF mutations could result in abnormal apoptosis and release of cell contents. In support of this hypothesis, Gottlieb and Dosanjh (95) reported that epithelial cells expressing F508 CFTR failed to undergo cytoplasmic acidification and DNA fragmentation in response to stimulation with cycloheximide or etoposide. Moreover, inhibition of CFTR with diphenylamine carboxylate delayed apoptosis in wild-type expressing cells, and treatment of F508 CFTR-expressing cells with propionic acid to acidify intracellular compartments significantly diminished resistance to cycloheximide-induced apoptosis (95). These data suggest that CF mutations may interfere with epithelial apoptosis, which in turn may promote release of undigested DNA and other cellular contents that are normally not released during apoptosis. In the airway and gut, release of DNA could increase the viscosity of secretions, and in the airway, a similar process in neutrophils could contribute to the release of proinflammatory cellular contents. Further studies are clearly necessary to address these hypotheses, specifically to determine whether abnormal DNA fragmentation leads to release of cellular contents and impacts on airway inflammation. Nevertheless, the notion that aberrant apoptosis occurs in CF provides a plausible mechanism for the apparent exuberant inflammatory response in the CF airway.

In addition to the potential role of apoptosis in the CF inflammatory response, recent work suggests that bacterial DNA may itself induce inflammatory responses in the lung and contribute to the persistence of airway inflammation in CF. The sputum in patients with CF has been found by numerous investigators to have an increased concentration of DNA (205), much of which is derived from bacteria. In addition to the apparent adverse effects of DNA on mucus rheology (205), it appears that unmethylated bacterial DNA and oligonucleotides recruit neutrophils to murine airway and induce the proinflammatory cytokines TNF-, IL-6, and macrophage inflammatory protein-2 (238). Specifically, unmethylated CpG motifs were proinflammatory, whereas prokaryotic DNA (in which unmethylated DNA is much less abundant), methylated DNA, oligonucleotides without CpG motifs, and amounts of lipopolysaccharide equal to those in the DNA preparations did not induce pulmonary inflammation. Similar inflammatory responses in BAL fluid were observed after instillation of purified DNA from the sputum of CF patients infected with Pseudomonas. These data suggest that bacterial DNA may itself be proinflammatory and, if confirmed in other species, may provide another mechanism for the exuberant inflammatory response observed in the CF airway.

Thus several mechanisms have been identified that may explain the persistent inflammatory response in the CF airway; however, the mechanism(s) for the initiation of bacterial infection remain to be clearly defined. In addition, the notion that inflammation precedes and/or is excessive to that induced by bacterial infection will require further investigation.

	VIII. SUMMARY

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In the context of the molecular, cellular, and lung physiology described above, a complicated pathophysiological cascade emerges, as summarized in Figure 8. Cystic fibrosis gene mutations impair CFTR function and permit bacterial infection of the airway via one or more mechanisms. First, as derived from CFTR's function as an apical membrane chloride channel, dysfunctional CFTR leads to abnormalities in the ASL composition or volume, which impair antimicrobial activity or mucociliary clearance, respectively, and create a permissive environment for bacterial infection. Alternatively, or in addition, CFTR's function in intracellular compartments and glycoconjugate processing suggests additional mechanisms whereby CFTR mutations permit bacterial infection. Abnormal glycosylation of secreted or membrane-bound glycoconjugates, particularly mucins and glycolipids, may promote mucus obstruction of the small airways and provide adhesive ligands for pathogenic bacteria.

View larger version (27K): [in this window] [in a new window]   FIG. 8.   Summary of proposed pathophysiological links between CF gene mutations and development of lung disease in CF. Defects in CFTR lead to abnormal airway surface fluid and/or abnormal glycosylation of secreted and membrane-bound glycoconjugates. Via mechanisms outlined, bacterial infection results and initiates a vicious cycle in which infection begets inflammation, which leads to bronchiectasis, induction of mucin gene(s), and further impairments in mucociliary clearance that promote maintenance of infection.

Once initiated, bacterial infection elicits an inflammatory response that contains infection to the airway lumen but does not eradicate the organisms. Mutations of CFTR may contribute to dysregulation of the inflammatory response, via the mechanisms discussed above, including opsonophagocytic mismatch, defective apoptosis, excessive oxidant formation, and impaired antioxidant secretion. Moreover, bacterial infection could then create a permissive environment for its persistence via a number of mechanisms, including induction of mucin genes whose products may be aberrantly processed, exoproduct-mediated epithelial damage with subsequent disclosure of bacterial adhesion sites and exposure of matrix proteins, and induction of an inflammatory response that persists abnormally because of defects in anti-inflammatory cytokine production. The vicious cycle of infection, inflammation, and impaired mucociliary clearance ultimately leads to bronchiectasis. However complex, the improved understanding of the pathogenesis of airway disease in CF has generated hypotheses to be experimentally tested in the coming years. We anticipate that further delineation of the pathophysiological cascade will assist in the development of new therapeutic strategies and refined end points for the evaluation of pharmacological and genetic therapies.

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