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PHYSIOLOGICAL REVIEWS

Vol. 79, Suppl., No. 1, January 1999Printed in U.S.A.

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 S216II. Genetics and Physiology of CFTR Gene Mutations S216

A. Cellular genotype-phenotype comparisons: classes of mutations S216B. Correlation of genotype with in vivo function S218C. Relation of CFTR mutations to disease severity S219D. Importance of CFTR in organ physiology and development of different organs S220

III. Clinical Course of Cystic Fibrosis: Turning Points in Pathogenesis S220A. Earliest pathological manifestations of pulmonary disease S220B. Airway infection and inflammation lead to bronchiectasis S221C. Role of inflammation in the progression of airway pathology S222

IV. Determinants of Airway Surface Liquid S223A. Basic principles S223B. Transport functions of proximal airways S225C. Other transport functions of CFTR S232D. Airway fluid transport and water permeability S233E. Composition and thickness of airway surface liquid S235F. Lessons from other genetic diseases S237

V. Mucociliary Clearance S238A. Factors contributing to normal clearance S238B. Mucociliary clearance and sputum properties in CF S239C. Effect of salt concentration on mucus transport S240D. Comparison with dyskinetic cilia syndromes S241

VI. Airway Infection S241A. Organisms and their mechanisms S241B. How the airway environment in CF permits infection S243

VII. Inflammatory Mechanisms S244A. Immune processes: defects in opsonization S244B. Defects in anti-inflammatory cytokines: interleukin-10 S244C. Oxidant environment and glutathione transport S245D. Defective apoptosis related to CF mutations S245E. Proinflammatory effects of bacterial DNA S245

VIII. Summary S246

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 conductanceregulator (CFTR), which accounts for the cAMP-regulated chloride conductance of airway epithelial cells. Lung diseaseis the chief cause of morbidity and mortality in CF patients. This review focuses on mechanisms whereby the deletionor impairment of CFTR chloride channel function produces lung disease. It examines the major themes of the channelhypothesis of CF, which involve impaired regulation of airway surface fluid volume or composition. Available evidenceindicates 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 periciliarysalt and water content, which impairs mucociliary clearance. In submucosal glands, loss of CFTR-mediated salt andwater 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, leadsto a progressive cycle of infection, inflammation, and declining lung function. Here, we provide the details of thispathophysiological cascade in the hope that its understanding will promote the development of new therapies forCF.

S2150031-9333/99 $15.00 Copyright q 1999 the American Physiological Society

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JOSEPH M. PILEWSKI AND RAYMOND A. FRIZZELL Volume 79S216

I. INTRODUCTION ture of mechanisms that control the physical and chemicalcomposition of the ASL, and they are therefore embodiedin the channel hypothesis of CF. In this review, we sum-In patients with cystic fibrosis (CF), pulmonary dis-

ease is the major cause of morbidity and mortality. With marize these functions of CFTR and refer, as appropriate,to other reviews in this supplement where these functionsthe development of treatments for intestinal obstruction

and pancreatic insufficiency, patients typically survive be- are described in greater detail.Why focus on CFTR’s function as an ion channel toyond infancy, and at some point, virtually all patients de-

velop chronic bacterial infection, abnormal airway secre- explain CF pathology? First, in exocrine tissues and intes-tine, the pathophysiology of CF appears to be explainedtions, and airway inflammation. This typically results in

progressive bronchiectasis, respiratory failure, and death. well by CFTR’s channel function. Intestinal obstructionin CF is due to a drying out of the luminal contents thatHowever, the question of how CF transmembrane conduc-

tance regulator (CFTR) mutations cause lung disease con- is caused by insufficient net fluid secretory activity. Theelevated salt in CF sweat and salivary secretions is ex-tinues to be one of the most perplexing and poorly under-

stood chapters in the story of CF and airway epithelial plained by a lack of anion channel function (218). Second,if one surveys different organs or across species, thecell pathophysiology. In short, we know that the CFTR is

a regulated anion channel that accounts for the cAMP- pathophysiology of CF generally varies with the capacityof different epithelia to express alternate ion channelregulated Cl conductance of the apical membranes of air-

way epithelial cells, and we know that CFTR mutations pathways. This appears to at least partly explain the rela-tive absence of lung pathology in CF mice, and it is theeither eliminate or markedly impair this conductance

pathway. But the question of how the loss of CFTR causes lead theory for why different strains of mice show differ-ent degrees of intestinal impairment when the CF geneCF remains incompletely understood. Several years ago,

there were educated guesses regarding the possible links is knocked out (see review by Grubb and Boucher, thissupplement). These findings suggest that other anionfrom CFTR to lung disease; today, there are well-reasoned

and testable hypotheses. Hopefully, the next several years channels can compensate for the loss of CFTR. Finally,most mutations that interfere only with the magnitude ofwill provide an understanding that provides for more ef-

fective treatments. the CFTR Cl conductance, as opposed to its processingand targeting to the plasma membrane, produce CF, butShould we focus on the Cl channel function of CFTR

and the loss of this function as the underlying source of usually in a milder form (254, see sect. IIC). Thus thesimplifying principle is that lung disease is manifest inCF pathophysiology? The reasons for doing so are embod-

ied in traditional, as well as more recent concepts of the some way because of the absence of a cAMP-regulatedanion channel in airway cells, and this leads either toconnecting links between CFTR and airway disease. The

channel hypothesis of CF now has two major themes: one physical or compositional impairment in the properties ofthe airway surface fluid that make the mucous gel moreis based on the role of CFTR in determining the volume

of the airway surface liquid (ASL). This concept holds difficult to clear and easier for bacteria to colonize.In this review, we first summarize the physiologicallythat a lack of fluid secretion, together with excessive fluid

absorption, leads to a reduction in the watery component relevant molecular genetics and clinical turning points inthe pathogenesis of CF lung disease. We then attempt toof the ASL, to a thickening of its mucous component, to

blocked submucosal gland ducts, and to impaired muco- relate the cellular functions that have been ascribed toCFTR to the properties of the ASL, its role in mucociliaryciliary clearance, infection, inflammation, and ultimately,

the tissue destruction characteristic of bronchiectasis. A clearance, and to aberrant inflammatory mechanisms thatmay explain how mutations in CFTR cause pulmonarysecond, more recent, concept is that this scenario arises

from an inherent ability of airway cells to fight against disease.bacterial infections that is compromised by the loss ofCFTR. This may be linked to changes in the composition

II. GENETICS AND PHYSIOLOGY OF CFTRof the ASL.

GENE MUTATIONSThere are also nonchannel or regulatory subthemes

that may contribute to CF airway disease. One featuresthe ability of CFTR to regulate other ion channels and A. Cellular Genotype-Phenotype Comparisons:

Classes of Mutationstherefore relates to formation of the ASL (see review bySchwiebert et al., this supplement). Another involvesfunctions of CFTR within intracellular compartments that To date, over 700 mutations in the CFTR gene have

been associated with a CF disease phenotype (S. FitzSim-lead to altered processing of macromolecules and the ap-pearance of glycoconjugates with different properties on mons, CF Foundation, personal communication). From a

physiological perspective, the grouping of mutations intothe airway surface (see review by Bradbury, this supple-ment). These regulatory events are part of the larger pic- five classes based on the primary mechanism responsible


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January 1999 ROLE OF CFTR IN AIRWAY DISEASE S217

FIG. 1. Classes of cystic fibrosis (CF)gene mutations. CF mutations can be di-vided into 5 classes that define the mecha-nism for defective chloride conductance.In normal epithelia (top panel), the CFgene is transcribed into mRNA, which istranslated in the endoplasmic reticulum(ER) to cystic fibrosis transmembraneconductance regulator (CFTR) protein.After translation, nascent CFTR is glyco-sylated in the Golgi apparatus before inser-tion in the cell membrane. In class 1 CFmutations, there is failure of CFTR transla-tion, typically due to stop mutations suchas G542X. In class 2 mutations, which in-clude the most common CF mutation,DF508, CFTR fails to mature and is de-graded by proteases in the ER. Class 3 mu-tations are fully processed and inserted inthe membrane, but mature protein is re-fractory to activation, as for example, theG551D mutation fails to conduct chloridein response to stimulation with protein ki-nase A. In class 4 mutations, the matureprotein is activated normally, but the chlo-ride conductance of channel is diminished.Finally, class 5 mutations are splice sitemutations that result in decreased abun-dance of full-length mRNAs, hence a de-crease in the quantity of fully functionalCFTR at cell membrane. [Modified fromTsui, L.-C., and P. Durie. Hosp. Pract. 32:115, 1997. Original illustration by SewardHung.]

for reduced CFTR Cl channel function has provided a detectable at the plasma membrane (51), but at 277C,some DF508 CFTR traffics to the cell membrane whereuseful framework for considering genotype-phenotype re-it forms partially functional Cl channels (50, 72, 171). Mostlationships. As proposed by Welsh and Smith (313) andlaboratories agree that DF508 CFTR exhibits a reducedsummarized in Figure 1, class 1 mutations, such as G542Xopen probability, however (72). Thus both class 1 and 2and R553X, are those in which stop codons or frameshiftmutations prevent sufficient CFTR expression at the cellmutations lead to premature termination of mRNA trans-membrane. As would be expected, clinical studies havelation, and thus essentially no protein production. In classconfirmed that these mutations are associated with typical2 mutations, CFTR protein fails to mature properly inmultiorgan disease, including male infertility, pancreaticthe biosynthetic pathway, with degradation of translatedinsufficiency, and progressive obstructive pulmonary dis-protein before it can progress past the endoplasmic reticu-ease.lum (see review by Kopito, this supplement). The DF508

mutation, the prototypic class 2 and the most common In contrast, class 3 and 4 mutations allow proteinproduction and transit to the apical surface, but they re-CF mutation, results in a temperature-sensitive defect in

protein processing; at 377C, little or no mature protein is sult in channels that are insensitive to activation or display


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altered Cl conductance. Class 3 mutations, such as G551D, of mutation paradigm, no Cl secretory response was ob-served in patients homozygous for DF508 or in those withare regulatory mutations in which single amino acid sub-

stitutions or deletions result in a properly processed pro- a DF508 mutation and a truncation mutation (G542X orR553X). Thus there is suggestive evidence for a correla-tein that is virtually insensitive to channel activation. For

example, reduced ATP binding to G551D CFTR results in tion between gene mutations and ion transport function;however, the sensitivity of the nasal potential differencea severely diminished macroscopic Cl conductance (173)

that would intuitively be associated with a severe disease assay for residual Cl secretory activity, and the correlationwith disease severity, remains to be determined.phenotype. Clinical studies have confirmed this predic-

tion, because there is no evidence that either pancreatic A second in vivo approach to correlate genotype withphysiology has been to examine the relationship betweenor pulmonary disease severity in these patients differs

significantly from patients with class 1 or 2 mutations genotype and carbachol-induced Cl secretion in rectal bi-opsies. Compared with patients with class 1 or 2 muta-(102). Class 4 mutations, such as R117H, R347P, and

R334W, respond to activation by cAMP agonists but ex- tions, patients with an A455E mutation had higher residualCl secretion. This was associated with a later age of CFhibit reduced Cl channel conductance or channel open

probability (254). As such, these mutations would be ex- diagnosis, a lower incidence of pancreatic insufficiency,and a higher achieved age (304). Collectively, the in vivopected to result in mild disease manifestations, and sev-

eral reports have confirmed that this is the case for pan- studies of nasal and rectal epithelial function support thein vitro observation that at least some class 4 mutationscreatic disease (11, 65, 90, 112, 159).

More recent studies have suggested that splice site permit residual Cl secretion that reduces disease severity.Of note is that residual Cl secretion was also observed inmutations, which affect the efficiency of normal mRNA

splicing and thereby alter the abundance of normally pro- a subset of homozygous DF508 patients. This suggeststhat other factors, such as the expression of other Clcessed and functional CFTR at the cell membrane, should

be considered a fifth class of CF mutations. The prototype channels, or the trafficking of some mutant DF508 CFTRto the membrane, or perhaps genetic polymorphisms, con-of this class is the 3849/10 kb C to T mutation, in which

a nucleotide substitution at a splice site reduces but does tribute to the variations in disease severity.Recently, several polymorphisms within the CFTRnot preclude correct mRNA splicing. Consequently, this

mutation yields at least some mRNA capable of producing gene have been identified and found to influence the pene-trance of some CFTR mutations. Chu and Cutting (55)functional protein that would be expected to be associ-

ated with mild disease. Clinical correlation with the lim- identified variations in the length of the polypyrimidinetract in the intron 8 splice acceptor site (the Tn locus)ited number of patients who have this mutation has docu-

mented a mild phenotype for pancreatic disease, and sur- and found that three length variants were associated withvarying efficiencies of exon 9 splicing. The 5 thymidineprisingly, this is true for the male genital tract as well

(109, 268). Congenital bilateral absence of vas deferens (5T) variant was associated with inefficient splicing andfrequent transcripts lacking exon 9, which is critical for(CBAVD) is a phenotype that is normally very sensitive

to mutation of CFTR (see sect. IID). formation of a functional CFTR protein (271). In a subse-quent study of the relationship between polypyrimidinevariants and the phenotype of patients with the R117H

B. Correlation of Genotype With In Vivo Function mutation, the 5T variant was most clearly associated withthe typical CF phenotype, whereas the 7T variant wasobserved both in patients with pancreatic sufficient CFSeveral approaches have been used to demonstrate

ion transport differences in vivo among the classes of and in patients with CBAVD alone (137). Thus it appearsthat the 5T variant often leads to lower levels of partiallyCFTR gene mutations, and in general, these have provided

evidence for residual Cl secretory capacity in some class functional R117H-CFTR, and hence clinical CF. The spliceefficiency of the 7T variant is not consistent; thus the4 mutations. One approach has been to measure transepi-

thelial potential difference across the nasal epithelium combination of R117H and 7T may or may not result inclinical disease.to assess amiloride-inhibitable Na transport and cAMP-

mediated Cl secretion (see Ref. 149 for review of tech- The presence of other polymorphic loci has beenproposed to account for the partial penetrance of poly-nique). In patients with gene mutations in which CFTR

traffics to the membrane (G551D, A455E, R117H), there morphic Tn loci and may also contribute to the observedphenotypic variability within CFTR mutations. Evidencewas significantly more residual Cl secretion than in pa-

tients with mutations that do not permit protein synthesis for partial penetrance of the 5T allele was recently de-rived from an analysis of the 5T allele in a large ethnicallyor trafficking (DF508, W1282X, Q493X) (111). Moreover,

there was a positive correlation between the amount of similar population. An increased frequency of the 5T al-lele was found in patients with CF or atypical CF whoresidual Cl secretion and the forced expiratory volume,

an indicator of airway function. As predicted by the class did not have CFTR mutations on the chromosome car-


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rying the 5T allele. Moreover, within families, the same vealed wide variation in pulmonary disease severity. Pa-tients with an A455E mutation had a higher likelihood of5T allele was associated with a wide range of clinical

presentations, from healthy fertile male to CBAVD to pancreatic sufficiency, better pulmonary function, and alower likelihood of airway colonization with Pseudomo-clinical CF, supporting the notion that the 5T allele is a

splice variant with partial penetrance (134). Two other nas aeruginosa (88). Interestingly, two studies have dem-onstrated that heterologous expression of A455E CFTRpolymorphisms were recently demonstrated to contrib-

ute to the variable penetrance of the 5T allele. Cuppens resulted in a diminished but significant halide permeabil-ity compared with wild-type CFTR (253, 308). In contrast,et al. (62) examined the effects of polymorphisms at the

(TG)m and M470V loci and found that a higher number of nasal potential difference studies to determine the pres-ence of a cAMP-mediated Cl secretory response in nasalTG repeats on the 5T allele was associated with disease,

whereas a low number was observed in healthy CF fa- epithelium of A455E patients revealed a modest responsein only one of five patients (308). Thus there appears tothers. Moreover, the number of TG repeats influenced the

exon 9 splice acceptor efficiency, and CFTR Cl channel be a correlation between in vitro Cl secretion and diseaseseverity for some class 4 mutations. The failure to discernactivity varied with the polymorphism at the 470 locus.

These data provide strong evidence that polymorphisms a nasal potential difference response could reflect a lowexpression level of CFTR in surface epithelium or indicatecontribute to the partial penetrance of the 5T allele and

suggest that such polymorphisms may contribute to het- that the nasal potential difference assay lacks sensitivityfor detection of residual Cl secretion.erogeneity in both CFTR Cl channel conductance and

disease phenotype among individuals with the same The larger study by the CF genotype-phenotypeconsortium (101) failed to identify differences in pul-CFTR mutation.monary disease severity between DF508 homozygouspatients and those who were compound heterozygotes

C. Relation of CFTR Mutations to Disease Severity for DF508 and several other mutations, includingG542X, R553X, W1282X, N1303K, 621 / 1G to T, 1717 –1G to A, and R117H. The wide intragenotype variationHeterogeneity in CF phenotype has raised the logical

question of whether the clinical variability could be ex- in pulmonary disease severity has been proposed toreflect environmental factors, the presence of poly-plained on the basis of genotypic differences, with the

hypothesis that mutations having residual CFTR function morphisms or modifier genes (see above), or differ-ences in therapy and/or patient compliance. Togetherwould be associated with milder phenotypes. Several ap-

proaches have been used to correlate genotype with phe- with the limited number of patients having a class 4 or5 mutation, the variability in pulmonary disease makesnotype, including the in vivo ion transport studies dis-

cussed above and a number of epidemiological studies. identification of mild pulmonary mutations more diffi-cult. A recent longitudinal analysis of pulmonary func-The large case control analysis from the multinational

CF genotype-phenotype consortium clearly demonstrated tion in CF patients revealed that DF508 homozygouspatients had a higher rate of pulmonary function de-that certain mutations are associated with pancreatic suf-

ficiency; however, a correlation between genotype and cline than patients who were heterozygous for DF508or had two non-DF508 mutations (60). Similar analysesseverity of pulmonary disease could not be identified.

