Loss of CFTR Chloride Channels Alters Salt Absorption by Cystic Fibrosis Airway Epithelia In Vitro

Transcription

1 Molecular Cell, Vol. 2, , September, 1998, Copyright 1998 by Cell Press Loss of CFTR Chloride Channels Alters Salt Absorption by Cystic Fibrosis Airway Epithelia In Vitro Joseph Zabner, 1,7 Jeffrey J. Smith, 2,7 Philip H. Karp, 1,3 Jonathan H. Widdicombe, 5 and Michael J. Welsh 1,3,6 1 Department of Internal Medicine salt and liquid absorption have been proposed to be increased, decreased, or equal to normal (Boucher et al., 1986; Jiang et al., 1993; Smith et al., 1994; Quinton, 1994; Welsh et al., 1995; Zhang et al., 1996). Likewise, 2 Department of Pediatrics estimates of airway surface liquid (ASL) ion concentra- 3 Department of Physiology and Biophysics tions have produced widely disparate results (Gilljam et 4 Howard Hughes Medical Institute al., 1989; Joris et al., 1993; Knowles et al., 1997). University of Iowa College of Medicine Limited knowledge of liquid and salt transport and Iowa City, Iowa ASL NaCl concentration ([NaCl]) in the airway are due to 5 Cardiovascular Research Institute several factors. Model systems with air-covered apical University of California at San Francisco surfaces that resemble in vivo airways have been diffi- San Francisco, California cult to develop and study. In addition, attempts to study CF in vivo are complicated by chronic airway infection and inflammation that could alter ion transport and ASL Summary electrolyte composition from values that exist very early in the disease. Technical limitations are also a significant Cystic fibrosis (CF) is caused by the loss of functional obstacle; the volume of ASL is very small and the pro- CFTR Cl channels. However, it is not understood how cess of removing and measuring ASL may change its this defect disrupts salt and liquid movement in the composition. Touching the epithelium with a filter paper, airway or whether it alters the NaCl concentration in an ion-selective electrode, or a bronchoscope as is re- the thin liquid film covering the airway surface. Using quired with current approaches could damage or irritate a new approach, we found that CF airway surface the epithelium, thereby altering ASL composition, ion liquid had a higher NaCl concentration than normal. transport, and gland secretion. For example, with an Both CF and non-cf epithelia absorbed salt and liquid; ASL depth of 20 m, 1 cm 2 of epithelium would be however, expression of CFTR Cl channels was recollect ASL, although this method also draws liquid from covered by 2 l. Several studies used filter paper to quired for maximal absorption. Thus, loss of CFTR elevates the salt concentration in CF airway surface the basolateral surface (Erjefält and Persson, 1990). One liquid and in sweat by related mechanisms; the elehigher ASL [NaCl] in CF than non-cf (Joris et al., 1993). study collected 1 10 nl from distal trachea and found vated NaCl concentration is due to a block in transcellular Cl movement. The high NaCl may predispose Three studies removed larger volumes of ASL; two found CF airways to bacterial infections by inhibiting endogsimilar [NaCl] in non-cf and CF (Knowles et al., 1997; enous antibacterial defenses. Hull et al., 1998), and we found inconsistent results be- tween CF and non-cf nasal liquid (Smith et al., 1996). Additional studies using aspiration (Gilljam et al., 1989) Introduction or tracheas filled with liquid (Goldman et al., 1997) reported higher salt concentrations in CF. In the genetic disease cystic fibrosis (CF), the loss of The current lack of understanding on how ASL compofunctional CFTR Cl channels disrupts Cl transport sition is regulated is particularly frustrating because al- across epithelia (Welsh et al., 1995). However, there is tered salt transport may be responsible for the clinical little knowledge of how loss of CFTR alters salt trans- hallmark of CF airway disease, chronic infections. Non- port in CF airways, a major siteof disease. Airway epitheincluding CF and CF ASL contain several antibacterial factors, lial ion transport has most often been evaluated with -defensins and lysozyme (Fleming, 1922; large volumes of liquid on both surfaces and with trans- Smith et al., 1996; Zhao et al., 1996; Goldman et al., 1997; epithelial voltage held constant at zero, the short-circuit McCray and Bentley, 1997). Activity of these factors is current (Isc) condition. Under these