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Physiol. Rev. 79: 215-255, 1999;
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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.

Should we focus on the Cl channel function of CFTR and the loss of this function as the underlying source of CF pathophysiology? The reasons for doing so are embodied in traditional, as well as more recent concepts of the connecting links between CFTR and airway disease. The channel hypothesis of CF now has two major themes: one is based on the role of CFTR in determining the volume of the airway surface liquid (ASL). This concept holds that a lack of fluid secretion, together with excessive fluid absorption, leads to a reduction in the watery component of the ASL, to a thickening of its mucous component, to blocked submucosal gland ducts, and to impaired mucociliary clearance, infection, inflammation, and ultimately, the tissue destruction characteristic of bronchiectasis. A second, more recent, concept is that this scenario arises from an inherent ability of airway cells to fight against bacterial infections that is compromised by the loss of CFTR. This may be linked to changes in the composition of the ASL.

There are also nonchannel or regulatory subthemes that may contribute to CF airway disease. One features the ability of CFTR to regulate other ion channels and therefore relates to formation of the ASL (see review by Schwiebert et al., this supplement). Another involves functions of CFTR within intracellular compartments that lead to altered processing of macromolecules and the appearance of glycoconjugates with different properties on the airway surface (see review by Bradbury, this supplement). These regulatory events are part of the larger picture of mechanisms that control the physical and chemical composition of the ASL, and they are therefore embodied in the channel hypothesis of CF. In this review, we summarize these functions of CFTR and refer, as appropriate, to other reviews in this supplement where these functions are described in greater detail.

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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A. Cellular Genotype-Phenotype Comparisons: Classes of Mutations

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 Delta 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 Delta F508 CFTR traffics to the cell membrane where it forms partially functional Cl channels (50, 72, 171). Most laboratories agree that Delta 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.


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  		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, Delta 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).

B. Correlation of Genotype With In Vivo Function

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 (Delta 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 Delta F508 or in those with a Delta 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 Delta F508 patients. This suggests that other factors, such as the expression of other Cl channels, or the trafficking of some mutant Delta 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.

C. Relation of CFTR Mutations to Disease Severity

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 Delta 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 Delta F508 homozygous patients and those who were compound heterozygotes for Delta 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 Delta F508 homozygous patients had a higher rate of pulmonary function decline than patients who were heterozygous for Delta F508 or had two non-Delta 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).

D. Importance of CFTR in Organ Physiology and Development of Different Organs

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.

 
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  		TABLE 1.   Relation of amount of functional CFTR and organ dysfunction for representative CFTR mutations

    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.

A. Earliest Pathological Manifestations of Pulmonary Disease

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.

 
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  		TABLE 2.   Turning points in the pathophysiology of CF lung disease

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).

B. Airway Infection and Inflammation Lead to Bronchiectasis

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.

C. Role of Inflammation in the Progression of Airway Pathology

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.


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  		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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A. Basic Principles

1. Cell types

The upper respiratory tract is lined by a pseudo-stratified, mostly ciliated epithelium that extends from the proximal trachea to the terminal bronchioles (see Fig. 3; Ref. 7). Ciliated and nonciliated columnar cells and goblet cells populate the surface epithelium. In the large airways, the ratio of ciliated columnar cells to goblet cells is ~5:1; the numbers of both cell types decrease in peripheral airways where the nonciliated cells become more numerous (230). Interspersed among the ciliated cells of the proximal airways are brush cells, which are also nonciliated (7, 337). Their microvilli, like those of the ciliated cells, measure ~1 µm in length (7). The cilia, on the other hand, have an average length of 6 µm (79, 249), which has been proposed as the minimal periciliary liquid depth (see sect. IVD1). Below the surface epithelium of the proximal airways are numerous submucosal glands, which contain mucous and serous secretory cells (123). Submucosal glands are found in the regions of proximal airway where there is cartilage (229). With increased airway branching, as one approaches the periphery, the pseudo-stratified structure of the epithelium is lost. In the distal bronchioles, the epithelial cells take on a cuboidal shape and are termed clara cells, ~40% of which are ciliated (39). Goblet cells are normally absent in this region.