Comparison of DF508 homozygous patients with a limited of larger cohorts of patients having ‘‘mild’’ mutations,or collection of survival data from large registries, mayspectrum of compound heterozygotes revealed that the

class 4 R117H mutation was associated with pancreatic eventually prove more fruitful than case control stud-ies for determining the influence of genotype on pul-sufficiency, later age at CF diagnosis, and lower sweat Cl

concentrations (101). Other studies have demonstrated monary function.Considered collectively, the studies of CFTR channelthat another class 4 mutation (A455E) and a class 5 muta-

tion (3849/10 kb C to T) are also associated with pancre- function in vitro and correlation with in vivo ion transportand disease phenotype suggest that the amount of residualatic sufficiency (109). These studies demonstrate that at

least some class 4 or 5 mutations are associated with mild Cl channel function of a given mutation influences diseaseseverity in the pancreas and, in some cases, the lung.pancreatic disease and thereby support the notion that

for pancreatic function, a class 4 or 5 mutation provides At least some class 4 mutations permit sufficient CFTRfunction to yield a less severe phenotype (159); however,enough residual CFTR Cl channel function to result in a

mild phenotype. other factors must be implicated to account for the widevariation in disease severity within a group of patientsCase control studies that have attempted to correlate

genotype with pulmonary disease severity have disclosed having the same mutation. Moreover, further studies arenecessary to determine whether residual CFTR Cl channelthat A455E (a class 4 mutant) is associated with mild

disease (88); however, studies of groups of patients with function causes a less severe disease phenotype orwhether such function is merely a marker of nonchannelother uncommon mutations, including siblings, have re-


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

Organ Dysfunction

Functional Vas SweatMutation (Class) CFTR, % deferens gland Lung Pancreas

Wild type/mutation (CF carrier) 50 0 0 0 05T/5T polymorphism with wild-type CFTR 10 / 0 0 0R117H (4) on 7T background Ç4–5 // 0 //0 0A455E (4) Ç4 // //0 //0 03849 / 10 kb C to T (5) Ç4 //0 0 //0 0R117H (4) on 5T background 1 // / / 0G551D (3) õ1 // // // //DF508 (2) õ1 // // // //G542X (1) õ1 // // // //

CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; //, early-onset disease, typically severe; /, late-onset ormild disease; //0, mild or clinically insignificant disease; 0, normal function.

CFTR function that may be more important in pathophysi- tation are estimated to have Ç4% of normal CFTR func-tion, since Ç8% of A455E CFTR reaches the cell surfaceology (see below).(253). Most of these patients have high sweat Cl concen-trations but milder CF pulmonary disease and pancreatic

D. Importance of CFTR in Organ Physiology and sufficiency. This suggests that lung and sweat duct dys-Development of Different Organs function occurs when the level of functional CFTR is less

than Ç5%. With the severe mutations (class 1, 2, or 3),Recent studies have suggested that variations in the the level of functional CFTR is generally õ1%. Patients

organ system manifestations of CF reflect differences in with two of these mutations typically present with pancre-the level of CFTR function necessary for normal organ atic insufficiency in addition to severe pulmonary disease,function. These variations are particularly intriguing for suggesting that pancreatic disease occurs withõ1% func-male genital tract disease. Over 95% of males with multiple tional CFTR. On the basis of this analysis, the rank orderorgan manifestations of CF suffer from infertility due to of organ susceptibility to CFTR mutations from the most-bilateral absence of the vas deferens. At the other end of to-least sensitive is the vas deferens, the sweat duct, thethe clinical spectrum are patients with CBAVD but no lung, and the pancreas. Exceptions to this generalization,recognized pulmonary, pancreatic, or sinus disease. Close such as the observation of fertility and significant butevaluation has revealed that more CBAVD patients are delayed onset pulmonary disease in male patients withheterozygous at the CFTR locus (50 vs. 4% in the general the splicing mutation 3849/10 kb C to T (268), suggestpopulation). In addition, several patients have two CFTR that other mechanisms, such as alternative splicing in dif-mutations, one of which is R117H (70). Other studies have ferent organs (281) or organ-specific modifier genes, con-found a high incidence of the 5T exon 9 splice variant on tribute to this already difficult effort of rigorously definingthe normal allele in the heterozygous CBAVD patients the correlation between genotype and phenotype.(52), suggesting that a reduction of functional CFTR toÇ10% of wild-type levels may result in genital tract dis-

III. CLINICAL COURSE OF CYSTIC FIBROSIS:ease. These observations suggest that CBAVD is a formTURNING POINTS IN PATHOGENESISof CF disease, and they further demonstrate that mild

mutations, such as R117H, may provide sufficient residualAs discussed briefly in section I, obstructive pulmo-Cl secretory capacity to prevent the development of typi-

nary disease is the major cause of morbidity and mortalitycal CF pulmonary and pancreatic disease.in CF. Before turning to a discussion of the pathogenesisThe correlation between the level of functional CFTRof CF airway disease, we review the clinical turning pointsand phenotype has been further extended to estimate theto place the subsequent discussion of ASL within areduction of functional protein necessary in the lung andbroader context.pancreas to cause disease [proposed by Davis et al. (64)

and summarized in Table 1]. Individuals carrying one mu-tant allele (heterozygotes or disease carriers) are ex- A. Earliest Pathological Manifestations

pected to express 50% of normal CFTR, and they have no of Pulmonary Disease

disease phenotype. This suggests that a 50% reduction inCFTR expression is developmentally and physiologically Clinical and pathological studies have suggested a

number of turning points in the generally progressiveinsignificant. Patients with A455E plus a severe CFTR mu-


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TABLE 2. Turning points in the pathophysiology of CF lungs (184). Collectively, these data suggest that CFTRlung disease does not play an important role during lung development,

presumably because of alternative secretory pathways,Mucus inspissation of submucosal glands such as the ClC-2 Cl channel (197). The importance andBacterial infection of the airway with Haemophilus influenzae or

mechanisms of secretory pathways in lung developmentStaphylococcus aureus

Airway inflammation is beyond the scope of this review; the reader is referredInfection with Pseudomonas aeruginosa and acquisition of mucoidy to a number of recent reviews in this area (28, 269).

B. Airway Infection and Inflammation Leadcourse of CF (summarized in Table 2). It is noteworthyto Bronchiectasisthat although the CF lung is macroscopically normal at

birth, subtle abnormalities in mucus secretion appear veryearly and may represent the first turning point in patho- Although it has been suggested recently that there is

a lack of causation between airway infection and inflam-genesis. Pathological descriptions of mucus inspissationin submucosal glands as early as the second trimester of mation in the CF airway (see sects. VI and VII), bacterial

infection and airway inflammation appear to be the sec-development imply that abnormal mucus secretion occursin CF in the absence of airway infection (203). Histopatho- ond and third turning points in the pathogenesis of CF

airway disease. The airways of CF patients are preferen-logical analysis of the conducting airways from six ofseven fetuses with CF revealed dilatation of the tracheal tially colonized by specific bacterial pathogens, often in

the first year of life. Studies of CF patients from the clini-submucosal glands with accumulation of inspissated mu-cus. Similar changes have been observed in CF newborns cally well newborn to the severely affected adult have

implicated airway inflammation as critical to the patho-dying of meconium ileus (76, 77); however, a separategroup was unable to confirm consistent submucosal gland physiology of CF lung disease, with most clinical studies

suggesting that bacterial infection drives the airway in-pathology in newborns (54). Moreover, the specificity ofgland dilatation to CF has been called into question by flammation. With the advent of newborn screening for CF,

patients were identified before the onset of overt pulmo-other investigators who found similar pathology in submu-cosal glands in newborns dying of other airway diseases nary disease, allowing for serial assessments of bacterial

colonization and an early evaluation of lower airway in-(201). These uncertainties may relate to differences be-tween the late fetal and postnatal lung. Among the patho- flammation. These studies have demonstrated an evolu-

tion of bacterial pathogens: Staphylococcus aureus (SA)logical studies, however, the studies of CF fetuses andnewborns imply a defect in mucus secretion that precedes and Hemophilus influenzae (HI) appear to inhabit the CF

airway early, often before the onset of clinical symptoms,infection.The link between CFTR and the histological findings while airway infection with Pseudomonas aeruginosa

(PA) almost universally follows these other pathogens (1).during lung development is unclear, because CF muta-tions do not appear to alter morphogenesis. The CFTR is The age at first positive culture for SA was significantly

earlier (mean of 12.4 mo) than for PA (mean of 20.8 mo),expressed in conducting airway during lung development.With the use of sensitive assays (reverse transcription and patients infected with PA typically had SA and HI

isolated from the airway before infection with PA. OtherPCR), CFTR mRNA was detected in the lung of 18-wkhuman fetuses (108). Subsequent studies in human and studies have confirmed this sequence of bacterial infec-

tion (133). Moreover, infants in whom PA could be iso-rabbit fetuses confirmed expression of CFTR mRNA inthe pseudoglandular stage of lung development and dem- lated were more likely to have chronic cough and had a

higher frequency of hospitalizations for respiratory dis-onstrated expression of CFTR protein from the pseudo-glandular stage through birth (184, 186). In situ hybridiza- ease than patients without PA in lower airway cultures

(1). This suggests that the presence of PA has pathophysi-tion revealed CF gene expression in both large bronchiand small airway epithelium throughout lung develop- ological significance (see below). Persistent bacterial iso-

lation from the CF airway has been considered coloniza-ment, with decreasing expression in distal epithelium inthe later developmental stages (283). Interestingly, al- tion, which implies a harmless interaction between host

and organism. However, the above clinical studies andthough CFTR is abundantly expressed in the serous cellsof submucosal glands in postnatal lung (74, 121), no CF the observation that inflammation and protease excess

persists through periods of clinical stability (see below)gene expression was observed in fetal submucosal glands(283). Moreover, despite differences in the volume of lung support the contention that bacterial persistence repre-

sents a harmful stimulus to the airway by driving inflam-secretions and transepithelial potential difference be-tween CF and non-CF second trimester fetal lung explants mation. In brief, the CF airway may most accurately be

perceived as chronically infected with bacterial pathogensmaintained in short-term organ culture, there were nogross morphological differences in second trimester fetal rather than colonized (44).


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The implication of these clinical observations is thatPA, particularly the mucoid strain, plays a major role inthe pathogenesis of CF airway disease and that acquisitionof mucoid PA be considered a fourth turning point inpathogenesis. An alternative explanation has been thatPA colonization is not itself pathogenic but merely reflectsthe severity of airway dysfunction. One attempt to resolvethis issue was to examine the relationship between coloni-zation with PA and the decline in pulmonary function. In alongitudinal 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% lowerlung function relative to patients in whom PA was notisolated. This suggested that airway infection with PA ispathogenic and not merely a marker of airway pathology.

Studies of the clinical course of CF suggest thatbacterial infection of the airway leads to an inflamma-tory response. Persistence of infection and inflamma-tion through periods of clinical stability (156) ulti-mately leads to bronchiectasis, that is, to abnormal and

FIG. 2. Typical clinical course of airway disease in CF. On left isgenerally irreversible dilatation of the airways. Morean approximate time line for the highlights in development of airwayrecent studies using samples from the lower airway ofdisease in the typical patient with CF. Although there is considerable

infants with CF have confirmed early airway infection variation in the timing of each event, with some patients not presentingwith lung disease until adulthood, the linear sequence is generally ob-with SA and supported the notion that bacterial infec-served.tion begets airway inflammation. As expected, the ma-

jority of infants who had greater than 105 cfu bacteria/ml of lower airway fluid had increased numbers of neu-

of Khan et al. (136) that suggest a discordance betweentrophils and higher total cell counts. The few infants

airway inflammation and infection remain to be con-with increased inflammatory cells without bacterial or

firmed.viral isolates were thought to have an alternative etiol-ogy, such as aspiration lung disease (12, 13). However,

C. Role of Inflammation in the Progressiona second study raised the question of whether inflam-of Airway Pathologymation precedes significant airway infection. Seven of

19 CF infants who had negative cultures for bacterialpathogens or common respiratory viruses had evi- Studies describing the sequence of pathological

changes in the lung indicate that the submucosal glandsdence of airway inflammation [increased leukocytesand concentration of the potent chemoattractant in- are involved uniformly and that airway disease involves

both proximal and distal airway segments. Morphologicalterleukin (IL)-8] (136). Although plausible explana-tions for this finding are that the sensitivity of broncho- changes in the airway wall and lumen occur shortly after

hyperplasia and obstruction of tracheal and bronchial sub-scopic sampling for detection of viruses and bacteriais suboptimal, or that inflammatory cells persist during mucosal glands (22, 267). Inflammatory infiltrates in the

airway submucosa and plugging of bronchi and bronchiolithe resolution phase of a subclinical infection, this ob-servation raised the intriguing hypothesis that airway with mucus and inflammatory cells were observed in the

majority of patients who died in the first 4 mo. However,inflammation may precede bacterial infection in theCF airway. A recent study by Armstrong et al. (13), the hallmark pathological change in CF, bronchiectasis,

was unusual at this age (22). In a subsequent morphologi-however, identified a larger cohort of infants lacking adetectable airway pathogen. In this larger population, cal study, changes in the more distal airways varied de-

pending on the age at death, with airway dilatation beingseveral markers of inflammation [bronchoalveolar la-vage (BAL) cell count, concentrations of IL-8, and elas- prominent in younger patients (267). More recent studies

have suggested that over time, chronic infection and in-tolytic activity] were no different from a control popu-lation of infants with stridor, a condition characterized flammation in the proximal airway lead to a destruction

of bronchial cartilage (200) that contributes to both expi-by upper airway obstruction (13). Moreover, serialBAL samples from the same individuals suggested a ratory airflow limitation and the progression of bronchiec-

tasis. Thus pathological and clinical studies support theclose correlation between inflammatory markers andthe presence of bacterial pathogens. Thus the findings pathophysiological sequence summarized in Figure 2 and


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FIG. 3. Schematized morphology ofproximal and distal airways. Elements ofmucociliary defense and clearance mech-anism are depicted. Shown is the conven-tional view that separate surface gel andpericiliary sol layers comprise the airwaysurface fluid. See text for further discus-sion.

posed of two phases, gel and sol, was first proposed bythe hypothesis that airway inflammation due to infectionLucas and Douglas (174) from transmission electron mi-is necessary for the development of bronchiectasis.crographs of the apical surface of tracheal epithelium.Airway surface liquid coats distal airways as well, but a

IV. DETERMINANTS OF AIRWAY corresponding mucous gel layer is minimal distally (337).SURFACE LIQUID Experimental estimates of the ASL thickness vary widely,

but most studies agree that its depth is Ç10–20 mm (14,298). For the most part, it is the gel layer that contributesA. Basic Principlesto variations in ASL thickness; the gel contains a varietyof macromolecular secretory products (21), including gly-1. Cell typescoproteins, proteoglycans, lipids, defense molecules (27,

The upper respiratory tract is lined by a pseudo-stra- 53, 73), DNA (59), and actin (303). These latter two com-tified, mostly ciliated epithelium that extends from the ponents are produced by cellular breakdown and bacteria,proximal trachea to the terminal bronchioles (see Fig. 3; and they can become a significant burden in the airwaysRef. 7). Ciliated and nonciliated columnar cells and goblet of patients with CF. The concept that disrupting the net-cells populate the surface epithelium. In the large airways, work of DNA and actin would enhance clearance of thethe ratio of ciliated columnar cells to goblet cells is Ç5:1; gel has led to the development of DNase and gelsolin inthe numbers of both cell types decrease in peripheral an attempt to liquify these tangled, complex structures (5,airways where the nonciliated cells become more numer- 251, 296, 303).ous (230). Interspersed among the ciliated cells of the The ASL is the first line of defense against inhaledproximal airways are brush cells, which are also noncili- pathogens, and it is mandatory for effective mucociliaryated (7, 337). Their microvilli, like those of the ciliated clearance (238). The upward movement of liquid throughcells, measureÇ1 mm in length (7). The cilia, on the other the trachea averages 10–100 ml/day, as deduced fromhand, have an average length of 6 mm (79, 249), which fluid collections from tracheostomy patients (285). Thehas been proposed as the minimal periciliary liquid depth division of the ASL into a periciliary liquid (sol) layer(see sect. IVD1). Below the surface epithelium of the prox- and a separate, overlying mucous (gel) layer (174, 214)imal airways are numerous submucosal glands, which provides an anatomic basis (perhaps bias) for one inter-contain mucous and serous secretory cells (123). Submu- pretation of how mucociliary clearance occurs (111, 307).cosal glands are found in the regions of proximal airway The concept is that the cilia can beat and clear the gelwhere there is cartilage (229). With increased airway more effectively when bathed by a liquid (sol) layer whosebranching, as one approaches the periphery, the pseudo- depth approximates the ciliary length. In this view, thestratified structure of the epithelium is lost. In the distal tips of the cilia extend to contact the mucous layer onbronchioles, the epithelial cells take on a cuboidal shape their forward stroke and return in a more folded mannerand are termed clara cells, Ç40% of which are ciliated to complete the beat cycle. Thus, if the periciliary liquid(39). Goblet cells are normally absent in this region. layer becomes either too deep or too shallow, mucociliary

The airway epithelium is bathed on its apical surface clearance will be impaired because the mucous layer iseither too far away to be contacted or it lays directly onby a thin liquid layer. The concept that this layer is com-


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the cilia, impeding their ability to beat productively. In clarified. First, it is important to identify the source of theliquid from distal regions that would be moving proxi-either case, the mechanics of ciliary interactions with the

mucous blanket are suboptimal. mally. The physical forces of hydrostatic and colloid os-motic pressures acting across the alveolus are poised toA more recent concept proposes that mucins of the

gel do not form a discrete blanket, or islands, but rather, a keep the air-space surface dry (81, 165). In addition, themost distal airway cells that have been examined experi-tangled, hydrated network (306). Arguments in favor of this

model rest on observations showing that mucins become mentally are found to be absorptive not secretory (94,178, 301). Second, it is important to know whether bothmore gel-like (structured) at higher glycoprotein concentra-

tions (185). Accordingly, the gel layer may be more concen- liquid and mucin are moving in response to the ciliaryclearance mechanism. Whether the mucins exist as a dis-trated near the air-liquid interface and more dispersed near

the epithelium where the cilia are beating. The details re- tinct physical layer or as a tangled network, the gel maybe moved preferentially to the periciliary sol by ciliarymain to be assessed, but it seems clear that the periciliary

region of the ASL is more liquid than mucin rich and that beating so that large volumes of water may not be movingproximally. Third, it is important to know the contributionthis optimizes ciliary activity and mucin clearance. Ac-

cording to both models, mucins of the gel are propelled of pulmonary surfactants and the forces of surface tensionto the determination of ASL depth (15). These physicalupward, whereas the periciliary liquid is assumed to be

relatively static, moving to and fro with ciliary beating, but forces are likely to differ in distal and proximal regionson a geometric basis, and this may contribute to theirwith little net transport. This concept has been tested re-

cently with the use of fluorescent markers of the gel and capacity to hold different amounts of liquid. Clearly, themechanisms that govern axial liquid flow need to be de-sol phases and is discussed in section IVE.