conditions, CFTRgested inhibited by high salt concentrations. Earlier work sug- dependent current is due to Cl secretion, and amilofactors, that a high [NaCl] in CF inhibits ASL antibacterial ride-sensitive current (Isc (Amil) ) is due to Na absorption. and this inhibition predisposes the CF airway In CF, disruption of Cl transport is measured as defechypothesis to infection (Smith et al., 1996). A key feature of this tive Cl secretion, and increased activity of apical Na is the assumption that CF ASL has an abnor- channels is measured as an increase in Isc (Amil) (Boucher mally high [NaCl]. et al., 1986; Stutts et al., 1995; Welsh et al., 1995). Under more physiologic conditions, the normal epithelium ab- Results sorbs both Na and Cl ; however, the consequences of absent transcellular Cl transport and increased Na NaCl Concentrations in ASL channel activity in CF are uncertain. For example, in CF, Because knowledge of the salt concentration at the air interface is critical to understanding CF airway disease, 6 To whom correspondence should be addressed ( mjwelsh we developed a new radiotracer method to ASL [Na] and [Cl]. We studied primary cultures of human 7 These authors contributed equally to this work. airway epithelia grown at the air-liquid interface under

2 Molecular Cell 398 Figure 1. Na and Cl Concentrations in ASL (A C) Comparison of [Na], [Cl], and volume in non-cf and CF ASL. n 15 for [Na], n 16 for [Cl], and n 31 for volume. Asterisk indicates p (D) Expression of CFTR corrected the ASL [Na] in CF epithelia. Treatment of CF epithelia with an adenovirus encoding CFTR increased bumetanide-sensitive Isc (Isc (Bumet) ) to A cm 2 from in CF epithelia treated with an adenovirus expressing -galactosidase. n 6 non-cf, 6 CF, and 6 CF treated with Ad/CFTR. Asterisk indicates p 0.01 compared to non-cf epithelia and double asterisk indicates p 0.01 compared to CF epithelia treated with Ad/ Gal. (E) Inhibiting Isc (Amil) increased ASL [Na] in non-cf epithelia. Epithelia were studied in the presence of benzamil in the medium or serum-free medium. Isc (Amil) was A cm 2 for control epithelia and A cm 2 for epithelia treated with serumfree medium. n 9 control, 9 benzamiltreated, and 9 serum-free epithelia. Asterisk indicates p 0.01 compared to control epithelia. conditions in which they differentiate, develop a ciliated benzamil, an amiloride analog (Kleyman and Cragoe, apical surface, and retain transepithelial electrolyte 1988), the ASL [Na] increased (Figure 1E). We also incu- transport by CFTR and Na channels (Yamaya et al., bated epithelia in serum-free medium for 2 days before 1992; Zabner et al., 1996). To measure ASL [Na], we the start of the study; this maneuver inhibits Na current added 22 Na to the basolateral medium together with without altering CFTR-dependent current (see below, 3 H 2 O. After the tracer content of ASL reached equilibrium, unpublished data, and Zabner et al., 1997). Figure 1E we removed ASL by rinsing the apical surface with shows that this intervention also increased the ASL [Na]. 100 l of medium. The basolateral medium was also These data indicate that both CFTR Cl channels and sampled and its [Na] was measured. The ratio of 22 Na Na channels are required to maintain ASL [NaCl] in the to 3 H 2 O in each compartment allowed us to calculate normal range. ASL [Na]. We performed similar experiments with 36 Cl. There were two key methodological details. First, after Relationship between Isc (Amil) and Liquid Absorption isotopes were added, non-cf and CF epithelia were At first inspection, the high [NaCl] in CF ASL may seem studied at the same time and sealed in the same cham- inconsistent with reports that the Isc (Amil) is increased ber. Second, the sealed container was humidified with across CF epithelia (Boucher et al., 1986). However, water that had the same specific activity of 3 H 2 O as Isc (Amil) only measures the rate of active transepithelial the culture medium. In this way, water vapor over the Na transport in the absence of any transepithelial ion surface of epithelia contained the same ratio of 3 H 2 O to or voltage gradients; thus, there is no requirement for Cl H 2 O as that in ASL and basolateral medium. to balance charge. To examine the relationship between Figures 1A and 1B show that in non-cf epithelia the Isc (Amil) and liquid absorption, we varied Isc (Amil) by incubating ASL [Na] was 50 4 mm and [Cl] was 37 6 mm, epithelia without