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  		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.

The airway epithelium is bathed on its apical surface by a thin liquid layer. The concept that this layer is composed of two phases, gel and sol, was first proposed by Lucas and Douglas (174) from transmission electron micrographs of the apical surface of tracheal epithelium. Airway surface liquid coats distal airways as well, but a corresponding mucous gel layer is minimal distally (337). Experimental estimates of the ASL thickness vary widely, but most studies agree that its depth is ~10-20 µm (14, 298). For the most part, it is the gel layer that contributes to variations in ASL thickness; the gel contains a variety of macromolecular secretory products (21), including glycoproteins, proteoglycans, lipids, defense molecules (27, 53, 73), DNA (59), and actin (303). These latter two components are produced by cellular breakdown and bacteria, and they can become a significant burden in the airways of patients with CF. The concept that disrupting the network of DNA and actin would enhance clearance of the gel has led to the development of DNase and gelsolin in an attempt to liquify these tangled, complex structures (5, 251, 296, 303).

The ASL is the first line of defense against inhaled pathogens, and it is mandatory for effective mucociliary clearance (238). The upward movement of liquid through the trachea averages 10-100 ml/day, as deduced from fluid collections from tracheostomy patients (285). The division of the ASL into a periciliary liquid (sol) layer and a separate, overlying mucous (gel) layer (174, 214) provides an anatomic basis (perhaps bias) for one interpretation of how mucociliary clearance occurs (111, 307). The concept is that the cilia can beat and clear the gel more effectively when bathed by a liquid (sol) layer whose depth approximates the ciliary length. In this view, the tips of the cilia extend to contact the mucous layer on their forward stroke and return in a more folded manner to complete the beat cycle. Thus, if the periciliary liquid layer becomes either too deep or too shallow, mucociliary clearance will be impaired because the mucous layer is either too far away to be contacted or it lays directly on the cilia, impeding their ability to beat productively. In either case, the mechanics of ciliary interactions with the mucous blanket are suboptimal.

A more recent concept proposes that mucins of the gel do not form a discrete blanket, or islands, but rather, a tangled, hydrated network (306). Arguments in favor of this model rest on observations showing that mucins become more gel-like (structured) at higher glycoprotein concentrations (185). Accordingly, the gel layer may be more concentrated near the air-liquid interface and more dispersed near the epithelium where the cilia are beating. The details remain to be assessed, but it seems clear that the periciliary region of the ASL is more liquid than mucin rich and that this optimizes ciliary activity and mucin clearance. According to both models, mucins of the gel are propelled upward, whereas the periciliary liquid is assumed to be relatively static, moving to and fro with ciliary beating, but with little net transport. This concept has been tested recently with the use of fluorescent markers of the gel and sol phases and is discussed in section IVE.

The periciliary fluid composition reflects the salt and water absorptive and secretory functions of the airway epithelium, which hydrate the mucous gel and influence its clearance (32, 33). Thus the ion and water transport properties of the epithelium have the opportunity to influence the volume and composition of the periciliary liquid layer, and accordingly, changes in cellular transport properties may result in composition and/or volume changes in the ASL that contribute to the development of airway disease. Our goal here is to summarize the transport properties of the epithelium and our knowledge of their influence on the volume and composition of this fluid compartment.

2. Fluid transport: radial and axial flow

A concept of airway fluid homeostasis that has implications for the volume and composition of the ASL is the idea that liquid moves axially between different regions of the lung. This concept, first proposed by Kilburn (138), was reviewed by Boucher (32). It recognizes the large disparity in the surface areas of the distal versus proximal airways. The fact that the thickness of the ASL is similar in these regions means that there is a large disparity in the amount of liquid contained at different levels of the lung. For example, we can estimate that there is ~700 ml of liquid residing in distal airways and air spaces (based on an ASL thickness of 10 µm) and only ~1 ml of liquid in the trachea. The idea that surface liquid is moving up the airways implies that the 0.7 liter present distally would need to be reabsorbed, given the geometry of the airway network, otherwise the proximal airways would be awash in liquid.