The periciliary fluid composition reflects the salt and fined. However, the small volume of sputum emergingfrom tracheostomies implies that at least the mucins willwater absorptive and secretory functions of the airway

epithelium, which hydrate the mucous gel and influence become more concentrated as they move proximally.The concept that emerges from the discussion aboveits clearance (32, 33). Thus the ion and water transport

properties of the epithelium have the opportunity to in- is that the thickness of the ASL, particularly the periciliaryliquid layer, is carefully regulated. Whether this is true isfluence the volume and composition of the periciliary liq-

uid layer, and accordingly, changes in cellular transport uncertain because it is difficult to test experimentally.This concept suggests that the surface epithelium some-properties may result in composition and/or volume

changes in the ASL that contribute to the development of how has the capacity to sense properties (e.g., salt con-centration, volume) of the liquid lying on its surface andairway disease. Our goal here is to summarize the trans-

port properties of the epithelium and our knowledge of to adjust those properties by appropriately adding or re-moving salt and water. Such mechanisms have not beentheir influence on the volume and composition of this

fluid compartment. identified. Indeed, regulation of the periciliary liquid maybe primarily local, i.e., occurring only over several or evensingle cells. Recent advances in the techniques for primary2. Fluid transport: radial and axial flowcell culture of airway surface cells, which can duplicatethe ciliated columnar cell morphology of the surface epi-A concept of airway fluid homeostasis that has impli-

cations for the volume and composition of the ASL is the thelium (180, 261; see Fig. 4), should be useful in providingexperimental assessment of the axial flow of liquid andidea that liquid moves axially between different regions

of the lung. This concept, first proposed by Kilburn (138), mucus and of the local (cell-mediated) controls over ASLvolume and composition. Nevertheless, these cultureswas reviewed by Boucher (32). It recognizes the large

disparity in the surface areas of the distal versus proximal lack innervation and eliminate any contribution of thesubmucosal glands to fluid formation or its regulation.airways. The fact that the thickness of the ASL is similar

in these regions means that there is a large disparity in The likelihood that submucosal glands add to the volumeand compositional properties of the ASL in proximal air-the amount of liquid contained at different levels of the

lung. For example, we can estimate that there is Ç700 ml ways has not received sufficient attention, and this is dis-cussed in more detail in section IVB2.of liquid residing in distal airways and air spaces (based

on an ASL thickness of 10 mm) and only Ç1 ml of liquidin the trachea. The idea that surface liquid is moving up 3. Sites of CFTR expressionthe airways implies that the 0.7 liter present distally wouldneed to be reabsorbed, given the geometry of the airway Soon after the identification of the CFTR gene, inves-

tigators determined its expression in human proximalnetwork, otherwise the proximal airways would be awashin liquid. lung tissue using protein and RNA detection methods.

Here, CFTR was found in both the surface epitheliumAlthough this is an intriguing concept, several keyelements and assumptions of this model need to be further and in submucosal glands (see Fig. 5). In bronchoscopy


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FIG. 4. Surface scanning electronmicrograph of human bronchial epithe-lium in primary culture at an air-liquidinterface. Presence of ciliated and noncil-iated cells mirrors the morphology ofproximal airway in vivo. The gel layerthat forms over the polarized, differenti-ated epithelium has been removed bywashing before processing. (Courtesy ofDrs. D. Devor, S. C. Watkins, and J. M.Pilewski.)

samples of surface epithelium from normal subjects, having known properties of CFTR is a more sensitiveassay than immunocytochemistry, and such studies leadquantitative PCR techniques were used to estimate that

only about one or two mRNA transcripts for CFTR were to the conclusion that CFTR is present at the apical mem-brane domain of ciliated surface cells (58, 322).expressed in each cell (290). Detection of protein in native

airway has been difficult because generation of high-affin- In contrast to the low levels of mRNA and protein ex-pression in the surface epithelium, CFTR is expressed atity, high-specificity antibodies against CFTR has not been

straightforward and because the protein is expressed at higher levels in subpopulations of cells in the submucosalglands (74, 121). The CFTR mRNA and protein have beenlow levels. Indeed, immunostaining of normal lung does

not always reveal CFTR expression in ciliated airway cells detected also in the striated ducts, where a small percentageof cells exhibit the highest levels of CFTR expression identi-(69, 74). Because CFTR is a Cl channel with a turnover

rate ofÇ21 106 ions/s, relatively few copies of the protein fied in the airways. Its function at this site is as yet unknown.In addition, serous cells at the base of the submucosal glandsare needed to provide the required apical membrane Cl

conductance. From the channel’s properties, estimates in- contain readily detectable levels of CFTR. In distal airways,a small population of cells also express relatively high levelsdicate that only several hundred to several thousand

CFTRs per cell are necessary to provide the diffusional of CFTR, as in submucosal gland ducts. They comprise onlyÇ1% of the nonciliated epithelial cell population so thatCl transport properties required at the apical membrane.

The identification of a cAMP-regulated Cl conductance the function of CFTR in this setting is difficult to defineexperimentally. This expression pattern has also been de-scribed for small intestine, where occasional cells appear tohave very high CFTR levels (9). Because the less differenti-ated crypt cells of the intestine are the highest sites of CFTRexpression (291), it is possible that these high expressingcells in both intestine and airway have strayed from a normaldevelopmental program, remaining in an undifferentiatedstate. In general, studies of CFTR gene expression suggesta role for CFTR at the apical membranes of ciliated surfaceepithelial cells throughout the airway and in the submucosalgland 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 theairway are derived primarily from examination of proxi-mal airway cells, studied as either excised airway seg-ments or as epithelial monolayers in primary culture (fordetailed review, see Refs. 32, 33). Considerable in vitrodata are available from human airway cells, derived pri-

FIG. 5. Proximal airway schematic showing sites of salt and watermarily from nasal or bronchial epithelia. As yet, there istransport and mucin secretion that lead to formation of airway surface

fluid. Relative level of CFTR expression is indicated by shading. no published transport data from human distal (noncarti-


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absorbed in response to the transepithelial potential dif-ference, and similar results are obtained from tissues ex-cised from nasal or bronchial regions (321). The transepi-thelial resistance of excised upper airway epithelia is rela-tively low (Ç300 Vrcm2; Ref. 148), and under short-circuitconditions, bidirectional Cl fluxes across excised airwaysegments are relatively large (148). This is consistent withthe concept that paracellular Cl permeability is high andthat Cl flow between the cells could provide the principalpathway for Cl absorption (32, 320, 322). The route of Clflow during NaCl absorption across the surface epithelium

FIG. 6. Cellular models for NaCl absorption (surface epithelium) should be better clarified, since direct estimates of cellularand secretion (serous cells of submucosal gland). CFTR is apical Clversus paracellular Cl flow have not been made.conductance shown in both cell types. The transepithelial electrical po-

tential difference (Vt) is lumen negative across both absorptive and Microelectrode studies of the apical membrane volt-secretory cells with physiological solutions at both surfaces. Vt arises age and intracellular ion activities detect a large electro-from differences in the apical and basolateral membrane voltages as

chemical driving force favoring Na entry (Ç60 mV) acrossshown; average values are given for open-circuit conditions.the apical membrane (61, 311, 323). In addition to entrythrough amiloride-sensitive channels, Na-coupled glucoseentry may contribute to Na absorption (128). This processlaginous) airway. Data on cultured epithelia may be compro-

mised by variability arising from the cell culture methods may scavenge glucose from the ASL that diffuses in fromthe plasma.used by different laboratories, but these techniques have

consistently improved and become more uniform. Although Microelectrode studies suggest that cell Cl is distrib-uted close to its equilibrium distribution across the api-the in vivo situation cannot be reproduced using cultured

cells, the morphology of epithelia grown on permeable colla- cal membrane; that is, there is not a significant drivingforce for diffusional Cl entry from the lumen (312, 322).gen supports with an air interface at the apical surface (98)

has become qualitatively similar to that of the native surface An equilibrium distribution of Cl across the apical mem-brane implies that there will be essentially no net Cl flowepithelium (see Fig. 4). Thus it is possible to obtain well-

differentiated epithelia in vitro that resembles the native into the cell during Na absorption. Accordingly, the Clconductance of surface epithelial cells should have mini-surface epithelium. Unfortunately, this has not yet been

achieved for the submucosal glands (see below). mal impact on the NaCl absorption rate because Cl isbeing absorbed between the cells, not through them (32).A) NaCl ABSORPTION. I) In vitro measurements. Studies

performed under standard physiological conditions indi- Thus, in CF, the absence of CFTR at the apical membranewould not markedly affect the NaCl absorptive proper-cate that proximal airway surface epithelia absorb Na, Cl,

and water (35, 124, 151) and that this occurs by the basic ties of the epithelium, except for its role as a regulatorof the apical Na conductance (see review by Schwiebertmechanism defined by Koefoed-Johnsen and Ussing (152)

almost five decades ago (see Fig. 6). According to this et al., this supplement). This leads us to a curious conclu-sion physiologically: the primary function of CFTR in themodel, Na enters across the apical membranes via amilo-

ride-sensitive, epithelial Na channels (ENaC), reviewed airway surface epithelium is not as a cellular Cl conduc-tance that provides a pathway for Cl flow during NaClby Garty and Palmer (89). Cell Na is extruded by the Na-

K pump (275), and K accumulated by the pump can be absorption; rather, its role is to regulate the activity ofthe apical Na channel. It remains to be seen whether theeither secreted or recycled to the interstitial space. Exit

of K down its electrochemical potential gradient across driving force on Cl flow at the apical membrane, identi-fied in excised airway segments and cell culture systems,the apical membrane may contribute to the relatively high

K concentration of the ASL (see below), but the responsi- applies also to the epithelium in vivo. Another uncer-tainty regarding transcellular Cl flow is the mechanismble apical K channel has not been identified. Most of the

K taken up by the pump is recycled to the submucosal of Cl transport across the basolateral membrane. Ordi-narily, there is a significant driving force for Cl exit fromsolution via K channels in the basolateral membranes (32).

From in vitro studies, the predominate active ion the cell in the absorptive direction, and in ion replace-ment studies, a small basolateral membrane Cl conduc-transport activity of either freshly excised (148) or cul-

tured (332) airway epithelia studied under short-circuit tance has been detected (322). However, the molecularidentity of the basolateral Cl conductance pathway hasconditions is electrogenic Na absorption (320). Under

open-circuit conditions, these tissues also show net Na not been defined. It is presumably not CFTR. The basolat-eral conductance properties are dominated by K-selec-absorption, with the magnitude of net Na transport, rela-

tive to the short-circuit condition, somewhat reduced by tive pathways.II) In vivo measurements. The experimental basisthe transepithelial (lumen-negative) voltage. Chloride is


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for identifying NaCl absorption as the major salt transportevent in the surface epithelium relies primarily on in vitromeasurements using electrophysiological and isotopicflux techniques (as above). Measurement of the transepi-thelial electrical potential difference in vivo detects, underbasal conditions, a voltage (Vt) across the proximal airwayepithelium of normal subjects of approximately 030 mV(lumen negative) (144–146); similar values have been de-tected in the proximal airways. The activity of the ENaCis the principal determinant of Na absorption rate, as re-flected by inhibition of Vt by amiloride. Superfusion ofamiloride onto the airway surface of normal subjects elim-inates Ç60% of the Vt (144, 146). The residual voltageunder these conditions may reflect stimulation of Cl secre-tion (see below), although electrogenic HCO3 secretionmay also contribute. In CF airway, the basal Vt is elevatedto about 060 mV, due largely to an increase in the amilo-ride-sensitive component of Vt (146). Most of this elevatedVt is amiloride sensitive (149). Results from in vitro mea-surements on excised tissues or cultured epithelia suggestthat the elevated Vt reflects enhanced activity of ENaC at

FIG. 7. Interactions of CFTR with other cellular events. Steps inthe apical membranes of airway surface cells (35, 61, 321).CFTR biosynthesis dictate its presence in intracellular compartments

The absence of an apical Cl conductance may contribute where its activity can contribute to their acidification. Phosphorylation-dependent regulation of CFTR leads to its insertion and retrieval atto the larger Vt across CF airways, but the leakier paracel-plasma membrane. Time constants for these CFTR trafficking reactionslular pathway would attenuate its contribution. In theare several minutes, consistent with time course of stimulation of trans-

sweat duct, Vt rises higher in CF because the cellular epithelial Cl current. Time constants of biosynthesis and degradationare Ç10 h. Activity of CFTR Cl channels in intracellular compartmentspathway is dominant for transepithelial Cl flow.may lead to alteration in processing of glycoproteins or in functionalIII) Regulation. Little is known about the acute orexpression of other Cl channels [e.g., outwardly rectifying Cl channels

chronic regulation of Na transport in airway surface cells. (ORCC)] or amiloride-sensitive Na channel (ENaC). PPase, protein phos-phatase. See text and reviews by Bradbury and Schwiebert et al. forEvidence from ENaC expression studies suggests thatdiscussion.cAMP stimulation enhances amiloride-sensitive Na cur-

rents in the absence of CFTR expression but that withCFTR coexpression, Na currents were smaller and were

is sufficiently hyperpolarized, which can be produced byinhibited by cAMP (273). These data are consistent withblocking apical Na entry with amiloride, a significant driv-the proposed role of CFTR as a negative regulator ofing force for Cl exit from cell to lumen is established (322).ENaC, an influence that would be removed in CF. Single-In the presence of amiloride, transcellular Cl secretion ischannel studies of ENaC expressed in fibroblasts suggestobserved, and its rate can be further increased by cAMP orthat CFTR alters Na current by changing ENaC open prob-Ca-dependent secretory agonists by enhancing the apicalability (276, see also review by Schwiebert et al., thismembrane Cl conductance (34).supplement). There are indications also that inflammatory

The cellular mechanism for Cl secretion, establishedmediators alter airway Na transport (56). At the basolat-from studies in airway and other secretory epithelia (85),eral membrane, cytokines or ATP can accelerate the rateis shown in Figure 7. Agents that raise cellular cAMPof Na absorption. However, at the apical surface, nucleo-are effective secretogogues, and under these conditions,tide triphosphates such as UTP are inhibitors of Na ab-CFTR is the principal Cl conductance pathway (58, 130,sorption (D. C. Devor, personal communication). Finally,277). Alternate Cl conductance pathways may contributesteroid hormones do not appear to be major regulatorsto Cl secretion in response to certain agonists or whenof Na transport rates across airway surface cells (147).CFTR is absent. Chloride secretion can be evoked by Ca-B) NaCl SECRETION. Under normal conditions, the air-mediated agonists, although this response is generallyway surface epithelium absorbs NaCl, and net salt secre-transient, in contrast to the more sustained response usu-tion is not observed. With Cl distributed at equilibriumally elicited by cAMP agonists. The molecular basis of theacross the apical membrane, agents that further increaseCl conductance activated by a cellular Ca rise in airwaythe Cl conductance (e.g., isoproterenol) do not yield netcells is still not clear. Chloride conductance pathwayssecretory activity. However, under some experimentalalternate to CFTR may explain the absence of significantconditions in vitro, Cl can be secreted across the surface

epithelium (151, 322). When the apical membrane voltage airway pathology in the CF mouse, where significant Ca-


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mediated Cl secretion is observed (see review by Grubb mucins and their clearance from the glands onto the air-way surface.and Boucher, this supplement). Luminal nucleotide tri-

phosphates are effective agonists for activation of a non- D) SUMMARY OF ION TRANSPORT PROCESSES IN SURFACE

EPITHELIUM. Thus, in surface epithelium, absorption ofCFTR apical Cl conductance (179, 274). The presence ofATP on the luminal side of the epithelium induces Cl NaCl and water is the significant physiological transport

process (see also fluid transport studies below). The ratesecretion, by activation of a P2y2 (P2u) receptor. Othernucleotides such as UTP are effective secretogogues, and of Na absorption is enhanced by CFTR deletion and is

reduced by CFTR stimulation under normal conditions.their nonhydrolyzable analogs can produce longer lastingresponses (166). Electrophysiological data indicate pri- Stutts and co-workers (273, 276) have related these effects

to changes in the probability that ENaC channels are inmary activation of a non-CFTR apical Cl conductance byATP/UTP, which would add to the activation by other the open conformation (Po); that is, stimulation of wild-

type CFTR decreased ENaC Po , whereas ENaC Po wassecond messenger pathways, including that of CFTR. Ithas been proposed that ATP may act as a coordinator of increased by cAMP stimulation in cells expressing DF508

CFTR. These findings are consistent with the discussionthe ASL volume and composition by virtue of its presencein airway secretions, including those derived from secre- above, which argues that the influence of the CFTR Cl

conductance on transepithelial salt transport is exertedtory cells during mucin release (33, 316).It should be emphasized that the influence of the primarily via CFTR regulation of ENaC, and not by alter-

ing transcellular Cl transport. The higher Na absorptionbasolateral membrane K conductance is critical in de-termining the magnitude of Cl secretion (160, 161, 183, rate in CF would be expected to reduce the salt concentra-

tion of the ASL if the epithelium is relatively water imper-264). By electrical coupling, this K conductance estab-lishes the electrical driving force for Cl exit across the meant. Alternatively, if water follows the enhanced salt

transport, then the volume of periciliary liquid would beapical membrane (87). In principle, this could occur evenin the face of Na absorption should the K conductance decreased relative to normal conditions. We consider

these issues in section IVE, after the water permeabilityrise sufficiently. Whether this occurs in vivo is not clear,but this phenomenon is demonstrable in human airway and fluid transport properties of the epithelium have been

reviewed. Stimulation of CFTR in the surface epitheliumcell cultures, and it may represent a means of controllingthe rate and direction of NaCl transport both physiologi- does not give rise to net fluid secretion. This CFTR-depen-

dent process is the province of the submucosal glands.cally and pharmacologically (see review by Schultz et al.,this supplement).