serum, with serum, or with se- concentrations lower than those in the basolateral me- rum plus camp agonists (20 hr). These treatments resulted dium where [Na] was 156 mm and [Cl] was 135 mm. in low, intermediate, and high values of Isc (Amil) ASL covering CF epithelia had [Na] and [Cl] that were but had minimal effects on transcellular Cl transport increased compared to non-cf, even though the basolateral (Figure 2A). We then placed 60 l (a depth of 1 mm) medium was identical. We also found that non- of isosmotic liquid on the apical surface and measured CF and CF epithelia had the same volume of ASL (Figure liquid absorption. In non-cf epithelia, as Isc (Amil) in- 1C). The measured volume corresponds to an average creased liquid absorption increased (Figure 2B). In contrast, ASL depth of approximately 20 m, in excellent agreement in CF epithelia, as Isc (Amil) increased liquid absorp- with microelectrode and microscopic measure- tion increased initially but then plateaued. Thus, at high ments (Johnson et al., 1993; Wu et al., 1998). values of Isc (Amil), CF epithelia showed a defect in liquid When we expressed wild-type CFTR in CF epithelia, absorption. the ASL [Na] decreased into the normal range (Figure This result indicates that CFTR Cl channels are re- 1D). Thus, the increased salt concentration in CF is due quired for maximal liquid absorption. To test this conclusion to the loss of CFTR function and not to some other further, we expressed CFTR in CF epithelia and factor. measured electrical properties and liquid absorption. Conversely, when we inhibited Na channel activity with CFTR increased Cl transport as measured in an Ussing

3 Control of Airway Surface Liquid in CF 399 Figure 2. Effect of Modifying Isc (Amil) on Rate of Liquid Absorption (A) Amiloride-sensitive and bumetanide-sensitive Isc (indices of transcellular Na and Cl transport, respectively) in non-cf and CF epithelia. Before study, airway epithelia were cultured either in the absence of serum for 4 days, in the presence of serum (Basal), or in the presence of serum plus camp agonists (10 M forskolin and 100 M IBMX) for 20 hr. camp agonists were present during the study. Data are from epithelia shown in (B). For each individual experiment (each point in [B]), 3 epithelia were used to measure electrical properties; bars show mean SEM from the epithelia studied. (B) Relationship between Isc (Amil) and liquid absorption for non-cf (left) and CF (right) epithelia. Prior to study, epithelia were cultured under basal conditions (closed circles), 20 hr of camp agonists (open circles), or serumfree medium (diamonds). Each point represents mean SEM for Isc (Amil) (n 3) and liquid absorption (n 4 8); some SEM bars are hidden by symbols. chamber but had little effect on Isc (Amil) (Figure3A). Impor- In the normal sweat gland duct, apical Na channels tantly, expression of CFTR increased liquid absorption and CFTR Cl channels absorb salt across the duct (Figure 3A). Figure 3B shows that inhibition of Na trans- epithelium. Because liquid cannot follow through the port with benzamil inhibited liquid absorption in both water-impermeable epithelium, the [NaCl] in the duct non-cf and CF epithelia. This result is consistent with lumen falls. In CF, the lack of CFTR blocks transcellular data in Figure 2B showing that when Isc (Amil) is near zero absorption of Cl. The demands of electroneutrality also liquid absorption is inhibited. These findings indicate prevent absorption of Na. As a result, the sweat [NaCl] that transcellular pathways both for Na and for Cl are is high, a diagnostic hallmark of the disease. required for normal liquid absorption. How does CFTR influence liquid absorption and [NaCl] in airway epithelia? First, consider CF epithelia. Active Transepithelial Cl Transport Na absorption occurs through the cell, with Na enter- When we measured liquid absorption as described ing through amiloride-sensitive, apical Na channels above, absorption was isosmotic; after 4 hr, the mea- and exiting via the basolateral membrane Na-K-ATPase sured osmolality of the apical liquid was mosm (Welsh et al., 1995). Because the apical membrane of (n 8) in non-cf epithelia, mosm in CF (n CF epithelia is Cl impermeable, Cl absorption must 9), and mosm in the basolateral medium. These be primarily through the paracellular pathway in reresults are consistent with the high water permeability sponse to the transepithelial