Although this is an intriguing concept, several key elements and assumptions of this model need to be further clarified. First, it is important to identify the source of the liquid from distal regions that would be moving proximally. The physical forces of hydrostatic and colloid osmotic pressures acting across the alveolus are poised to keep the air-space surface dry (81, 165). In addition, the most distal airway cells that have been examined experimentally are found to be absorptive not secretory (94, 178, 301). Second, it is important to know whether both liquid and mucin are moving in response to the ciliary clearance mechanism. Whether the mucins exist as a distinct physical layer or as a tangled network, the gel may be moved preferentially to the periciliary sol by ciliary beating so that large volumes of water may not be moving proximally. Third, it is important to know the contribution of pulmonary surfactants and the forces of surface tension to the determination of ASL depth (15). These physical forces are likely to differ in distal and proximal regions on a geometric basis, and this may contribute to their capacity to hold different amounts of liquid. Clearly, the mechanisms that govern axial liquid flow need to be defined. However, the small volume of sputum emerging from tracheostomies implies that at least the mucins will become more concentrated as they move proximally.

The concept that emerges from the discussion above is that the thickness of the ASL, particularly the periciliary liquid layer, is carefully regulated. Whether this is true is uncertain because it is difficult to test experimentally. This concept suggests that the surface epithelium somehow has the capacity to sense properties (e.g., salt concentration, volume) of the liquid lying on its surface and to adjust those properties by appropriately adding or removing salt and water. Such mechanisms have not been identified. Indeed, regulation of the periciliary liquid may be primarily local, i.e., occurring only over several or even single cells. Recent advances in the techniques for primary cell culture of airway surface cells, which can duplicate the ciliated columnar cell morphology of the surface epithelium (180, 261; see Fig. 4), should be useful in providing experimental assessment of the axial flow of liquid and mucus and of the local (cell-mediated) controls over ASL volume and composition. Nevertheless, these cultures lack innervation and eliminate any contribution of the submucosal glands to fluid formation or its regulation. The likelihood that submucosal glands add to the volume and compositional properties of the ASL in proximal airways has not received sufficient attention, and this is discussed in more detail in section IVB2.


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  		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.)

3. Sites of CFTR expression

Soon after the identification of the CFTR gene, investigators determined its expression in human proximal lung tissue using protein and RNA detection methods. Here, CFTR was found in both the surface epithelium and in submucosal glands (see Fig. 5). In bronchoscopy samples of surface epithelium from normal subjects, quantitative PCR techniques were used to estimate that only about one or two mRNA transcripts for CFTR were expressed in each cell (290). Detection of protein in native airway has been difficult because generation of high-affinity, high-specificity antibodies against CFTR has not been straightforward and because the protein is expressed at low levels. Indeed, immunostaining of normal lung does not always reveal CFTR expression in ciliated airway cells (69, 74). Because CFTR is a Cl channel with a turnover rate of ~2 × 106 ions/s, relatively few copies of the protein are needed to provide the required apical membrane Cl conductance. From the channel's properties, estimates indicate that only several hundred to several thousand CFTRs per cell are necessary to provide the diffusional Cl transport properties required at the apical membrane. The identification of a cAMP-regulated Cl conductance having known properties of CFTR is a more sensitive assay than immunocytochemistry, and such studies lead to the conclusion that CFTR is present at the apical membrane domain of ciliated surface cells (58, 322).


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  		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.

In contrast to the low levels of mRNA and protein expression in the surface epithelium, CFTR is expressed at higher levels in subpopulations of cells in the submucosal glands (74, 121). The CFTR mRNA and protein have been detected also in the striated ducts, where a small percentage of cells exhibit the highest levels of CFTR expression identified in the airways. Its function at this site is as yet unknown. In addition, serous cells at the base of the submucosal glands contain readily detectable levels of CFTR. In distal airways, a small population of cells also express relatively high levels of CFTR, as in submucosal gland ducts. They comprise only ~1% of the nonciliated epithelial cell population so that the function of CFTR in this setting is difficult to define experimentally. This expression pattern has also been described for small intestine, where occasional cells appear to have very high CFTR levels (9). Because the less differentiated crypt cells of the intestine are the highest sites of CFTR expression (291), it is possible that these high expressing cells in both intestine and airway have strayed from a normal developmental program, remaining in an undifferentiated state. In general, studies of CFTR gene expression suggest a role for CFTR at the apical membranes of ciliated surface epithelial cells throughout the airway and in the submucosal gland serous cells of cartilaginous airway regions (Fig. 5).