C) HCO3 SECRETION. A transport process of proximal 2. Submucosal glandsairway that has not received sufficient attention is trans-epithelial HCO3 transport. Evidence consistent with air- Submucosal glands are found in the cartilaginous air-

ways. The glands are composed of both mucous and se-way HCO3 secretion has emerged from several studieswhich show that the rate of Na transport is often signifi- rous cells, and their distribution and density varies among

species. The proportion is Ç60% serous cells and 40%cantly smaller than the measured transepithelial current(the short-circuit current or ISC), even in the absence of mucous cells in humans (280). Whereas mucous cells are

present in the surface epithelium, virtually all airway se-bath Cl. The unidentified current component appears tobe carried by HCO3 secreted into the lumen (262). Inter- rous cells are found in the submucosal glands. The secre-

tory products of the mucous cells are high-molecular-estingly, in CF epithelia, ISC was attributable to the rateof Na absorption, implying that HCO3 secretion was im- weight glycoproteins, which are sialylated and sulfated.

The serous cells contain fewer secretory granules, whichpaired in CF. This process may also contribute to theresidual, amiloride-insensitive voltage across non-CF air- are somewhat smaller in size than those in mucous cells

(122).way in vivo where, in general, Vt across CF epithelia isentirely amiloride sensitive. The transport mechanisms Studies of submucosal gland secretion are not numer-

ous, and the approaches have been relatively indirectthat would be responsible for HCO3 secretion across prox-imal airway cells have not been adequately identified, but (295). In excised airway tissues, the application of a gland

secretory agonist (acetylcholine) to the submucosal sur-the presence of HCO3 transport mechanisms may alsoexplain the lack of complete bumetanide sensitivity of the face induces an electrically silent secretion of NaCl, which

can be detected by isotopic flux methods but is not associ-ISC response to cAMP-mediated agonists. Moreover, thecontribution of HCO3 secretion to the volume and compo- ated with a change in transepithelial voltage (148). The

absence of a secretion-linked voltage change probably re-sition of the ASL is unclear at present, but a similar pro-cess likely resides in the submucosal glands (see sect. sults from dissipation of the secretory current by the cable

properties of the collecting duct that leads to the airwayIVB2). It is interesting to speculate that luminal alkalinity,particularly in the submucosal glands, may be an im- lumen.

Other studies have focused on isolated cell and cellportant determinant of the physical properties of secreted


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culture systems to assess gland secretions (71, 331). Cell Calu-3 epithelia or whether other explanations for HCO3

transport should be sought. Studies of the pancreatic ductculture techniques have been devised to primarily encour-age the proliferation of submucosal gland cells, but they (119, 120) offer even more possibilities to ponder in rela-

tion to HCO3 secreting tissues where the cells have theproduce cells having a mixed serous-mucous phenotype.These cells are identified as primarily secretory, and they capacity to raise luminal HCO3 concentration to high lev-

els (approaching 150 mM). These tissues express a baso-express several submucosal gland markers. However,they also express an amiloride-sensitive Na current, which lateral membrane HCO3 entry mechanism that is Na cou-

pled (42, 233). In contrast to predictions of the anionis somewhat unexpected (see below). In cultured submu-cosal gland monolayers grown on permeable supports, exchanger model cited above, alkalinization of the pancre-

atic lumen was not inhibited by low luminal Cl concentra-agonists induce relatively short-lived (õ1 min) secretoryresponses. Effects of both Ca- and cAMP-mediated secretion or by the CFTR blocker glibenclamide (119, 120).

These findings suggest that a new paradigm may be re-tagogues are evident (329); the response to Ca-mediatedagonists is larger and is the only significant response that quired to explain transepithelial HCO3 secretion, in partic-

ular, the exit of HCO3 across the apical membrane andremains in gland cultures from CF patients (330).The lung adenocarcinoma cell line Calu-3 appears to the role of CFTR in that process. It is too early to know

whether there are parallels between airway submucosalbe a good model for the submucosal gland serous cell(108). Calu-3 cells express many markers of serous cell glands and the pancreatic duct in this respect, but the

studies cited above raise many interesting questions forfunction, including lysozyme and lactoferrin (252). Thecells express high levels of CFTR and show secretory future investigations.

The structure of the submucosal glands suggests ancurrent responses to both cAMP-and Ca-mediated ago-nists (108). Calu-3 monolayers in which the basolateral important role of serous cell liquid secretion in the elabo-

ration of the secretory product (Fig. 3). Serous cells linemembrane is permeabilized with nystatin show Cl conduc-tance responses to elevation of cAMP but not cell Ca the most distal acinar structures, whereas the mucous

cells are located primarily in the more proximal secretory(194). This finding suggests that CFTR may provide theapical Cl conductance pathway for secretory responses ducts (189, 190). Thus secretion of liquid from serous cells

provides the vehicle that moves mucins toward the airwaymediated by Ca-dependent agonists, a situation similar tothat observed in intestinal cells (10). The results of recent lumen (316). In addition, if Na channels (ENaC) are ex-

pressed in the tubules and collecting ducts of the submu-studies suggest that the net secretory current stimulatedby cAMP-mediated agonists across Calu-3 cells is actually cosal glands, they may serve to reduce the NaCl concen-

tration of the secreted fluid (41). In view of the potentialcarried by HCO3 rather than Cl (167; R. J. Bridges, per-sonal communication). The effects of ion replacement for gland secretory rate to exceed the surface absorptive

rate by a factor of Ç6, this could markedly decrease theconditions, transport inhibitors, and transepithelial isoto-pic flux determinations are consistent with this view. The NaCl concentration of the ASL when the glands are active.

The relative contributions of surface and gland mu-role of HCO3 secretion, or the high luminal pH which itinfers, in airway gland secretion physiology is unknown. cous cells to the ASL gel volume is uncertain. However,

estimates of total gland cell volume relative to the volumeGiven our uncertainty regarding the transport mecha-nisms involved in submucosal gland salt secretion, it is of goblet cells in the surface epithelium suggest that the

gland cells predominate by a factor of Ç40 (229). It isnot surprising that the role of CFTR in this process hasnot been adequately clarified. It has been generally as- estimated that in adults there are Ç100 submucosal

glands/cm2 tracheal surface (286). Gland secretion ratessumed that the salt secretion mechanisms of submucosalserous cells resemble those of the surface epithelium and (125) are regulated, and during maximal secretion, a sin-

gle gland produces fluid at a rate ofÇ10 nl/min (217, 295).other secretory epithelia (see above and Fig. 7) and aredue to secondary-active Cl secretion. However, newer evi- This implies that 1 cm2 of upper airway during maximal

stimulation can generate Ç60 ml fluid/h. In contrast, thedence indicates that Calu-3 cells secrete HCO3, and be-cause they express submucosal markers, the same may rate of fluid absorption across cultured surface epithe-

lium, measured in the studies of Jiang et al. (124), wasoccur in submucosal gland serous cells. Unfortunately,this makes the role of CFTR in gland secretion less clear. Ç5 mlrcm02

rh01. In the steady state, the composition ofthe ASL will be determined by the balance of gland secre-One model that features a key role for CFTR in this pro-

cess is derived from studies of pancreatic duct cells (97, tion and surface reabsorption. Apart from the secretedliquid, the glands are generally considered to contribute99). Here an apical Cl/HCO3 exchange mechanism, op-

erating in parallel with apical CFTR, produces CFTR-de- the majority of glycoconjugates secreted onto the airwaysurface (228). Thus the secretion of liquid from the sub-pendent net HCO3 secretion by recycling the Cl that enters

the cell via the anion exchanger through CFTR. Appro- mucosal glands, at least in the upper airway, is perhapsa more important factor controlling the water content ofpriate inhibitor and ion replacement studies should be

performed to determine whether this model applies to airway secretions than is transport by the surface epithe-


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lium. In this context, it seems reasonable to view the ions is contained within the secretory granules of serouscells (306). Secretion of mucins from some epithelial cellupper airway as a functional secretory-absorptive unit,

composed of a submucosal gland and duct that connects types is mediated by cAMP pathways (163, 169), and thisraises the possibility that CFTR is involved in the secre-with the surface epithelium.

In analogy with the structure-function relations of tory process. For example, it is possible that CFTR Clchannels in mucin granules assist in disrupting the bal-exocrine glands, the airway consists of a distal secretory

component (the mucous and serous cells of the submuco- ance that maintains mucins in a condensed state. Forstneret al. (84) have shown that mucin secretion from T84 cellssal glands) in series with a more proximal absorptive com-

ponent (the gland collecting duct and surface epithelium). is stimulated by cAMP, which therefore likely depends onstimulation of CFTR. Evidence has been presented thatThe loci of expression of CFTR and ENaC, detected by

in situ hybridization, are consistent with this view (41, 74). CFTR plays a role in mucin secretions by gallbladder epi-thelial cells (162, 163). In the submandibular gland, anti-Duct cells are reported to express relatively high levels of

ENaC, which suggests that they may play a role in NaCl bodies to CFTR inhibited mucin secretion stimulated byb-adrenergic agonists (192). In human tracheal epithelialabsorption. The duct cells and surface epithelium together

could modify the composition of the liquid secreted from cells, protein kinase A (PKA) stimulated glycoconjugaterelease by a mechanism that involves CFTR; that is, secre-the glands as it travels to the surface. The final secretory

product, and thus the regional composition of the ASL, tion could be altered by manipulating CFTR expressionlevels (188). The presence of CFTR in the secretory gran-would be expected to vary in its salt composition and

tonicity, depending on the reabsorptive activity of the duct ules of serous cells (121) suggests that CFTR contributesmechanistically to glycoprotein secretion, either by pro-and surface epithelia and their water permeabilities. Duc-

tal salt reabsorption combined with a low H2O permeabil- viding a regulated Cl conductance in secretory granulemembranes or by contributing to cAMP-mediated secre-ity would yield a hypotonic solution with lower NaCl con-

centration than that of the primary secretion, as occurs tory vesicle exocytosis (see below).in the sweat or salivary gland.

Although current evidence for this view is sparse, 3. Involvement of submucosal glands in CFrecent work from Knowles et al. (150) has suggested thatthe secretions of submucosal glands may be hypotonic. There is ample reason to believe that malfunction of

submucosal gland serous cells may be a primary sourceThey sampled liquid from the bronchial epithelium usinga filter paper technique and found that the collected liquid of CF pathology (125). Changes in the morphology of the

submucosal glands are among the earliest features of al-was hypotonic, resembling that collected from nasal mu-cosa during strong stimulation of gland secretion. Accord- tered airway morphology in CF patients (272). Generally,

the gland lumens become engorged with mucus and dilate.ingly, changes in the composition of the ASL, evoked bya hypotonic submucosal gland secretory product, may be In the ensuing inflammatory environment, the glands con-

tinue to enlarge and become hyperplastic, increasing theirimportant for the activity of defense molecules in thegland secretions (see sect. VIB). apparent secretory capacity (202). Serous cells have been

termed the primary defensive cell of the airway mucosaSecretory products of submucosal glands play an im-portant role in defending the airway against inhaled patho- (21). They discharge a variety of bacteriocidal products

that defend against a broad spectrum of pathogens. Thegens. Accordingly, impaired gland secretion in CF maycompromise airway defense. Mucins trap bacteria and origin of most nonmucin submucosal gland secretory

products is the airway serous cell. Its secretory productsparticulates (see below), and the ASL gel may contain asignificant proportion of secreted defense molecules include lysozyme, lactoferrin, secretory IgA, peroxidase,

protease inhibitors, proline-rich proteins, and albumin(since mucins bear a net negative charge and defensinsare polycationic). Little is known about the mechanism (21). Serous cells also secrete several defensins, salt-sen-

sitive antimicrobial substances whose importance in air-of macromolecular secretions by airway cells and whatrole CFTR may have in this process. Within the cell, mu- way defense has been noted by several investigators (93,

261, 342).cus granules are condensed and dehydrated relative totheir physical state following secretion (280). Verdugo Sulfated proteoglycans and various cationic secre-

tory products coexist within serous cell secretory gran-(305) has proposed that Ca ions contained within mucousgranules act to neutralize the negative charge of highly ules (226). As discussed in sect. IVB2, ionic interactions

between cationic proteins, Ca and large polyanions allowscondensed mucins. Serous cells react poorly with alcianblue, despite the fact that they synthesize highly sulfated for condensation of macromolecular secretory products

through charge shielding, which would exclude the waterproteoglycans. The formation of a complex between poly-saccharides and divalent cations causes proteoglycans to that would otherwise be required for their hydration (305,

306). Proteoglycans are conjugated with sulfate. Theselose their affinity for alcian blue (195), and the lack ofstaining may thus imply that a high level of divalent cat- charged macromolecules are present in respiratory secre-


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tions and are part of the mucin network that lies at the kinin and histamine (129, 139). The secretory responseof cultured gland cells to b-agonists is mediated by ad-airway surface (75). The high charge density of proteogly-

cans, due to their heavy sulfation, may be important in renergic receptors of the b2-subtype (176) which en-hance secretion through elevation of intracellular cAMPregulating the hydration of this mucin network. Because

the overall charge density on proteoglycans is higher than (82). This process leads to membrane fusion and exo-cytosis with a release of serous cell secretory productsthat on mucins (21), the secretory products of serous cells

tend to be more watery. The combined secretions of the as well as secretion of salt and water. The possibilitythat CFTR is localized in the secretory granules sug-serous and mucous cells probably contribute to the hydra-

tion and rheological properties of the mucin gel by influ- gests that it may participate in the secretory responsein a manner similar to the cAMP-dependent control ofencing the ratio of secreted proteoglycans to the less

highly charged mucins (21). This concept has clear impli- exocytosis observed in other cell types (see below andreview by Bradbury, this supplement).cations for CF if the absence of CFTR from the serous

cells of submucosal glands alters either the composition The work of Ballard and colleagues highlights theimportance of submucosal gland secretion to liquid ho-or volume of their secretions. In addition, a different pro-

teoglycan-to-glycoprotein ratio emerges from stimulation meostasis in proximal airways. Ballard et al. (17) com-pared the transepithelial secretory response, deter-of the submucosal glands than that which emerges from

goblet cells in the surface epithelium. If the glands are mined from electrical measurements, with the numbersof submucosal gland duct openings in different airwaydysfunctional or their connecting ducts are blocked in CF,

serous cell products will be depleted from the ASL gel. regions. Their data imply a direct relationship betweenthe magnitude of Ca-stimulated Cl secretion and theAccordingly, the mucus secreted by the surface epithe-

lium may be less hydrated by virtue of its low proteogly- numbers of submucosal glands. Airway segments werealso fixed and stained to estimate ductal mucin contentcan content.

If the absence of fluid secretion due to CFTR dele- during stimulation. Tissues were exposed to ion trans-port inhibitors in an attempt to implicate known trans-tion were associated with a reduction in the net secre-

tion of hydrated sulfonated proteoglycans, the visco- port processes in the acetylcholine-evoked secretoryresponse (118). Pretreatment of distal bronchi withelasticity of the mucous gel lying at the airway surface

would be altered. Mucus elasticity is a major determi- blockers of Cl and HCO3 secretion before stimulationresulted in ductal mucus accumulation. This impliesnant of mucociliary clearance (216, 319, see below), and

it is critically dependent on the degree of hydration of that inhibition of gland salt and water secretion leadsto mucus obstruction of submucosal gland ducts,the surface gel layer (259). In addition, if absorption of

fluid from the airway surface is elevated in CF, then the which, as discussed above, is one of the early pathologi-cal findings in the CF airway (272). The relation of sub-mucous gel will become dehydrated, mucins within the

gel will be concentrated, and mucociliary clearance will mucosal gland ion transport activity to mucin clearancefrom the ducts is consistent with this conclusion, al-be impaired.

As pointed out earlier, the serous cells of the submu- though the effects of DIDS may not be selective for Cl/HCO3 exchange processes (see review by Schultz et al.,cosal glands are the predominant site of CFTR expression

in the airways (74, 121). Thus deletion of CFTR is ex- this supplement). However, another caution with re-spect to these studies is the use of full-thickness tissues,pected to have profound effects on the functions of these

cells, which will include a reduction in salt and water which could impair viability and alter transport eventsin the submucosal glands, at least quantitatively.secretion, and perhaps also, in the secretions of molecules

involved in microbial defense and mucin hydration. The In summary, alterations in submucosal glands couldcontribute to the pathology of CF in two important ways.loss of these products may occur either as a result of

CFTR’s involvement in serous cell secretion, of blockage First, submucosal glands contribute to the ion and watercomposition and volume of the ASL. The absence of aof the ducts, or both. As pointed out by Grubb and Bou-

cher (this supplement), CF mice fail to develop airway watery secretion from the submucosal glands (particu-larly from serous cells) would reduce the volume of air-pathology and are difficult to colonize with bacteria (265).