voltage. However, paracel- of airway epithelia (Folkesson et al., 1996). When taken lular pathways (Bradley and Purcell, 1982; Schneeberger together with the ability of non-cf epithelia to absorb and Lynch, 1992) usually have a Na to Cl selectivity more liquid, these findings suggested that non-cf epi- greater than 1. Thus, the relative paracellular Na to thelia should have a higher rate of salt absorption. To Cl selectivity will determine, in part, the rate of liquid test this directly, we measured unidirectional 36 Cl fluxes absorption and the transepithelial [NaCl] gradient that in epithelia treated chronically with camp agonists. Con- can be maintained. In contrast to CF, in normal epithelia sistent with the liquid absorption measurements, net Cl the presence of CFTR in the apical membrane and an absorption was greater in non-cf than in CF epithelia incompletely defined Cl channel in the basolateral (Figure 4). membrane (Willumsen et al., 1989) provide an additional transcellular Cl -selective pathway that allows for more Discussion liquid absorption when apical volume is large and a lower ASL [NaCl] when the ASL volume is small. The data show that in an in vitro model, CF epithelia have The ability of sweat duct epithelia to reduce apical a defect in liquid and salt absorption that is corrected [NaCl] is explained by its water impermeability; water by expression of CFTR. This abnormality leads to an cannot osmotically follow salt absorption and therefore elevated [NaCl] in ASL. Thus, both normal sweat duct the apical [NaCl] falls to low values. In contrast, airway and normal airway epithelia can lower the luminal [NaCl], epithelia have a significant water permeability (Folkes- and in CF this process is defective. son et al., 1996). How then can airway epithelia generate The consequences of losing CFTR function are most transepithelial ion concentration gradients? There must readily appreciated in the sweat gland (Quinton, 1990). be some force to counter osmotic pressure generated

4 Molecular Cell 400 Figure 4. Cl Fluxes across Non-CF and CF Epithelia Open squares indicate basolateral to apical flux and closed squares apical to basolateral flux. Lines are linear least-squares fit. Net absorption (dashed line) is difference between the two unidirectional fluxes. Net absorption was greater in non-cf than CF epithelia, p n In some cases, SEM bars are hidden by symbols. by the ion concentration difference. Impermeable osmo- lytes in the small volume of ASL might be responsible. In addition, the air liquid interface introduces surface tensions not present in liquid-covered epithelia. As sug- gested by Widdicombe and Widdicombe (1995), capillary forces might oppose a transepithelial osmotic pres- sure. Once osmotically driven absorption lowers ASL to the tips of the cilia, the closely packed cilia and microvilli could generate a large surface tension countering the osmotic pressure and thereby preventing further liquid absorption. Thus, the level of ASL would not fall below the tips of cilia. In addition, an apical mucus gel (Bas- baum and Finkbeiner, 1989) could hold water in the lumen; as liquid is absorbed, capillary pressure would increase and counter an ionic osmotic pressure. With ASL volume effectively prevented from decreasing to zero by one or a combination of these forces, active epithelial transport could reduce the ASL [NaCl]. These considerations are consistent with our finding that non- CF and CF epithelia have the same volume of ASL. Figure 3. Effect of Expressing CFTR and Inhibiting Na Transport on Liquid Absorption (A) Expression of CFTR in CF epithelia increases the rate of liquid absorption. CF epithelia were treated with adenovirus expressing -galactosidase (Ad/ Gal) or CFTR (Ad/CFTR) before study. All epi- thelia were treated with camp agonists for 20 hr before study as well as during study. Asterisks indicate p 0.001; in each group n 6 for Isc and 21 for absorption. Note that expression of CFTR increased liquid absorption even though Isc (Bumet) increased to values only 1/6 those in non-cf epithelia studied under similar conditions (compare with Figure 2A). (B) Inhibition of Na transport with benzamil inhibits liquid absorp- tion. Epithelia were studied under basal conditions (closed circles) or following 20 hr treatment with camp agonists (open circles). Benzamil (1 M) was included in apical and basolateral solutions as indicated. Each data point is mean from 