B. Transport Functions of Proximal Airways

1. Surface epithelium

Our concepts of electrolyte and liquid handling in the airway are derived primarily from examination of proximal airway cells, studied as either excised airway segments or as epithelial monolayers in primary culture (for detailed review, see Refs. 32, 33). Considerable in vitro data are available from human airway cells, derived primarily from nasal or bronchial epithelia. As yet, there is no published transport data from human distal (noncartilaginous) airway. Data on cultured epithelia may be compromised by variability arising from the cell culture methods used by different laboratories, but these techniques have consistently improved and become more uniform. Although the in vivo situation cannot be reproduced using cultured cells, the morphology of epithelia grown on permeable collagen supports with an air interface at the apical surface (98) has become qualitatively similar to that of the native surface epithelium (see Fig. 4). Thus it is possible to obtain well-differentiated epithelia in vitro that resembles the native surface epithelium. Unfortunately, this has not yet been achieved for the submucosal glands (see below).

A) NACL ABSORPTION. I) In vitro measurements. Studies performed under standard physiological conditions indicate that proximal airway surface epithelia absorb Na, Cl, and water (35, 124, 151) and that this occurs by the basic mechanism defined by Koefoed-Johnsen and Ussing (152) almost five decades ago (see Fig. 6). According to this model, Na enters across the apical membranes via amiloride-sensitive, epithelial Na channels (ENaC), reviewed by Garty and Palmer (89). Cell Na is extruded by the Na-K pump (275), and K accumulated by the pump can be either secreted or recycled to the interstitial space. Exit of K down its electrochemical potential gradient across the apical membrane may contribute to the relatively high K concentration of the ASL (see below), but the responsible apical K channel has not been identified. Most of the K taken up by the pump is recycled to the submucosal solution via K channels in the basolateral membranes (32).


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  		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.

From in vitro studies, the predominate active ion transport activity of either freshly excised (148) or cultured (332) airway epithelia studied under short-circuit conditions is electrogenic Na absorption (320). Under open-circuit conditions, these tissues also show net Na absorption, with the magnitude of net Na transport, relative to the short-circuit condition, somewhat reduced by the transepithelial (lumen-negative) voltage. Chloride is absorbed in response to the transepithelial potential difference, and similar results are obtained from tissues excised from nasal or bronchial regions (321). The transepithelial resistance of excised upper airway epithelia is relatively low (~300 Omega ·cm2; Ref. 148), and under short-circuit conditions, bidirectional Cl fluxes across excised airway segments are relatively large (148). This is consistent with the concept that paracellular Cl permeability is high and that Cl flow between the cells could provide the principal pathway for Cl absorption (32, 320, 322). The route of Cl flow during NaCl absorption across the surface epithelium should be better clarified, since direct estimates of cellular versus paracellular Cl flow have not been made.

Microelectrode studies of the apical membrane voltage and intracellular ion activities detect a large electrochemical driving force favoring Na entry (~60 mV) across the apical membrane (61, 311, 323). In addition to entry through amiloride-sensitive channels, Na-coupled glucose entry may contribute to Na absorption (128). This process may scavenge glucose from the ASL that diffuses in from the plasma.