The relative absence of pathology may be due to an ab- way secretion and eliminate a liquid component that isimportant in airway clearance. Accordingly, the salt con-sence of submucosal glands in rodent airways.

The regulation of submucosal gland secretion has centrations of the ASL in the absence of this componentcould be higher, having implications for airway defensebeen characterized primarily as a purinergic or cholin-

ergic response; however, these cells are also responsive against microbes (see sect. VI). Second, the macromolecu-lar composition of the surface gel would lack a significantto b-adrenergic agonists (125). Mixed primary cultures

of submucosal gland cells secrete in response to the component of submucosal gland secretions. This couldlead to a less hydrated (and harder to clear) gel in thecAMP-mediated agonists, isoproterenol and prostaglan-

dins, as well as the classic Ca-mediated agonists, brady- airway lumen.


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C. Other Transport Functions of CFTR CFTR is rapidly endocytosed under basal conditions(213). 6) cAMP increases CFTR immunofluorescence inthe apical membrane domain of secretory epithelia (289).In addition to its ion channel function, CFTR influ-This staining pattern, and the Cl current response, wereences other cellular processes. These include changes ininhibited by reagents that disrupt microtubules. 7) CFTRthe activities of other channels (see review by Schwiebertactivation stimulates endosome-endosome fusion, sug-et al., this supplement), the posttranslational processinggesting its participation in vesicle trafficking processesof glycoproteins, and regulation of the trafficking of mem-(25). As these findings indicate, cAMP/PKA would alterbrane vesicles at the epithelial apical surface. A centralCFTR trafficking so as to increase cell surface content oftheme that may link these events to CFTR activity is theCFTR, providing an important mechanism for enhancingability of CFTR to function in intracellular compartmentsapical Cl conductance. Accordingly, one manner in which(reviewed by Bradbury, this supplement). In essence, byphosphorylation of CFTR controls the Cl conductance ofacting as a Cl conductance within internal membranes,the apical membranes of secretory cells is by controllingor by an as yet undefined non-ion channel function, thethe insertion and retrieval of the channel at the surfaceimplication is that CFTR influences processes other thanmembrane.the plasma membrane Cl conductance. Accordingly, these

Apart from the acute regulation of apical Cl conduc-events will be altered in cells from CF patients.tance, regulated membrane trafficking and recycling ofCFTR have implications for the manner in which this pro-

1. Regulation of CFTR traffic at the apical membranetein interacts with other channels and processes, particu-larly if they are residents of trafficking membranes. Thus,Cells acutely regulate transport across their plasma

membranes using two general mechanisms: they alter for example, the presence of amiloride-sensitive Na chan-nels or other Cl channels in compartments through whichturnover rates or the numbers of transporters exposed at

the membrane surface. There is no question that the Po of CFTR traffics, or changes in their compartment distribu-tion in CFTR deficient cells, could explain some of theCFTR can be regulated by phosphorylation at the plasma

membrane. However, there is now ample evidence that interactions observed between CFTR and other ion chan-nels or processes. In addition, the processing of mem-the apical membrane content of CFTR is regulated also

by cAMP/PKA in epithelial cells. The list of transporters brane glycoproteins or glycolipids may occur in intracellu-lar compartments containing CFTR. This could affect thethat undergo regulated trafficking has grown steadily.

Many channels and other transport proteins undergo regu- structure and function of these proteins at the cell surfaceand may influence their interaction with pathogenic bacte-lated trafficking as the chief mechanism governing trans-

port rate (for review see Bradbury and Bridges, Ref. 36). ria (242, 340; and see below). Thus the trafficking of CFTRmay influence not only the Cl conductance of the apicalSubstantial evidence has accumulated that the process of

regulated insertion-retrieval applies also to CFTR. membrane but also other transport and compositionalproperties of the epithelium.There are now seven independent lines of evidence

which indicate that acute regulation of CFTR insertion-retrieval is an important mechanism controlling apical Cl 2. Acidification of intracellular organellesconductance. 1) CFTR regulates plasma membrane turn-over. Expression of CFTR confers cAMP-dependent exo- As integral membrane proteins, Cl channels such as

CFTR are at least transient residents of virtually all intra-cytosis and endocytosis on cells in which it is expressed.These processes can be monitored by uptake of fluid- cellular compartments through which protein biogenesis

and secretion occur. Functional studies have suggestedphase markers or by cell surface labeling (38, 212). 2)Immunogold labeling studies have identified CFTR in both that Cl channels are present in virtually all intracellular

membranes, including Golgi, zymogen granules, endo-plasma membrane and submembrane vesicles (310). 3)Significant CFTR levels have been identified in clathrin- somes, and CCVs (36). The presence of a regulated Cl

conductance may facilitate the acidification of these intra-coated membrane vesicles (CCVs), indicating the selec-tive retrieval of CFTR from the cell surface by clathrin- cellular organelles. As first proposed by Barasch et al.

(20) and Mulberg et al. (196), a Cl channel in parallel withmediated endocytosis. The identity of CFTR in CCVs wasconfirmed by Western blot, phosphopeptide mapping, and an electrogenic proton pump would provide counter-ion

for vesicular HCl accumulation and intravesicular acidifi-reconstitution of functional CFTR into planar lipid bi-layers (37). 4) Parallel measurements of Cl conductance cation, and the extent of acidification would vary with the

anion permeability.and membrane capacitance indicate that CFTR-express-ing cells increase their surface area during cAMP-depen- Bae and Verkman (16) showed that renal cortex en-

dosomes contain a Cl conductance whose activation bydent Cl current stimulation (248, 278). 5) Surface labelingof CFTR followed by immunoprecipitation suggests that PKA enhances endosomal acidification. Similar findings

have been obtained from CCVs by Mulberg et al. (196). Inat least 50% of CFTR in T84 cells is intracellular and that


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several CFTR-expressing cells types, cAMP stimulation partments and thereby leads to decreased sialylation ofsecreted proteins. Inasmuch as these enzymes have pHhas been proposed to enhance secretory vesicle ion con-

ductance and acidification (20). In contrast, no effect of optima of Ç6 (336), small changes in compartment pHcould influence their activities significantly, presumablyCFTR expression on endosomal pH was detected in L

cells transfected with CFTR in the presence or absence leading to the changes in protein composition reportedabove. Jilling and Kirk (126) provided evidence supportingof Cl or cAMP (234). However, the relative contribution

that CFTR makes to total vesicle ion conductance is likely this view, showing that the sialylation of a1-antitypsin se-creted by CFTR-expressing colonic cells was cAMP de-to vary among cell types and from compartment to com-

partment. Identification of other ion channels and ion pendent and Cl dependent.Other posttranslational processing reactions may betransport processes that may influence the acidification

process is required (see Ref. 299). Thus the impact that affected by pH along the protein secretory pathway, eitherby a mechanism where enzymatic rates are altered or byCFTR, or its deletion, may have within a given compart-

ment may vary from cell to cell and from organelle to changes in enzyme distribution within these compart-ments associated with deletion of CFTR. This hypothesisorganelle.

A role for CFTR phosphorylation in the rate of vesicle would then allow for changes in bacterial adherence andcolonization by alteration of the sialoglycoconjugate com-acidification has been implicated in two studies. Both

were performed using heterologous expression systems position of the luminal surface of CF airway cells. Baraschand Al-Awqati (19) have also proposed that the distribu-and, thus, are subject to uncertainties regarding overex-

pression and the appropriate distribution of CFTR, espe- tion of sialotransferase in epithelial cells extends beyondthe Golgi and that this would provide for continued sialiccially in relation to the complement of other transport

proteins that would normally influence vesicle composi- acid addition to proteins migrating from Golgi to plasmamembrane. This hypothesis could explain how a smalltional properties. Studies by Lukacs et al. (175) and by

Biwersi and Verkman (26) showed that the rate of vesicle difference in vesicle pH within this extended compart-ment could be amplified to produce significant changesacidification could be enhanced by prestimulation of cells

with forskolin before preparation of membrane vesicles. in the chemistry of cell surface proteins. In this way, im-paired acidification could affect multiple cellular pro-The effects of stimulation were eliminated by exposure

of vesicles to low-Cl media, suggesting that the extent of cesses related to protein trafficking and processing. Theseprocesses are likely to be epithelial cell specific becauseacidification depends on activation of the CFTR Cl con-

ductance. they would occur primarily in the apical compartmentsof epithelial cells, and they may not be mimicked in heter-ologous expression systems. For a more complete discus-3. Glycoprotein processingsion of this area, see the review by Bradbury (this supple-ment).Alterations in vesicle composition, such as lack of

appropriate acidification, imply that compositionalchanges may alter the manner in which proteins within

D. Airway Fluid Transport and Water Permeabilitythese compartments are processed (see Ref. 20). Studiesof airway epithelial cells have suggested that sulfation of

1. Fluid transport across cultured airway cellsglycoproteins is increased in CF cells (49, 341). This isintriguing because high levels of CFTR expression are Defining the role of CFTR in determining the salt

composition and osmolarity of the surface lining fluid re-normally observed in serous cells of the submucosalglands as noted previously. The mechanism by which ex- quires knowledge of the fluid transport and water perme-

ability properties of airway epithelia. Several measure-cess sulfation occurs has not been delineated, but otherstudies suggest that CF cells do not have abnormalities in ment methods have been employed to examine these is-

sues, and studies have been conducted in human airwaysulfate transport or in intracellular sulfate content (193).Changes in membrane protein expression as detected by epithelia both in vitro and in vivo.

Smith and Welsh and co-workers (260, 263) measuredthe ability of lectins to associate with specific sialatedglycoproteins at the airway surface membrane have also fluid and electrolyte transport across cultured human na-

sal epithelia by monitoring changes in the composition ofbeen implicated. These studies imply that the formationof sialoglycoconjugates of specific types, particularly the the ASL present at the air-liquid interface of cultured air-

way epithelia. They added Ç100 ml of media at the apicalgangliosides, are impaired in CF cells (242).The model of Barasch and Al-Awqati (19) can account surface, covered this solution with oil, sampled the fluid

remaining after 24 h, and compared its composition withfor alterations in the chemistry of secreted and plasmamembrane glycoproteins in CF. This model postulates that of the submucosal solution. In the absence of added

agonists, they found that cultured nasal epithelia ab-that an alteration in vesicle pH causes changes in theactivities of sialotransferases in Golgi and post-Golgi com- sorbed Na and water. Fluid and Na absorption were inhib-


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ited by addition of amiloride to the apical solution, sug- leak rates balance; under these conditions, there are notime-dependent changes in luminal composition or vol-gesting that an ENaC-mediated absorptive process was

responsible. Net Cl movement across the epithelium un- ume. One might argue that this approximates conditionsin vivo, where the epithelium is bathed by a solution ofder basal conditions was not detected, implying that some

other anion accompanied the absorbed Na, but its identity low volume (i.e., a periciliary liquid layer only Ç10 mmthick). However, alterations in the composition of the lu-was not resolved. When cAMP agonists were added in

the presence of amiloride, NaCl and fluid secretion were minal compartment will also influence the epithelium’stransport properties quantitatively, and perhaps qualita-stimulated. Stimulation by cAMP in the absence of amilo-

ride led to variable results. In some preparations, net fluid tively, as gradients arise. Indeed, Widdicombe and Widdi-combe (317) have argued that the minimal ASL volumesecretion was stimulated; in others, fluid absorption was

augmented. The source of this variability was not identi- will be determined by physical forces, such as surfacetension, that would develop when ASL is reduced to thefied. Nasal epithelia were found to secrete protons and to

absorb K under basal conditions. Using this approach, ciliary height (Ç6 mm). Whether this occurs, particularlyin the presence of a surface gel layer, is uncertain andthey also examined the properties of CF airway mono-

layers (260). Interestingly, there was no difference be- will require additional study. In any event, the steady-statevolume and composition will be determined by both thetween epithelia derived from CF and normal subjects in

the rate of fluid absorption, whereas previous studies of active transport and passive (leak) properties of the epi-thelial monolayer.nasal epithelia under short-circuit conditions have demon-

strated that electrogenic Na absorption is increased in CF The fluid transport studies of Jiang et al. (124) wereconducted using large fluid volumes under conditions ofairway (see sect. IVB1).

These findings contrast somewhat with the results of level flow (no gradients). Their results support prior trans-epithelial current and electrolyte flux determinations, per-Jiang et al. (124) in ways that may be related to differences

in methodology. Their measurements were made using formed under similar conditions. Epithelia from CF pa-tients showed hyperabsorption of fluid and an absence ofcultured bronchial epithelia in chambers where both sur-

faces of the monolayer were bathed by solutions of the fluid secretion in response to cAMP stimulation (151).This provides support for the concept that CF pathologysame composition throughout the transport measure-

ments. In other words, the conditions are like those em- (reduced mucociliary clearance) stems from a reductionin the volume of liquid that lines the airway. The largerployed in previous transepithelial transport studies of cul-

tured airway cells, except that the epithelia were not volt- amiloride-induced decrease in fluid absorption observedin CF cultures is consistent with the idea that the hyper-age clamped (i.e., open-circuit conditions). As in the other

study, Jiang et al. (124) observed fluid absorption under absorption of Na and fluid in CF is mediated by amiloride-sensitive apical membrane Na channels and with the con-basal conditions, which was inhibited by amiloride. In the

presence of amiloride, cAMP stimulated fluid secretion in cept that Na channels are upregulated in the absence offunctional CFTR. Interestingly, fluid secretion, althoughepithelia derived from normal subjects; these findings are

in agreement with prior Cl transport measurements (148). unresponsive to cAMP, could be induced in CF epitheliaby addition of UTP or ATP, presumably operating throughAs in the studies of Smith and Welsh (263), the effects of

cAMP in the absence of amiloride were variable. cAMP an alternate Cl conductance pathway that is operationalin CF epithelia. However, the fluid secretion stimulatedfailed to increase fluid secretion across CF epithelia. In

agreement with previous results (151), but in contrast to by UTP was transient, as are its effects on the ISC acrossairway cell monolayers (274).the results of Smith et al. (260), Jiang et al. (124) found

that the rate of amiloride-sensitive fluid absorption wasgreater in CF than in normal airway epithelia, and they 2. Airway water permeabilityshowed that amiloride inhibited this elevated fluid absorp-tion. A direct assessment of the water permeability proper-

ties of airway epithelia was made recently by FolkessonThe major technical difference in these studies liesin the volume of solution present at the apical surface et al. (83). These investigators dissected small airways

(100–200 mm diameter) from guinea pig lung and perfusedduring the transport assays. The lower rates of fluid trans-port measured by Smith and Welsh (263) probably result these airway segments in vitro using glass pipettes, as is

done conventionally for renal tubules or sweat ducts. Thefrom the approach to ‘‘static head’’ conditions as fluidabsorption proceeds; that is, only a small volume (100 ml) airway lumen was perfused with solutions containing an

impermeant fluorophour (fluorescein sulfonate) so thatof fluid was placed initially on the airway surface, and asa result, alterations in the volume and composition in changes in luminal fluorescence between the proximal

and distal ends of the perfused segment report transepi-this fluid may occur due to the transport activity of theepithelium. Static head conditions will be reached at thelial water flow. This provided a measure of the osmotic

water permeability (Pf) of the epithelial segment, deter-steady state, when gradients have formed and pump and


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mined from the water flow response to an imposed os- in submucosal gland acini and whether the osmotic prop-erties of this liquid can be altered (e.g., salt concentrationmotic gradient. The Pf values obtained, Ç5 1 1003 cm/s

(room temperature), were independent of the magnitude lowered due to low ductal water permeability) as liquidprogresses to the airway surface. When liquid arrives atand direction of the imposed osmotic gradient. There was

no saturation or rectification of fluid flow detected over the surface, it does not appear that solute gradients willbe maintained, given the osmotic permeability propertiesthe range of osmolarity employed. The temperature de-

pendence of Pf provided an estimate of the activation discussed above. Recent studies suggest new candidatesfor airway water permeation pathways (4, 140, 168, 198).energy for water flow, and the high value observed is

consistent with the concept that water crosses the airway A detailed knowledge of these pathways and their locationis needed to form an integrated view of the regulation ofepithelium via water channels. The Pf was not affected by

conditions that elevate cellular cAMP, and this implies ASL composition and the contributions of both surfaceand gland epithelia to its properties. This is vital, since asthat a regulated water channel mechanism (like the aqua-

porin-2 insertion process of distal nephron cells) is not we see from the following discussion, determination ofASL composition is methodologically difficult and a con-present in airway. Staining by antibodies to aquaporin 4

suggested that this isoform is expressed at the basolateral sensus view regarding ASL salt concentrations in CF andnon-CF airways has not emerged.membranes. Water channels that might be responsible for

apical membrane water flow have not yet been identified.The osmotic water permeability of the airways, simi-

E. Composition and Thickness of Airwaylar to values observed for the alveolus or the descendingSurface Liquidlimb of Henle’s loop in the kidney, indicates that the trans-

epithelial water permeability is high. This would facilitatethe osmotic absorption of fluid during NaCl transport. The composition and volume of the ASL are critical

in determining how changes in cellular ion transport prop-This high Pf also implies that airway epithelia cannot sus-tain large transepithelial osmotic pressure differences or erties associated with the loss of CFTR lead to pulmonary

disease. There are basically two views of the mechanismsolute concentration gradients. Such gradients would bedissipated by osmotic water flow. Using estimates of the by which altered salt transport in CF can lead to a break-

down of lung defenses. One view relates primarily to thetotal airway surface area ofÇ1.4 m2, and from an estimateof the rate of evaporative water loss, Folkesson et al. (83) volume of the airway lining fluid. It proposes that an in-

crease in the rate of liquid absorption (and/or a decreasecalculated that an osmotic gradient of 14 mosM couldexist across the proximal airway (e.g., a NaCl concentra- in liquid secretion) changes the volume of the ASL, de-

creases mucociliary clearance, and allows bacteria andtion difference of 7 mM assuming that these are imperme-ant solutes). Nevertheless, it is apparent that relatively mucus to accumulate (32, 33). The contrasting view is

based on differences in composition of the ASL. It holdslarge osmotic gradients, such as those predicted duringhyperventilation, cannot be maintained across distal air- that changes in ASL salt composition alter the ability of

airway defense mechanisms to kill bacteria and that thisways. For example, given a surface area of 1.4 m2 and anevaporative water loss of 1.0 ml/min, the corresponding leads to bacterial colonization and airway obstruction

(261). Reports of elevated ASL salt concentration in CFtransepithelial volume flow is 3.8 mlrcm02rh01 to main-

tain steady-state water balance in the lung. This rate of (see below), together with the known salt dependence ofairway defense molecules, suggest that these mechanismswater flow is similar to the fluid transport rates estimated

by Jiang et al. (124) under level flow conditions (absence fail because CF airway epithelia do not have the capacityto lower surface salt concentration, as do normal airwayof osmotic gradients). Thus the passive and active (solute-

driven) water flows across the airway are of similar magni- epithelia. Thus the volume and composition of the ASLbecomes a key issue in resolving the mechanism of airwaytude and suggest that salt gradients between ASL and

interstitial space would not be maintained. defense and how it is compromised in CF.It is almost embarrassing that we do not know theIt is clear that we need a better molecular under-

standing of the water channels that are responsible for ASL salt composition with certainty. Despite its impor-tance in helping to resolve the issues raised above, reportsthe apparent high transepithelial water permeability of

airway epithelia (181). Knowledge of the aquaporin iso- of ASL composition are few. This is due to difficulties insampling the very thin liquid layer at the airway surfaceforms present in both surface epithelia and submucosal

glands would guide our thinking regarding the formation and to likely changes in its volume and composition pro-duced by the sampling process. Most approaches haveand modification of airway fluids by CFTR-dependent pro-

cesses and the constraints on their composition at sites utilized an ASL collection technique based on the applica-tion of filter paper planchetes to the airway surface. Theof salt absorption and secretion. It is important to know

whether pathways for water flow are arranged so as to results from several reports suggested that the Cl concen-tration of ASL in CF patients was higher than that foundfacilitate the formation of an isosmotic primary secretion


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in normal subjects (92, 127). In earlier reports (see below), et al. (114). They measured ASL salt concentrations in thelower trachea in infants undergoing bronchoscopy alsosalt concentration in CF was slightly hypertonic to plasma

and exceeded values determined from non-CF airways, using a porous membrane sampling technique. They cor-related their findings with the extent of airway inflamma-which were hypotonic. As indicated by Quinton (219),

there is no known fluid transport process in flat simple tion. No difference in ASL Na concentration was detectedbetween CF and non-CF infants without pulmonary in-epithelia that would allow for the formation of a primary

secretion that is hypotonic to plasma, as implied from flammation; however, ASL Cl concentration was signifi-cantly lower in CF (a Ç30% reduction). In CF subjectsthe measurements in normal subjects; rather, it has been

suspected that evaporative water loss occurring during with inflammation, ASL Cl concentrations were higherand not different from non-CF values. The role of inflam-the measurement or during sample processing may pro-

duce falsely high values of NaCl concentration (127). mation in this effect is unclear; nevertheless, these resultsdo not support the concept that an increase in ASL saltA recent study by Knowles et al. (150) highlights the

problems associated with these measurements. First, how concentrations in CF is the basis of impaired airway de-fense against pulmonary pathogens.does one define the composition of a 5- to 10-mm-thick

periciliary liquid layer without perturbing it? A liquid layer One important consideration in the study of Knowleset al. (150) was the possibility that gland secretions con-10 mm thick would contain onlyÇ20% of the liquid volume

collected during sampling. Is the reminder of the sampled tribute to the volume and composition of liquid on airwaysurfaces. They explored the possibility that the filter paperliquid derived from axial or radial flow? The sampling

process, as well as any cellular damage which may occur, sampling technique, the common method for the lowerairway, elicits liquid secretion from submucosal glands.will draw liquid across or along the epithelium. Thus the

measurement is likely to assess a combination of the They attempted to separate the contributions of surfaceand gland epithelia by collecting accumulated liquid fromsteady-state composition of the ASL, plus aspects of the

salt and water permeability properties of the underlying the nose. Reflex stimulation of nasal glands was evokedby having subjects chew chili peppers, and this resultedepithelium. The serum protein levels and K concentra-

tions in the collected liquid are low, and this is used as an in a similar increase in the volume of fluid collected inboth CF and normal subjects. They interpreted this toargument against cell damage. However, another possible

complication arises from the mechanical stimulation of indicate that non-CFTR-dependent secretory events un-derlie this component of fluid secretion. Stimulation ofepithelial reflexes during sampling, which may evoke

transport responses from the surface or gland epithelia nasal fluid secretion diluted the concentrations of themeasured ions; the Na plus K decreasedÇ30–40 mM with(see below). This is a good example of Heisenberg’s prin-

ciple, where the system’s properties are likely perturbed a corresponding decrease in estimated osmolarity. Thisfinding raises the possibility that secretion from the sub-in the measurement itself. These perturbations may am-

plify or even generate differences in the liquid at the sur- mucosal glands of a hyposmotic solution can modify ASLcomposition. As discussed in section IVB2, these findingsface of CF and non-CF airways.

The nasal mucosa provides an accessible site for as- suggest that we should model the submucosal glands likeother exocrine glands that elaborate an isosmotic primarysessing the salt concentrations of the ASL, and liquid can

be sampled as it accumulates during nasal occlusion. Us- secretion that is modified by salt transport in the ductsleading to the airway surface. The ducts would need toing this approach, Knowles et al. (150) found a time-de-

pendent increase in liquid volume, accompanied by a re- absorb NaCl without water to produce a hypotonic secre-tory product and are therefore predicted to have a lowduction in salt concentration. After equilibration, Na con-

centration (109 mM) was lower than the plasma value, water permeability (absence of aquaporins) and a mecha-nism for Na reabsorption from dilute solutions. This con-the Cl concentration was similar to that of plasma, and K

was higher (Na plus K was similar to plasma). They found cept is consistent with the expression of amiloride-sensi-tive Na channels at this site, as reported from the in situno difference in the ionic composition of liquid obtained

from CF and normal subjects. Importantly, the measured hybridization studies of Burch et al. (41). Thus a hy-posmotic ASL, obtained from filter paper sampling of thesalt concentrations exceed values that are optimal for

the activity of salt-dependent defensins in the respiratory lower airways, could result from a reflex stimulation ofgland secretion. Consistent with this idea, Knowles et al.tract. These studies were extended to the lower airway,

using a filter paper sampling technique. Here, ion concen- (150) found that as the volume of fluid collected in-creased, its osmolarity fell. This could be a consequencetrations were significantly lower than in the nasal liquid

collection studies. The sum of Na plus K suggested that of stimulating secretion of a hyposmotic fluid. BecauseNa plus K did not differ in CF and normal ASL, the secre-the solution on bronchial surfaces is hyposmotic to

plasma. Again, no CF-related differences in Na, Cl, or K tion from the glands stimulated in this manner does notdiffer between normal and CF subjects. If this is true,concentrations of ASL were detected.

Similar findings have been reported recently by Hull then the Cl permeability properties of the duct, on which


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the reabsorption of NaCl to form a hypotonic secretory meability and would not favor maintenance of a low ASLsalt concentration. These findings argue for changes inproduct depend, would have to be independent of CFTR

and must therefore rely on some other Cl transport pro- mucociliary clearance that stem from reduced ASL vol-ume in CF; that is, enhanced salt and water absorption atcess. The submucosal gland properties would differ from

those of sweat ducts in this respect but would be similar the airway surface impairs the ciliary clearance mecha-nism.to salivary ducts, where a hypotonic secretory product is

formed (339).This discussion indicates that the composition of the

F. Lessons From Other Genetic DiseasesASL in lower airways remains relatively uncertain. Thefindings of Knowles et al. (150) suggest that its measure-

Alterations in mucociliary clearance could occur asment may be compromised by stimulation of gland secre-

a result of non-CFTR-related disease processes, and thesetion and, due to the size of the sampled compartment, the

would provide important insights into the specific rolemeasurement itself may perturb cellular transport pro-

of CFTR in CF. Findings characteristic of immotile ciliacesses. However, we should bear in mind that the forma-

syndrome are discussed in section VD. The hyperabsorp-tion of a hypotonic secretory product in submucosal gland

tion of Na characteristic of CF airway surface epitheliaducts could lead to activation of defense molecules. Be-

suggests that other genetic diseases that affect the activitycause the volume of liquid secreted by the glands can

of the amiloride-sensitive Na channel might lead also toexceed the transport rates of surface cells by about sixfold

pathophysiology. Would primary hyperabsorption of Na(see above), the secretion of a hypotonic solution con-

lead to the pulmonary manifestations observed in CF pa-taining defense molecules could have significant protec-

tients? Genetic diseases in which Na absorption is eithertive effects for the glands themselves and for the surface

chronically enhanced or reduced have been traced to pri-microenvironment at the site of secretion. However, this

mary alterations in the structure of the amiloride-sensitiveeffect would be transient, since a high water permeability

channel, ENaC.of the surface epithelium is expected to equilibrate thetonicity of secreted liquid.

1. Liddle’s syndromeFinally, a recent study performed using well-differen-

tiated proximal airway cells in primary culture implies In Liddle’s hypertension, truncation of the COOH ter-minus of the b- and g-subunits of ENaC are associatedthat the axial transport of sol and gel layers may be linked

(180). This study used fluorescent markers for gel and sol with salt-sensitive hypertension due to a primary increasein Na channel activity (104, 256). Introduction of theseto track their individual axial transport rates. In contrast

to expectation, both layers moved at the same rate. Re- mutations into ENaC expressed in heterologous systemsresults in severalfold increases in the macroscopic amilo-moving the mucus layer reduced lateral liquid transport

Ç80%, inferring that movement of the sol fluid depends ride-sensitive Na currents relative to that associated withwild-type ENaC expression. Because the gating propertiesin some way on the transport of mucus by the cilia. These

findings are consistent with prior measurements of muco- and selectivity of these channels were not altered by Lid-dle’s mutations, the increase in Na transport was ascribedciliary clearance, which suggest that the mucus itself plays

a role in ciliary transport (91, 238, 318). As mentioned to an increase in the number of Na channels in the plasmamembrane (266). This was supported by expression ofearlier, most prior studies have assumed that the sol layer

is relatively static and that mucins ride atop this layer, ENaC subunits bearing Liddle’s mutations in epithelialcells. As a consequence of enhanced Na absorption bypropelled by the ciliary beat. However, a recent study

suggests that periciliary liquid moves with mucus and that airway cells, we could entertain the hypothesis that thevolume or salt concentrations of the ASL would be re-its movement depends on mucus clearance. As summa-

rized by Matsui et al. (180), the amount of fluid trans- duced. Although there have been no estimates of the saltcomposition of the ASL in these patients, there are noported axially by ciliary clearance can be estimated from

the area of ciliated epithelium (Ç2,400 cm2, Ref. 181), the apparent abnormalities in pulmonary function. Accord-ingly, enhanced absorption of salt (and water) alone ap-ciliary length (Ç5 mm), and a mucociliary transport rate

of 0.5 cm/min (333) to be Ç860 ml/day. This is approxi- pears insufficient to reproduce the airway pathology ofCF. Presumably, a lowering of NaCl concentration in themately the volume of fluid thought to reside in the distal

airways (see sect. IVA2). It is consistent with the concept ASL would only enhance the activities of salt-sensitivedefensins. Perhaps the antimicrobial capacity would bethat there is a continual migration of liquid from distal to

proximal regions, and because of regional differences in improved over that present normally. On the other hand,NaCl and water hyperabsorption should reduce the thick-ASL volume, this liquid must be absorbed by the surface

cell salt and water transport processes discussed earlier. ness of the ASL, but there is no impairment evident inthe clearance of secretions from these patient’s airways.This implies that water absorption accompanies salt ab-

sorption, which would require a high epithelial water per- These patients are expected to show increased Na absorp-


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tive rates like those encountered in CF, but this has not secondarily, when this mechanism fails or is overloadedwith secretions, by cough clearance. Many of the factorsyet been demonstrated. The relation of Liddle’s mutations

to ENaC function in the airways must be examined before that regulate mucociliary clearance have been examinedin CF, yet the specific role of mucociliary clearance inits relation to CF pathology can be inferred.the pathophysiology of lung disease in CF remains to be

2. Pseudohypoaldosteronism defined. The ciliary propulsion of mucus up the airwaysis a crucial component of mucociliary clearance, as re-A second and perhaps more informative genetic con-

dition, characterized by impaired Na absorption, is that flected by the airway infection and bronchiectasis thatresult from defects in ciliary function that occur in ciliarywhich occurs in pseudohypoaldosteronism (PHA). This is

an inherited disorder that features excessive salt loss, dyskinesia syndromes (see sect. VD). It is clear that ciliarydysfunction is not a primary defect in CF, because exwith a lack of responsiveness of Na absorbing epithelia

to mineralocorticoids (209). Mutations in either the a- vivo studies of ciliary structure and beat frequency inCF epithelia have been normal (47, 235, 237). Althoughor b-subunits of the amiloride-sensitive Na channel are

observed in these patients (48); these are generally mis- Pseudomonal exoproducts and inflammatory mediators,such as neutrophil elastase, may adversely affect ciliarysense mutations that disrupt ENaC function (270). Heter-

ologous expression of ENaC subunits containing these beat frequency (reviewed in Ref. 309), and thereby con-tribute to the persistence of bacterial infection, there ismutations yields reduced macroscopic Na channel cur-

rents but no significant differences in the level of surface no evidence that abnormalities in ciliary function contrib-ute to the initiation of infection.expression of the mutant protein (99). This suggests that

PHA mutations reduce channel open probability. In amouse model of PHA, impaired Na channel activity results 2. Mucus rheology

in failure of newborns to clear lung fluid at birth, and thisA second factor that affects mucociliary clearance is

leads to death in the neonatal period (113). This diseasethe rheological properties of mucus (reviewed in Refs.

phenotype can be rescued by expression of wild-type141–143, 334). In general, ciliary transport is inversely

ENaC. There is a correlation with increased cholelithiasis,related to mucus viscosity (resistance to flow) and, except

suggesting impaired salt absorption in the biliary tract (6).at low extremes, to mucus elasticity (ability to store ap-

In an early report, subjects with PHA showed in-plied energy). Increases in mucus viscosity, or the ratio

creased sweat and saliva salt concentrations, and they areof viscosity to elasticity, decrease ciliary transport rates,

reported to have frequent lower respiratory tract infec-presumably via dissipation of the mechanical energy ap-

tions (101). Accordingly, one might postulate that the re-plied to mucus by beating cilia (reviewed in Ref. 143). As

duction in ENaC activity in PHA does not permit reductiondiscussed below, the relationship between ion transport

of the NaCl concentration of the ASL to levels necessaryand mucus viscosity, if any, remains to be clearly defined.

for effective defensin activity. However, a more extensiveHowever, it appears that increases in calcium concentra-

study was performed recently by Kerem et al. (131), whotion increase the viscosity of mucus, whereas monovalent

found no evidence of chronic airway infection or bronchi-ions do not alter mucus viscosity in vitro (177).

ectasis. Using nasal potential difference measurements,they detected no significant amiloride-sensitive Na ab-

3. Adhesivity and surfactantssorption and a large increase in ASL, assessed by filterpaper sampling. Isotopic lung clearance methods sug- In addition to viscoelasticity, the surface properties,

or adhesivity, of mucus appear critical to normal mucocili-gested that PHA patients compensate for their excess ASLvolume by accelerated clearance of liquid from the airway ary clearance. Conceptually, adhesivity represents the

ability of mucus to adhere to a surface and is determinedsurface. The collected fluid was isotonic, with Na plus Kconcentration only slightly elevated (Ç10%) relative to at least in part by surface tension and osmolality. In-

creases in adhesivity due to increases in surface tension,non-PHA subjects. This high salt concentration arguesthat an isotonic surface fluid per se is not sufficient to or to hyperosmolality, are proposed to reduce mucociliary

clearance. Although the precise determinants of surfacecause airway infection with the pathogens characteristicof CF. tension remain to be defined, phospholipids such as sur-

factant, derived from the alveolar space or secreted byairway epithelial cells (164, 314), appear to play an im-V. MUCOCILIARY CLEARANCE

portant role. Recent work has demonstrated a surfactant,or lipid, layer at the interface between the sol and gelA. Factors Contributing to Normal Clearance

layers at the ciliary interface (246), and both in vitro (8)1. Ciliary beat and in vivo (67) studies have demonstrated that addition

of surfactant increases mucociliary clearance. In additionClearance of inhaled particulates and organisms fromthe airway occurs primarily by mucociliary clearance, and to phospholipids, adhesivity is dependent on the concen-


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tration of mucins and the osmolality of their environment. that manipulation of ion transport adversely affects thephysical properties of mucus derived from stimulated air-This has not been investigated thoroughly, but increases

in osmolality have been demonstrated to increase adhesi- way segments. Additional studies will need to determinewhether these effects are stimulus dependent andvity in canine mucus (206). In these studies, dehydration

of tracheal pouch mucus from Ç320 to 430 mosmol/ whether primary defects in CFTR-mediated secretorypathways have effects on mucus rheology that are similarkgH2O, similar to CF sputum osmolality ofÇ460 mosmol/

kgH2O, significantly increased both viscosity and adhesi- to those stimulated by cholinergic pathways. We mustalso be mindful that the glands are comprised of differentvity. Moreover, osmolality of canine mucus was inversely

related to Cl concentration, but not related to Na, K, or cell types having different secretory products (as dis-cussed in section III). Therefore, transport inhibitors couldCa concentrations. These data suggest a pathophysiologi-

cal link between luminal Cl concentration and mucus preferentially affect different components of the secretoryproduct and alter its rheological properties in this manner.properties: luminal Cl is proposed to control water trans-

port, which in turn regulates osmolality. Defects in Cl(and water) secretion that would result in lower luminal