Microelectrode studies suggest that cell Cl is distributed close to its equilibrium distribution across the apical membrane; that is, there is not a significant driving force for diffusional Cl entry from the lumen (312, 322). An equilibrium distribution of Cl across the apical membrane implies that there will be essentially no net Cl flow into the cell during Na absorption. Accordingly, the Cl conductance of surface epithelial cells should have minimal impact on the NaCl absorption rate because Cl is being absorbed between the cells, not through them (32). Thus, in CF, the absence of CFTR at the apical membrane would not markedly affect the NaCl absorptive properties of the epithelium, except for its role as a regulator of the apical Na conductance (see review by Schwiebert et al., this supplement). This leads us to a curious conclusion physiologically: the primary function of CFTR in the airway surface epithelium is not as a cellular Cl conductance that provides a pathway for Cl flow during NaCl absorption; rather, its role is to regulate the activity of the apical Na channel. It remains to be seen whether the driving force on Cl flow at the apical membrane, identified in excised airway segments and cell culture systems, applies also to the epithelium in vivo. Another uncertainty regarding transcellular Cl flow is the mechanism of Cl transport across the basolateral membrane. Ordinarily, there is a significant driving force for Cl exit from the cell in the absorptive direction, and in ion replacement studies, a small basolateral membrane Cl conductance has been detected (322). However, the molecular identity of the basolateral Cl conductance pathway has not been defined. It is presumably not CFTR. The basolateral conductance properties are dominated by K-selective pathways.

II) In vivo measurements. The experimental basis for identifying NaCl absorption as the major salt transport event in the surface epithelium relies primarily on in vitro measurements using electrophysiological and isotopic flux techniques (as above). Measurement of the transepithelial electrical potential difference in vivo detects, under basal conditions, a voltage (Vt) across the proximal airway epithelium of normal subjects of approximately -30 mV (lumen negative) (144-146); similar values have been detected in the proximal airways. The activity of the ENaC is the principal determinant of Na absorption rate, as reflected by inhibition of Vt by amiloride. Superfusion of amiloride onto the airway surface of normal subjects eliminates ~60% of the Vt (144, 146). The residual voltage under these conditions may reflect stimulation of Cl secretion (see below), although electrogenic HCO3 secretion may also contribute. In CF airway, the basal Vt is elevated to about -60 mV, due largely to an increase in the amiloride-sensitive component of Vt (146). Most of this elevated Vt is amiloride sensitive (149). Results from in vitro measurements on excised tissues or cultured epithelia suggest that the elevated Vt reflects enhanced activity of ENaC at the apical membranes of airway surface cells (35, 61, 321). The absence of an apical Cl conductance may contribute to the larger Vt across CF airways, but the leakier paracellular pathway would attenuate its contribution. In the sweat duct, Vt rises higher in CF because the cellular pathway is dominant for transepithelial Cl flow.

III) Regulation. Little is known about the acute or chronic regulation of Na transport in airway surface cells. Evidence from ENaC expression studies suggests that cAMP stimulation enhances amiloride-sensitive Na currents in the absence of CFTR expression but that with CFTR coexpression, Na currents were smaller and were inhibited by cAMP (273). These data are consistent with the proposed role of CFTR as a negative regulator of ENaC, an influence that would be removed in CF. Single-channel studies of ENaC expressed in fibroblasts suggest that CFTR alters Na current by changing ENaC open probability (276, see also review by Schwiebert et al., this supplement). There are indications also that inflammatory mediators alter airway Na transport (56). At the basolateral membrane, cytokines or ATP can accelerate the rate of Na absorption. However, at the apical surface, nucleotide triphosphates such as UTP are inhibitors of Na absorption (D. C. Devor, personal communication). Finally, steroid hormones do not appear to be major regulators of Na transport rates across airway surface cells (147).

B) NACL SECRETION. Under normal conditions, the airway surface epithelium absorbs NaCl, and net salt secretion is not observed. With Cl distributed at equilibrium across the apical membrane, agents that further increase the Cl conductance (e.g., isoproterenol) do not yield net secretory activity. However, under some experimental conditions in vitro, Cl can be secreted across the surface epithelium (151, 322). When the apical membrane voltage is sufficiently hyperpolarized, which can be produced by blocking apical Na entry with amiloride, a significant driving force for Cl exit from cell to lumen is established (322). In the presence of amiloride, transcellular Cl secretion is observed, and its rate can be further increased by cAMP or Ca-dependent secretory agonists by enhancing the apical membrane Cl conductance (34).