B. Mucociliary Clearance and SputumCl concentrations, hyperosmolality of airway mucins, and,

Properties in CFultimately, increased mucus adhesivity would decreaseclearance. However, as discussed above, the relationship

1. Properties of CF sputumbetween luminal NaCl concentration and CF remains un-certain. The accumulated evidence suggests that Cl con- Several approaches have been used to address the

important question of whether defects in mucociliarycentrations in ASL are increased or the same in CF com-pared with controls so that this effect of Cl concentration clearance contribute directly to the initiation of airway

disease or merely represent a secondary disturbance thaton mucus adhesivity would not be expected to contributeto impaired mucus clearance in CF. Moreover, recent is permissive for infection. A first approach to this issue

has been provided by studies of the rheological propertiesstudies in dog and baboon airway have shown that acuteaerosol administration of hypertonic saline increased tra- of CF sputum. These studies suffer from problems associ-

ated with the source of this material, insofar as sputumcheal mucus velocity and mucociliary clearance (326) andthat inhibition of the Na-K-2Cl cotransporter with furose- samples have been obtained from patients infected with

bacteria. Rheological abnormalities may derive from themide abrogated a dry air-induced decrease in mucociliaryclearance (325). These data suggest that compensatory effects of inflammatory products rather than primary de-

fects in the biophysical properties of mucus. Nevertheless,responses to hypertonic ASL minimize any adverse effectsof ASL hypertonicity on adhesivity, such that the effects studies of CF sputum have indicated that when corrected

for the amount of inflammatory cells and DNA, the visco-of increases in ASL tonicity on mucociliary clearance arevery transient. elasticity is not significantly different from sputum ob-

tained from patients with inflammatory airway diseasessuch as chronic bronchitis (170, 205, 215). As demon-4. Relation of mucociliary clearance to salt secretionstrated in vitro, bacterial and leukocyte-derived DNA andactin filaments in infected sputum increase sputum viscos-As discussed in section IVB3, recent studies by Bal-

lard and colleagues (118, 292) have provided experimental ity, since treatment of CF sputum with either DNase (251)or gelsolin (303) reduces sputum viscosity. Moreover, evi-evidence that blockade of anion secretion adversely af-

fects the rheological properties of gland secretions. In a dence that increases in mucus sulfation increase the vis-cosity of mucus secretions (191) suggests that alterationspig distal airway model of airway gland secretion, inhibi-

tors of both Cl and bicarbonate secretion (bumetanide, in mucus sulfation may decrease mucociliary clearanceand indirectly impair bacterial clearance. However, theacetazolamide, dimethylamiloride, and DIDS) significantly

altered both the water content and viscoelasticity of mu- lack of obvious changes in sputum viscoelasticity in CFand the idea that the ASL is dehydrated in CF have ledcus. Pharmacological inhibition of ion transport before

acetylcholine stimulation resulted in a threefold increase to the notion that decreased sputum hydration increasessputum adhesivity without necessarily altering viscosity.in mucus solids, higher mucus viscosity, and lower mucus

recoil than in the absence of inhibitors. Histological analy- Evidence to support sputum dehydration is limited tostudies of sputum water content, which demonstrated in-sis of porcine bronchi under the same conditions revealed

filling of the mucus ducts with mucuslike material in the creased solid content of CF sputum compared with spu-tum derived from patients with tracheostomies (29). Al-airways treated with the cocktail of anion inhibitors (117,

118). Although the investigators cannot be certain that though supportive, the inability to sample uninfected se-cretions and control for the presence of bacteria andthese observations are due to specific effects of the anion

inhibitors on salt secretion, and not to indirect effects of leukocytes preclude a firm conclusion as to whether spu-tum dehydration leads to abnormal mucociliary clearancethese agents, these data provide experimental evidence


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and the initiation of airway infection. Whether uninfected bronchi (57), primary defects in mucus properties mayhave more significant physiological effects in this regionCF mucus exhibits increases in viscosity or adhesivity

that could impair mucociliary clearance remains to be of the airway. In support of a defect in peripheral mucocil-iary clearance, a recent study demonstrated an Ç45% re-determined.duction in peripheral clearance of iron oxide particles inCF patients compared with normal volunteers (23).2. In vivo clearance measurements

In contrast to the indirect approach of measuringsputum properties, a second approach has been to deter-

C. Effect of Salt Concentrationmine mucociliary clearance in vivo. Several methods have

on Mucus Transportbeen developed, each with its own advantages and defi-ciencies. The earliest studies used radiolabeled albuminand reported radioaerosol clearance rates similar to those Although there is considerable controversy over the

ionic concentrations of ASL in CF (see sect. IVE), recentin normal adults (243, 282). However, aerosol depositionpatterns were dramatically different between the popula- evidence suggests that increases in the salt concentration

of sputum increase ciliary transportability and mucocili-tions, which makes interpretation of these data difficult. Asubsequent study by Yeates et al. (335) used radiolabeled ary clearance. Using the mucus-depleted bovine trachea

model, Wills et al. (318) examined the effects of salt con-albumin microspheres with an inhalation pattern designedto enhance proximal airway deposition. Transport rates in centration on the ciliary transportability of sputum ob-

tained from CF and non-CF bronchiectasis patients. Nota-CF patients were highly variable, and there was significantoverlap with the rates observed in normal subjects. Subse- bly, there was no difference in transportability between

CF and non-CF sputum, nor were there differences in thequently, Wood et al. (328) directly observed the movementof 0.68-mm Teflon discs in the trachea to assess mucus response to changes in salt concentration. Incubation of

sputum in either excess isotonic or hypertonic salineclearance. Compared with normal volunteers, the CF pa-tients had significantly reduced disc movement rates. (200–600 mosM PBS) or solid NaCl (to increase NaCl

concentrationÇ90 mM) significantly increased the ciliaryHowever, there was no correlation between clearancerate and either clinical status or pulmonary function. transportability and decreased the viscosity and elasticity

of both CF and non-CF sputum without affecting ciliaryThe lack of emerging correlation and the disparatemethods from these early studies prompted Regnis et al. beat frequency. In contrast, increasing the hydration of

sputum by incubation in hypotonic solutions reduced cili-(227) to utilize more refined nuclear imaging techniquesand a larger patient population to determine mucociliary ary transportability. Based on these observations, and

those of previous investigators (106), this group suggeststransport rates in CF. Radiolabeled sulfur colloid was de-livered with a protocol that maximized proximal airway that ciliary transportability depends on saline content and

osmolality of sputum. In CF, excessive Na absorptiondelivery. By correlating clearance with deposition pat-terns, clearance from peripheral airways could be ex- could reduce the salt concentration of sputum, thereby

increasing mucus viscoelasticity and decreasing ciliarycluded from the analyses. The percent clearance of radio-aerosol at 60 min was significantly less in the CF popula- transportability.

What data exist to support the suggestion that spu-tion compared with a control population of similar age.In the five patients with normal small airway function, tum from CF patients has lower salt concentrations than

normal? Few studies have attempted to measure the saltthree had reduced clearance, and clearance rates de-creased with increasing disease severity. Moreover, multi- concentration of sputum itself and those that have relied

on sputum from patients with laryngectomies for ‘‘nor-ple regression analysis revealed a significant reduction inclearance rates for the CF population when corrected for mal’’ sputum. Matthews et al. (182) reported significantly

higher salt concentrations in laryngectomy samples com-severity of airflow obstruction, suggesting that the de-creased clearance rates are related to CF and are not pared with CF patients (165 vs. 101 mM for Na, 162 vs.

75 mM for Cl). More recent studies have also reportedmerely reflecting airway obstruction. However, becauseeach of the CF patients in this study had bacterial coloni- hypotonic Na and Cl concentrations in CF sputum (284).

However, comparisons of CF to non-CF bronchiectasiszation, this well-controlled in vivo study does not addresswhether defects in mucociliary clearance are due to pri- sputum revealed small differences (319), raising the ques-

tion of whether changes in salt concentration are due tomary defects in mucus transport or are secondary to air-way infection. This question will be difficult to address chronic infection and inflammation rather than solely to

CF mutations. Thus, as with the ASL, the effect of CFTRdefinitively, because there are currently no reliable meth-ods to assess mucociliary clearance in infants before the mutations on sputum salt concentration, and a role for

altered NaCl concentration on mucociliary clearance, re-development of airway infection. Because ciliary beat fre-quency is slower in small airways than the trachea and mains to be defined.


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D. Comparison With Dyskinetic Cilia Syndromes 1. PA and SA binding to epithelia and

airway secretions

Comparison of the clinical course and physiological Several studies have carefully localized SA and PA inderangement in mucociliary clearance between CF and the lung of CF patients and described the interaction ofthe dyskinetic cilia syndromes strongly suggests that de- these bacteria with normal and injured airway in vitro.fects in mucociliary clearance cannot solely account for Baltimore et al. (18) used immunohistochemistry on lungthe progressive obstructive lung disease in CF. Patients tissue obtained from autopsy to demonstrate the endo-with dyskinetic cilia syndromes, in which structural defor- bronchiolar location of PA and lack of PA outside themities in the ciliary motor apparatus impair ciliary move- airway lumen. Notably, PA was found primarily embeddedment, have a longer life span and better preserved pulmo- within intraluminal inflammatory exudates, and there wasnary function than patients with CF. This occurs despite no evidence that organisms were present in epithelialthe fact that mucociliary clearance is more severely im- cells. Other studies using organ culture of human airwaypaired in dyskinetic cilia syndromes than in CF (153). demonstrated that adherence of PA to epithelial cells oc-Moreover, in patients with bronchiectasis due to diseases curred only in areas where there was destruction of theother than CF, PA colonization is less frequent and ap- ciliated epithelial cells (204). Pseudomonas aeruginosapears to occur much later in the course of the disease, was found adherent to exposed areas of basement mem-when airway obstruction is severe (78). Moreover, pa- brane and to the mucus layer, but not adherent to thetients with dyskinetic cilia have a near-normal life expec- apical membrane of intact epithelial cells (208). In areastancy (3) compared with the current Ç30-yr median sur- of injury, PA was occasionally detected in intercellularvival for CF patients. These observations suggest that al- spaces, and within granulocytes, but not within epithelialthough mucociliary clearance is generally reduced in CF cells. Similarly, a recent study using native lung specimens(see sect. VB), the increased susceptibility to bacterial and an in vitro model of differentiated airway epitheliuminfection is disproportionate to the severity of mucociliary demonstrated by immunohistochemistry and electron mi-clearance impairment. This implies that other mecha- croscopy that SA bound primarily to intraluminal and cell-nisms contribute to pathogenesis of the progressive air- associated mucus. There was little binding directly to dif-way obstruction in CF. ferentiated airway epithelia and no significant difference

in binding between cultured CF and non-CF nasal epithe-lia (297). Collectively, these studies suggest that bacterial-

VI. AIRWAY INFECTIONmucin interactions are critical to the clearance of bothPA and SA, cast doubt on the importance of epithelial-bacterial adhesion, and raise the possibility that epithelialA. Organisms and Their Mechanismsingestion of PA is largely an artifact of in vitro culturesystems.

Numerous investigators have attempted to define theinitial mechanisms for infection of the CF airway with PA,

2. Interactions of bacteria and epithelial cellsand to a lesser extent HI and SA. Although a numberof hypotheses have been proposed and experimentally Evidence to support an important role for bacterial

adhesion to epithelial membranes suffers from uncertain-tested, this remains an important unresolved issue and inmany ways is at the crux of the pathophysiological CF ties regarding cell culture models, particularly when con-

sidered in the context of the bacterial localization studiesconundrum. Although the most parsimonious hypothesisto account for initial airway infection in CF is impaired discussed above. There is evidence, however, to suggest

that defects in CFTR enhance the adhesion of bacterialmucociliary clearance related to defects in apical mem-brane salt and water transport, as discussed above, a num- pathogens to airway epithelia, and recent progress has

been made in defining a number of bacterial adhesinsber of complementary hypotheses have emerged that mayexplain the initiation or persistence of airway infection. and epithelial cell ligands that may contribute to bacterial

infection (reviewed in Refs. 100, 211). On the epithelialThese include 1) abnormal epithelial cell ligands for spe-cific bacteria, 2) abnormal secreted mucins that impair cells, HI, SA, and PA appear to bind to a tetrasaccharide

containing a GalNAcb1–4Gal sequence that is exposed inmucociliary clearance or act as receptor sites for bacteria,3) exposure of extracellular matrix proteins that serve the less sialylated form of the glycolipid ganglioside 1

asialo-ganglioside M1 (asialoGM1) (116). As asialoGM1 isto promote adhesion, 4) impaired epithelial ingestion ofbacteria, and 5) inactivation of epithelial-derived bacterio- expressed more on CF than non-CF cells (242), and CFTR

complementation experiments show a reduction in bacte-cidal activity. Before the consideration of the evidencesupporting some of these mechanisms, pathological stud- rial binding, it appears likely that CFTR plays an important

role in the ability of CF pathogens to bind to epithelialies that have localized SA and PA in the airway meritconsideration. membranes in these cell culture systems. In addition to


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alterations in sialylation of membrane glycolipids that re- studies addressing the importance of mucin glycosylationfor PA binding have demonstrated PA binding to type 1sult from CFTR defects, Pseudomonal exoproducts such

as neuraminidase have been reported to have enhanced (Galb1–3GlcNAc) and type 2 (Galb1–4GlcNAc) disaccha-ride subunits of O-linked glycans in secreted non-CF tra-activity in CF airways. This could contribute to the cre-

ation of more asialoGM1 and further enhance bacterial cheobronchial mucins (220). In addition, there is limitedevidence for protein-protein interaction between a nonpi-adhesion (43, 241). The importance of these observations

for the intact airway remains unclear, since although asia- lus component of PA and the peptide moiety of a highlypurified human non-CF tracheobronchial mucin (224).loGM1 has been isolated from extracts of parenchymal

lung tissue (157, 158), its cellular and subcellular distribu- Nevertheless, subsequent studies that have attempted toidentify specific PA receptors on mucins have been unsuc-tion in the airway of humans has not been clearly estab-

lished. More importantly, these observations have been cessful, leaving open the possibility that the interactionbetween PA and mucins is largely nonspecific (224, 239,made on immortalized airway cells (IB-3 cells) and on

primary airway cells that established a transmembrane 240). In addition, the GalNAcb1–4Gal sequence, whichappears in the receptor sequence for PA that is presentresistance but lacked other morphological features of dif-

ferentiated airway cells. Finally, the lack of significant in asialoGM1 and asialoGM2, has not been observed insecreted mucins. Thus studies of bacterial interactionsnumbers of bacteria bound to differentiated epithelia in

native airway sections (18, 297), in contrast to significant with secreted mucins lack consensus. Some of the uncer-tainties may be related to differences in the sources andbinding to injured epithelia (66, 204, 208, 293), suggests

that direct epithelial-bacterial cell interactions require an- purification methods of mucins as well as to differencesin adhesion assays utilized. With the recent cloning oftecedent epithelial injury. Thus bacterial-epithelial adhe-

sion may be important once infection has caused epithe- specific mucin genes, it appears likely that the role ofmucins in the initiation of bacterial infection in CF willlial injury but appears less likely to play a critical role in

the initiation of infection. be clarified in the near future.The importance of mucin sulfation for PA binding

has not been evaluated as rigorously as sialylation, and it3. Interactions of bacteria and mucinsremains unclear whether the reported alterations in mucinsulfation in CF contribute to the initiation of infection. InSeveral studies using mucin from a variety of sources

have provided evidence that mucins bind PA and that one study, increased mucin sulfation, as reported to occurin cultured CF epithelia (49), resulted in less PA binding.carbohydrate modifications are important for PA binding

(reviewed in Ref. 245). Pseudomonas aeruginosa appears In contrast, another group of investigators was unable toidentify a correlation between sulfated cell surface glyco-to bind neutral and sialylated forms of mucin with little

binding to the sulfated forms (221). The pivotal role of conjugates and PA attachment (207). Thus the importanceof mucus sulfation for bacterial adhesion remains unclear.sialic acid in the binding of PA to mucins in vitro has been

demonstrated in several studies in which cleavage of sialic Mucin binding of HI and SA has not been studied asextensively as PA. Several nontypeable strains of HI ap-acid from mucin by neuraminidase abrogated the ability

of mucin to bind PA (223, 307). Other studies using free pear to bind purified airway mucins in suspension or onsolid phase, whereas encapsulated and other nontypeablesialic acid as a competitive inhibitor have corroborated

an important role for sialic acid in PA binding to mucins strains do not interact with mucins (63). The adhesinsand receptors responsible for HI and SA binding to mu-(46, 307). Moreover, aggregation of PA by CF and non-CF

salivary mucins has also been reported to be dependent cins and the role of posttranslational processing have notbeen determined to date. With regard to SA, specific bind-on sialylation, as exogenous and endogenous neuramini-

dase activity reduced mucin binding (154). These studies ing to immunoglobulin A-salivary mucin complexes andhuman nasal mucin via the interaction of mucin carbohy-have suggested that sialic acid may be a component of a

mucin receptor for PA, or alternatively, may alter tertiary drate moiety-SA proteins has been demonstrated (244,257); however, the exact nature of this binding has notstructure to enhance exposure of PA receptors on mucins.

These studies imply that undersialylation of secreted mu- been studied in detail.In addition to cell surface and mucin adhesion, therecins could reduce the efficiency of bacterial binding and

contribute to decreased bacterial clearance. is ample evidence that extracellular matrix proteins maycontribute to bacterial infection. For example, SA binds toSeveral studies have attempted to clarify the nature

of the interaction between PA and either CF or non-CF many matrix glycoproteins, including laminin, fibronectin,vitronectin, and fibrinogen (reviewed in Ref. 231). In con-mucin. Sajjan et al. (240), comparing the binding of pili-

ated PA to mucin purified from CF sputum and from non- trast, fibronectin does not appear to influence the interac-tion of PA with epithelial cells (2). As extracellular matrixCF intestinal mucin, were unable to detect any increase

in PA binding to CF or intestinal mucin compared with components become exposed or are released in the pro-cess of airway injury, it appears likely that they play aalbumin or gelatin in solid-phase assays. However, other


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more important role in the maintenance rather than the cationic proteins secreted by epithelia and immune cells.Like other airway antimicrobials, their ability to kill bacte-initiation of SA infection in the airway. Recent evidence

suggests that CFTR may affect the sulfation of glycosami- ria is salt sensitive. Their activity is usually markedly di-minished at NaCl concentrations that approximate plasmanoglycans (110); however, there is to date no evidence to

suggest that matrix components contribute to the infec- values (e.g., 100–140 mM NaCl). Accordingly, Smith et al.(261) added salt to the solution washed from the normaltions characteristic of the CF airway.

In summary, the components of normal mucociliary airway surface and found that this reduced its potency.Conversely, diluting the fluid at the apical surface of CFclearance have been elucidated over the last two decades,

and a number of potentially important alterations in the cells enhanced its bacterial killing activity. The importantconclusion from these observations was that if the ASLphysical environment of the CF airway have been identi-

fied. This has led to a number of highly plausible and had an abnormally high NaCl concentration in CF, thishigher concentration of salt would inactivate defensinsnonexclusive hypotheses as to how mutations in CFTR

alter the airway environment and create a permissive envi- and generally reduce the ability of endogenous antimicro-bials to kill bacteria. This was a property of the apicalronment for bacterial infection with specific pathogens.

Although there is good evidence that bacterial adhesion surface; no activity against bacteria was found in the solu-tion at the basolateral side of epithelium.to mucin occurs and is an important component of airway

clearance, it remains unclear whether deletion of CFTR In a subsequent series of experiments, Goldman etal. (93) basically replicated these findings using the repop-function alters the biochemical composition of mucins

and, if so, whether it is mucus clearance and/or bacterial ulated mouse xenograft model. They implicated a particu-lar human defensin, hBD-1, as the major bacteriocidalbinding that is primarily affected.factor. This observation was based on the ability of anti-sense oligonucleotides, which targeted the synthesis of

B. How the Airway Environment in CF hBD-1, to eliminate the ability of normal epithelia to killPermits Infection bacteria.

There are several questions that attend these im-portant observations. First, is a single agent or a combina-The traditional view of this problem is that the build-

up of mucins, perhaps abnormally sialylated or sulfated tion of agents responsible for the bacteriocidal activity ofthe ASL? Can a single factor such as hBD-1 explain theones, provides an environment that is permissive for infec-

tion of CF airways. Alterations in airway surface glycopro- capacity of the airway surface to kill bacteria? A mixtureof antimicrobials having different mechanisms of actionteins may provide attachment sites for specific microor-

ganisms, but, as discussed above, the overall importance is normally present. If these substances work together tocontrol the microbial population, how would the effectsof bacterial-epithelial adhesion remains unclear. Conse-

quently, in recent years, our attention has been shifted to of antisense suppression of hBD-1 alone be explained? Itis possible that combinations of bacteriocidal factors havethe inherent defense mechanisms of airway epithelial cells

and the possibility that these mechanisms are impaired synergistic effects. For example, lysozyme also shows saltsensitivity and operates by digesting bacterial cell walls.in the CF airway. This concept holds that epithelial cells

condition their microenvironment to make it inhospitable Lysozyme together with an agent that disrupts bacterialion gradients, like a b-defensin, may therefore have pro-for microbial infection and that their ability to do this

depends somehow on a normally functioning CFTR. found effects on colonization. Thus the antisense experi-ments might be explained by the synergism between anti-The important studies of Smith and Welsh (261) used

polarized airway cells cultured from normal subjects to microbials that have different mechanisms of action. Itappears unlikely that a single substance like hBD-1 canshow that these epithelia killed bacteria when they were

placed on the apical surface. Cystic fibrosis epithelia did account for antimicrobial activity of the airway, but itselimination may remove its synergistic effects with othernot. The bacterial challenge was 1,000 cfu or less. The

killing generally occurred in Ç10 h, and if the bacterial defense substances. Second, we do not understandwhether cultured cells fully replicate the in vivo condition.load exceeded Ç1,000 cfu, the system was overwhelmed.

The fluid washed from the apical surface of normal airway Submucosal glands are missing from both the epithelialmonolayer cultures and from the repopulated xenograftsepithelia possessed bacteriocidal activity, but the fluid

washed from CF cells did not kill bacteria in vitro. This studied by Smith et al. (261) and Goldman et al. (93).However, the glands are a major source of defense sub-suggested that some defense factor was present on the

surface of normal but not CF monolayers, or that the stances, and clogging of their ducts would physically elim-inate a major component of the antimicrobials that arefactor might be secreted by both normal and CF epithelia,

but that differences in the environment at the airway sur- normally available at the airway surface. Finally, we stilldo not know whether salt concentrations are altered inface affected its potency.

The defensins are a group of low-molecular-weight the ASL from CF patients, as discussed above. Given the


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relatively low bacterial load where this mechanism is op- Although the relative importance of opsonophagocytic de-fects in the CF airway remain to be defined, the localizederative, this line of defense is likely breached early in the

disease process so that mucociliary clearance and cell- impairment of some components of the complement andhumoral defense systems appears to contribute to bacte-mediated inflammatory mechanisms quickly come into

play. rial persistence.

VII. INFLAMMATORY MECHANISMS

B. Defects in Anti-inflammatory Cytokines:Mechanisms of airway inflammation in CF have been

Interleukin-10investigated extensively, but only recently has there beenevidence of CFTR-related dysregulation in the inflamma-tory response. In general, it appears that the coordinated As reviewed in section I, a major early feature ofnetwork of cytokines and cell adhesion molecules that the histopathology in CF is neutrophilic bronchitis andregulate the inflammatory response to bacterial infections bronchiolitis; however, the appropriateness of the in-in other airway diseases are intact in CF. A detailed dis- flammatory response relative to the bacterial burden re-cussion of these mechanisms is beyond the scope of this mains unclear. The long-standing paradigm, summarizedarticle and is available in a number of recent reviews (68, in Figure 2, is that bacterial infection of the airway initi-135, 156). The following discussion focuses on aspects of ates an inflammatory response that successfully containsthe inflammatory response that may be unique to CF. the infection in the airway lumen but generally fails to

eradicate the bacteria. Analyses of cytokines (IL-1b) andchemoattractants (leukotriene B4) in BAL from CF pa-A. Immune Processes: Defects in Opsonizationtients have revealed elevated levels compared withhealthy controls and levels comparable to other diseasesBefore isolation of the CF gene, individual compo-in which there is bacterial infection of the lung (324). Annents of the inflammatory response were evaluated inalternative paradigm suggested by the finding of inflam-search of an explanation for chronic airway infection. Themation without typical viral and bacterial pathogens (136)normal host response to bacterial infection can be viewedis that the CF gene defect itself somehow contributes toas a series of events that allows for eradication of bacteria.excessive airway inflammation, such that the neutrophilicEach event, recruitment of neutrophils, opsonization andinflammatory response exceeds that driven by bacterialphagocytosis of bacteria, humoral and cell-mediated im-infection. The data to support this notion are limited tomunity, contributes to the elimination of bacterial infec-the observation that there is decreased production of thetion. Evidence accumulated to date suggests that defectsanti-inflammatory cytokine IL-10 in CF (30). Cytokinein opsonization and in secretion of the anti-inflammatoryanalysis of BAL fluid and macrophages from CF patientscytokine IL-10 may permit the maintenance of bacterialand healthy control subjects demonstrated elevated con-infection, but there do not appear to be defects in thecentrations of the proinflammatory cytokines tumor ne-inflammatory response that would contribute to the initia-crosis factor-a (TNF-a), IL-1b, IL-8, and IL-6, but de-tion of infection.creased concentrations of IL-10 (31). Normal macro-Phagocytic function in CF neutrophils from periph-phages stimulated with lipopolysaccharide expressederal blood is normal, and humoral antibody responsesintracellular cytokines to a similar extent as macrophagesare intact (reviewed in Ref. 156). However, cleavage offrom CF patients, suggesting that the cytokine productionopsonic receptors on neutrophils by elastase may impairin the CF airway is in part driven by lipopolysaccharidephagocytosis in the CF airway lumen. Antibody attach-(31). The same investigators determined that IL-10 is con-ment to bacterial surfaces normally activates the comple-stitutively produced by bronchial epithelial cells from nor-ment cascade, with enzymatic cleavage of complementmal individuals but that freshly isolated CF epithelial cellscomponent C3 generating C3b and iC3b. In turn, C3b andfail to produce IL-10 (30). The mechanism for these obser-iC3b serve as ligands for complement receptors 1, 3, andvations remains unclear. The simplest interpretation is4, respectively, on neutrophils and thereby promotesthat the inflammatory milieu in the CF airway downregu-phagocytosis of bacteria. In the airway of CF patients,lates IL-10 production and that CFTR does not itself alterproteolytic enzymes such as elastase appear to over-the ability of macrophages or epithelial cells to producewhelm the endogenous antiprotease activity. Bronchoal-IL-10. Nevertheless, the notion that airway inflammationveolar lavage fluid from CF patients has been shown toin CF may continue unabated because of a deficiency incleave iC3b and complement receptor 1 (24), resulting inthe production of anti-inflammatory cytokines like IL-10‘‘opsonin-receptor mismatch’’ (288), and elastase disruptsmerits further investigation, including a comparison withother opsonin-receptor interactions by cleaving the neu-

trophil antibody receptor FcgRIIIB (287) and IgG (80). other diseases characterized by chronic airway infection.


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C. Oxidant Environment and Glutathione D. Defective Apoptosis Related to CF Mutations

Transport

In addition to the above potential mechanism of neu-trophil dysfunction in CF, recent reports suggest that mu-

A recent study of oxidant formation in CF neutrophils tations in CFTR may lead to abnormal programmed cellsuggests a CFTR-related abnormality that may contribute death, or apoptosis, in epithelial cells and leukocytes,to the pathogenesis of CF airway disease. Neutrophil oxi- thereby contributing to DNA release and mucus viscosity.dant function is regulated by NADPH oxidase activation Acidification of intracellular compartments accompaniesand myeloperoxidase activity. Activation of NADPH leads apoptosis, raising the possibility that defective intracellu-to the generation of superoxide anion and hydrogen per- lar acidification related to CF mutations could result inoxide. Myeloperoxidase in neutrophil granules utilizes hy- abnormal apoptosis and release of cell contents. In sup-drogen peroxide to generate hydrochlorous acid, which port of this hypothesis, Gottlieb and Dosanjh (95) re-reacts with amines to produce chloramines, which have a ported that epithelial cells expressing DF508 CFTR failedlong half-life relative to other oxidants. As excess oxidant to undergo cytoplasmic acidification and DNA fragmenta-activity in the CF airway could contribute significantly to tion in response to stimulation with cycloheximide orairway injury (reviewed in Ref. 40), investigators com- etoposide. Moreover, inhibition of CFTR with diphenyl-pared the neutrophil oxidant activities of CF children amine carboxylate delayed apoptosis in wild-type express-without active infection to their parents and healthy con- ing cells, and treatment of DF508 CFTR-expressing cellstrols. There were no differences in NADPH oxidase activ- with propionic acid to acidify intracellular compartmentsity among the three groups; however, both myeloperoxi- significantly diminished resistance to cycloheximide-in-dase-dependent oxidant activity and chloramine release duced apoptosis (95). These data suggest that CF muta-were increased in CF patients and, to a lesser extent, the tions may interfere with epithelial apoptosis, which inparents compared with controls (327). Treatment of CF turn may promote release of undigested DNA and otherneutrophils with amiloride or choline buffer reduced in- cellular contents that are normally not released duringtracellular myeloperoxidase-dependent oxidant genera- apoptosis. In the airway and gut, release of DNA couldtion and extracellular myeloperoxidase release, sug- increase the viscosity of secretions, and in the airway, agesting that altered myeloperoxidase activity in CF neu- similar process in neutrophils could contribute to the re-trophils may be regulated in part by intracellular pH or lease of proinflammatory cellular contents. Further stud-ion concentrations (327). Because CF gene expression has ies are clearly necessary to address these hypotheses, spe-been reported in neutrophils (338), and CFTR has been cifically to determine whether abnormal DNA fragmenta-implicated in the regulation of pH in intracellular organ- tion leads to release of cellular contents and impacts onelles (see discussion above), these data raise the possibil- airway inflammation. Nevertheless, the notion that aber-ity that CF gene mutations may contribute to exuberant rant apoptosis occurs in CF provides a plausible mecha-myeloperoxidase activity, and hence excessive oxidant nism for the apparent exuberant inflammatory responsegeneration that may promote airway injury through lipid in the CF airway.peroxidation. However, further studies are necessary todemonstrate that the observed differences are not duesolely to activation of circulating neutrophils, as has been E. Proinflammatory Effects of Bacterial DNA

suggested by previous investigations (96, 225, 232).In addition to possible neutrophil oxidant excess in In addition to the potential role of apoptosis in the

CF, a recent study provided evidence that CFTR may be CF inflammatory response, recent work suggests that bac-involved in regulating the level of glutathione, an im- terial DNA may itself induce inflammatory responses inportant antioxidant, in ASL. Using membrane patches the lung and contribute to the persistence of airway in-from cells transfected with CFTR and CFTR channel flammation in CF. The sputum in patients with CF hasblockers, Linsdell and Hanrahan (172) determined that been found by numerous investigators to have an in-CFTR is permeable to both glutathione and oxidized gluta- creased concentration of DNA (205), much of which isthione from the intracellular solution. This suggests that derived from bacteria. In addition to the apparent adverseCFTR may provide a conductive pathway for glutathione effects of DNA on mucus rheology (205), it appears thatto reach the ASL and account for both the reportedly high unmethylated bacterial DNA and oligonucleotides recruitlevels of glutathione in bronchoalveolar fluid from normal neutrophils to murine airway and induce the proinflam-lung (45) and the reduced levels reported in CF patients matory cytokines TNF-a, IL-6, and macrophage inflamma-(236). Thus CFTR mutations may directly impair glutathi- tory protein-2 (238). Specifically, unmethylated CpG mo-one transport, thereby disabling an important oxidant de- tifs were proinflammatory, whereas prokaryotic DNA (infense mechanism and increasing susceptibility of the CF which unmethylated DNA is much less abundant), methyl-

ated DNA, oligonucleotides without CpG motifs, andairway to oxidative injury.


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FIG. 8. Summary of proposed patho-physiological links between CF gene mu-tations and development of lung diseasein CF. Defects in CFTR lead to abnormalairway surface fluid and/or abnormal gly-cosylation of secreted and membrane-bound glycoconjugates. Via mechanismsoutlined, bacterial infection results andinitiates a vicious cycle in which infectionbegets inflammation, which leads to bron-chiectasis, induction of mucin gene(s),and further impairments in mucociliaryclearance that promote maintenance ofinfection.

amounts of lipopolysaccharide equal to those in the DNA ance, respectively, and create a permissive environmentpreparations did not induce pulmonary inflammation. for bacterial infection. Alternatively, or in addition,Similar inflammatory responses in BAL fluid were ob- CFTR’s function in intracellular compartments and glyco-served after instillation of purified DNA from the sputum conjugate processing suggests additional mechanismsof CF patients infected with Pseudomonas. These data whereby CFTR mutations permit bacterial infection. Ab-suggest that bacterial DNA may itself be proinflammatory normal glycosylation of secreted or membrane-bound gly-and, if confirmed in other species, may provide another coconjugates, particularly mucins and glycolipids, maymechanism for the exuberant inflammatory response ob- promote mucus obstruction of the small airways and pro-served in the CF airway. vide adhesive ligands for pathogenic bacteria.

Thus several mechanisms have been identified that Once initiated, bacterial infection elicits an inflam-may explain the persistent inflammatory response in the matory response that contains infection to the airway lu-CF airway; however, the mechanism(s) for the initiation men but does not eradicate the organisms. Mutations ofof bacterial infection remain to be clearly defined. In addi- CFTR may contribute to dysregulation of the inflamma-tion, the notion that inflammation precedes and/or is ex- tory response, via the mechanisms discussed above, in-cessive to that induced by bacterial infection will require cluding opsonophagocytic mismatch, defective apoptosis,further investigation. excessive oxidant formation, and impaired antioxidant se-

cretion. Moreover, bacterial infection could then create apermissive environment for its persistence via a number

VIII. SUMMARY of mechanisms, including induction of mucin genes whoseproducts may be aberrantly processed, exoproduct-medi-ated epithelial damage with subsequent disclosure of bac-In the context of the molecular, cellular, and lungterial adhesion sites and exposure of matrix proteins, andphysiology described above, a complicated pathophysio-induction of an inflammatory response that persists ab-logical cascade emerges, as summarized in Figure 8. Cys-normally because of defects in anti-inflammatory cytokinetic fibrosis gene mutations impair CFTR function and per-production. The vicious cycle of infection, inflammation,mit bacterial infection of the airway via one or more mech-and impaired mucociliary clearance ultimately leads toanisms. First, as derived from CFTR’s function as anbronchiectasis. However complex, the improved under-apical membrane chloride channel, dysfunctional CFTRstanding of the pathogenesis of airway disease in CF hasleads to abnormalities in the ASL composition or volume,

which impair antimicrobial activity or mucociliary clear- generated hypotheses to be experimentally tested in the


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