The cellular mechanism for Cl secretion, established from studies in airway and other secretory epithelia (85), is shown in Figure 7. Agents that raise cellular cAMP are effective secretogogues, and under these conditions, CFTR is the principal Cl conductance pathway (58, 130, 277). Alternate Cl conductance pathways may contribute to Cl secretion in response to certain agonists or when CFTR is absent. Chloride secretion can be evoked by Ca-mediated agonists, although this response is generally transient, in contrast to the more sustained response usually elicited by cAMP agonists. The molecular basis of the Cl conductance activated by a cellular Ca rise in airway cells is still not clear. Chloride conductance pathways alternate to CFTR may explain the absence of significant airway pathology in the CF mouse, where significant Ca-mediated Cl secretion is observed (see review by Grubb and Boucher, this supplement). Luminal nucleotide triphosphates are effective agonists for activation of a non-CFTR apical Cl conductance (179, 274). The presence of ATP on the luminal side of the epithelium induces Cl secretion, by activation of a P2y2 (P2u) receptor. Other nucleotides such as UTP are effective secretogogues, and their nonhydrolyzable analogs can produce longer lasting responses (166). Electrophysiological data indicate primary activation of a non-CFTR apical Cl conductance by ATP/UTP, which would add to the activation by other second messenger pathways, including that of CFTR. It has been proposed that ATP may act as a coordinator of the ASL volume and composition by virtue of its presence in airway secretions, including those derived from secretory cells during mucin release (33, 316).


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  		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.

It should be emphasized that the influence of the basolateral membrane K conductance is critical in determining the magnitude of Cl secretion (160, 161, 183, 264). By electrical coupling, this K conductance establishes the electrical driving force for Cl exit across the apical membrane (87). In principle, this could occur even in the face of Na absorption should the K conductance rise sufficiently. Whether this occurs in vivo is not clear, but this phenomenon is demonstrable in human airway cell cultures, and it may represent a means of controlling the rate and direction of NaCl transport both physiologically and pharmacologically (see review by Schultz et al., this supplement).

C) HCO3 SECRETION. A transport process of proximal airway that has not received sufficient attention is transepithelial HCO3 transport. Evidence consistent with airway HCO3 secretion has emerged from several studies which show that the rate of Na transport is often significantly smaller than the measured transepithelial current (the short-circuit current or ISC), even in the absence of bath Cl. The unidentified current component appears to be carried by HCO3 secreted into the lumen (262). Interestingly, in CF epithelia, ISC was attributable to the rate of Na absorption, implying that HCO3 secretion was impaired in CF. This process may also contribute to the residual, amiloride-insensitive voltage across non-CF airway in vivo where, in general, Vt across CF epithelia is entirely amiloride sensitive. The transport mechanisms that would be responsible for HCO3 secretion across proximal airway cells have not been adequately identified, but the presence of HCO3 transport mechanisms may also explain the lack of complete bumetanide sensitivity of the ISC response to cAMP-mediated agonists. Moreover, the contribution of HCO3 secretion to the volume and composition of the ASL is unclear at present, but a similar process likely resides in the submucosal glands (see sect. IVB2). It is interesting to speculate that luminal alkalinity, particularly in the submucosal glands, may be an important determinant of the physical properties of secreted mucins and their clearance from the glands onto the airway surface.

D) SUMMARY OF ION TRANSPORT PROCESSES IN SURFACE EPITHELIUM. Thus, in surface epithelium, absorption of NaCl and water is the significant physiological transport process (see also fluid transport studies below). The rate of Na absorption is enhanced by CFTR deletion and is reduced by CFTR stimulation under normal conditions. Stutts and co-workers (273, 276) have related these effects to changes in the probability that ENaC channels are in the open conformation (Po); that is, stimulation of wild-type CFTR decreased ENaC Po , whereas ENaC Po was increased by cAMP stimulation in cells expressing Delta F508 CFTR. These findings are consistent with the discussion above, which argues that the influence of the CFTR Cl conductance on transepithelial salt transport is exerted primarily via CFTR regulation of ENaC, and not by altering transcellular Cl transport. The higher Na absorption rate in CF would be expected to reduce the salt concentration of the ASL if the epithelium is relatively water impermeant. Alternatively, if water follows the enhanced salt transport, then the volume of periciliary liquid would be decreased relative to normal conditions. We consider these issues in section IVE, after the water permeability and fluid transport properties of the epithelium have been reviewed. Stimulation of CFTR in the surface epithelium does not give rise to net fluid secretion. This CFTR-dependent process is the province of the submucosal glands.

2. Submucosal glands

Submucosal glands are found in the cartilaginous airways. The glands are composed of both mucous and serous cells, and their distribution and density varies among species. The proportion is ~60% serous cells and 40% mucous cells in humans (280). Whereas mucous cells are present in the surface epithelium, virtually all airway serous cells are found in the submucosal glands. The secretory products of the mucous cells are high-molecular-weight glycoproteins, which are sialylated and sulfated. The serous cells contain fewer secretory granules, which are somewhat smaller in size than those in mucous cells (122).

Studies of submucosal gland secretion are not numerous, and the approaches have been relatively indirect (295). In excised airway tissues, the application of a gland secretory agonist (acetylcholine) to the submucosal surface induces an electrically silent secretion of NaCl, which can be detected by isotopic flux methods but is not associated with a change in transepithelial voltage (148). The absence of a secretion-linked voltage change probably results from dissipation of the secretory current by the cable properties of the collecting duct that leads to the airway lumen.

Other studies have focused on isolated cell and cell culture systems to assess gland secretions (71, 331). Cell culture techniques have been devised to primarily encourage the proliferation of submucosal gland cells, but they produce cells having a mixed serous-mucous phenotype. These cells are identified as primarily secretory, and they express several submucosal gland markers. However, they also express an amiloride-sensitive Na current, which is somewhat unexpected (see below). In cultured submucosal gland monolayers grown on permeable supports, agonists induce relatively short-lived (<1 min) secretory responses. Effects of both Ca- and cAMP-mediated secretagogues are evident (329); the response to Ca-mediated agonists is larger and is the only significant response that remains in gland cultures from CF patients (330).

The lung adenocarcinoma cell line Calu-3 appears to be a good model for the submucosal gland serous cell (108). Calu-3 cells express many markers of serous cell function, including lysozyme and lactoferrin (252). The cells express high levels of CFTR and show secretory current responses to both cAMP-and Ca-mediated agonists (108). Calu-3 monolayers in which the basolateral membrane is permeabilized with nystatin show Cl conductance responses to elevation of cAMP but not cell Ca (194). This finding suggests that CFTR may provide the apical Cl conductance pathway for secretory responses mediated by Ca-dependent agonists, a situation similar to that observed in intestinal cells (10). The results of recent studies suggest that the net secretory current stimulated by cAMP-mediated agonists across Calu-3 cells is actually carried by HCO3 rather than Cl (167; R. J. Bridges, personal communication). The effects of ion replacement conditions, transport inhibitors, and transepithelial isotopic flux determinations are consistent with this view. The role of HCO3 secretion, or the high luminal pH which it infers, in airway gland secretion physiology is unknown.

Given our uncertainty regarding the transport mechanisms involved in submucosal gland salt secretion, it is not surprising that the role of CFTR in this process has not been adequately clarified. It has been generally assumed that the salt secretion mechanisms of submucosal serous cells resemble those of the surface epithelium and other secretory epithelia (see above and Fig. 7) and are due to secondary-active Cl secretion. However, newer evidence indicates that Calu-3 cells secrete HCO3 , and because they express submucosal markers, the same may occur in submucosal gland serous cells. Unfortunately, this makes the role of CFTR in gland secretion less clear. One model that features a key role for CFTR in this process is derived from studies of pancreatic duct cells (97, 99). Here an apical Cl/HCO3 exchange mechanism, operating in parallel with apical CFTR, produces CFTR-dependent net HCO3 secretion by recycling the Cl that enters the cell via the anion exchanger through CFTR. Appropriate inhibitor and ion replacement studies should be performed to determine whether this model applies to Calu-3 epithelia or whether other explanations for HCO3 transport should be sought. Studies of the pancreatic duct (119, 120) offer even more possibilities to ponder in relation to HCO3 secreting tissues where the cells have the capacity to raise luminal HCO3 concentration to high levels (approaching 150 mM). These tissues express a basolateral membrane HCO3 entry mechanism that is Na coupled (42, 233). In contrast to predictions of the anion exchanger model cited above, alkalinization of the pancreatic lumen was not inhibited by low luminal Cl concentration or by the CFTR blocker glibenclamide (119, 120). These findings suggest that a new paradigm may be required to explain transepithelial HCO3 secretion, in particular, the exit of HCO3 across the apical membrane and the role of CFTR in that process. It is too early to know whether there are parallels between airway submucosal glands and the pancreatic duct in this respect, but the studies cited above raise many interesting questions for future investigations.

The structure of the submucosal glands suggests an important role of serous cell liquid secretion in the elaboration of the secretory product (Fig. 3). Serous cells line the most distal acinar structures, whereas the mucous cells are located primarily in the more proximal secretory ducts (189, 190). Thus secretion of liquid from serous cells provides the vehicle that moves mucins toward the airway lumen (316). In addition, if Na channels (ENaC) are expressed in the tubules and collecting ducts of the submucosal glands, they may serve to reduce the NaCl concentration of the secreted fluid (41). In view of the potential for gland secretory rate to exceed the surface absorptive rate by a factor of ~6, this could markedly decrease the NaCl concentration of the ASL when the glands are active.

The relative contributions of surface and gland mucous cells to the ASL gel volume is uncertain. However, estimates of total gland cell volume relative to the volume of goblet cells in the surface epithelium suggest that the gland cells predominate by a factor of ~40 (229). It is estimated that in adults there are ~100 submucosal glands/cm2 tracheal surface (286). Gland secretion rates (125) are regulated, and during maximal secretion, a single gland produces fluid at a rate of ~10 nl/min (217, 295). This implies that 1 cm2 of upper airway during maximal stimulation can generate ~60 ml fluid/h. In contrast, the rate of fluid absorption across cultured surface epithelium, measured in the studies of Jiang et al. (124), was ~5 ml·cm-2·h-1. In the steady state, the composition of the ASL will be determined by the balance of gland secretion and surface reabsorption. Apart from the secreted liquid, the glands are generally considered to contribute the majority of glycoconjugates secreted onto the airway surface (228). Thus the secretion of liquid from the submucosal glands, at least in the upper airway, is perhaps a more important factor controlling the water content of airway secretions than is transport by the surface epithelium. In this context, it seems reasonable to view the upper airway as a functional secretory-absorptive unit, composed of a submucosal gland and duct that connects with the surface epithelium.

In analogy with the structure-function relations of exocrine glands, the airway consists of a distal secretory component (the mucous and serous cells of the submucosal glands) in series with a more proximal absorptive component (the gland collecting duct and surface epithelium). The loci of expression of CFTR and ENaC, detected by in situ hybridization, are consistent with this view (41, 74). Duct cells are reported to express relatively high levels of ENaC, which suggests that they may play a role in NaCl absorption. The duct cells and surface epithelium together could modify the composition of the liquid secreted from the glands as it travels to the surface. The final secretory product, and thus the regional composition of the ASL, would be expected to vary in its salt composition and tonicity, depending on the reabsorptive activity of the duct and surface epithelia and their water permeabilities. Ductal salt reabsorption combined with a low H2O permeability would yield a hypotonic solution with lower NaCl concentration than that of the primary secretion, as occurs in the sweat or salivary gland.

Although current evidence for this view is sparse, recent work from Knowles et al. (150) has suggested that the secretions of submucosal glands may be hypotonic. They sampled liquid from the bronchial epithelium using a filter paper technique and found that the collected liquid was hypotonic, resembling that collected from nasal mucosa during strong stimulation of gland secretion. Accordingly, changes in the composition of the ASL, evoked by a hypotonic submucosal gland secretory product, may be important for the activity of defense molecules in the gland secretions (see sect. VIB).

Secretory products of submucosal glands play an important role in defending the airway against inhaled pathogens. Accordingly, i