4 6 epithelia; in some cases SEM bars are hidden by symbols. Basal Isc was A cm 2 in non-cf epithelia and A cm 2 in CF epithelia; following treatment with benzamil, Isc decreased to A cm 2 in non-cf and A cm 2 in CF epithelia. The data show that the lack of a cellular Cl conductive pathway is responsible for the increased ASL [NaCl] in CF. Thus, while active amiloride-sensitive Na transport is necessary to generate a low ASL [NaCl] and liquid absorption, in the absence of CFTR, Isc (Amil) is not directly related to the rate of liquid absorption. Perhaps the simplest way to think about this is to consider that an epithelium could have a significant Isc (Amil), but if there were no cellular or paracellular pathways for Cl flow then there would be no net transepithelial movement of Cl,Na,orH 2 O. In other words, the demands of electroneutrality only permit net Na movement if there is also a pathway for anion movement. Our approach to measuring ASL [NaCl] has several advantages: non-cf and CF epithelia were studied simultaneously under identical conditions; we measured the ratio of tracers after rapidly collecting ASL so that we minimized the chance that altered epithelial transport or ASL composition would influence our results; there was no infection or inflammation that could secondarily alter ASL ion concentrations; we studied well-differenti- ated, ciliated epithelia; and the epithelial surface was covered with air and a thin film of liquid throughout the study. A limitation of this study is that it is done in vitro. This model does not have submucosal glands, which are present in vivo in large airways. In this respect, our model may more closely resemble the distal airways, which lack submucosal glands but which are an important site of disease in CF. Although at present it is not clear how to measure ASL ion composition in vivo without altering either ion transport or the collected sample, our in vitro measures should provide a reasonably accu- rate reflection of conditions at the apical surface. Our results may also explain discrepant findings in earlier reports. Previous work suggested that liquid absorption by non-cf epithelia was less than or similar to CF epithelia (Jiang et al., 1993; Smith et al., 1994). These earlier studies by our laboratories were done at a time when airway epithelia were being cultured under conditions that did not produce well differentiated, ciliated epithelia, and the epithelia did not have a substantial Isc (Amil). Our data show that only in epithelia with a high Isc (Amil) is liquid absorption greater in non-cf epithelia. Thus, an important conclusion is that depending on absolute

5 Control of Airway Surface Liquid in CF 401 values of Isc (Amil) (as shown by the curves in Figure 2B), CF epithelia may have a lower, similar, or higher rate of liquid absorption than non-cf. The physiologic significance of this in different airway regions is not known. Finding that the ASL [NaCl] is elevated in CF supports a mechanism by which dysfunction of the CFTR Cl channel may contribute directly to the pathogenesis of airway infections; the high [NaCl] may inhibit antibacterial factors in ASL (Smith et al., 1996; Goldman et al., 1997; McCray and Bentley, 1997). The data also suggest that interventions that increase apical membrane Cl conductance could have a beneficial effect; such maneuvers might include, for example, activation of other apical Cl channels with agents such as inhaled nucleotides (Knowles et al., 1991), enhancing mutant CFTR function (Rubenstein and Zeitlin, 1998), incorporation of exogenous Cl channels into the apical membrane (El- Etri and Cuppoletti, 1996), or, as we show here, gene transfer of CFTR (Flotte et al., 1993; Hyde et al., 1993; Crystal, 1995). Experimental Procedures Epithelial Culture Airway epithelial cells were isolated from nasal, tracheal, and bron- chial tissue obtained from 7 CF and 12 non-cf people. Cells were seeded onto collagen-coated, semi-permeable membranes (0.6 cm 2 Millicel-HA; Millipore, Bedford, MA) and grown at the air liquid interface as previously described (Yamaya et al., 1992; Smith et al., 1996; Zabner et al., 1996). Culture medium, a 1:1 mixture of Dulbecco s modified Eagle s medium and Ham s F12 medium (DME/F12), was supplemented with 2% Ultroser G (BioSepra; Villeneuve, France) and initially with 100 mu/ml penicillin, 100 g/ml streptomycin, 50 g/ml gentamicin, 15 g/ml colimycin, 125 g/ml ceftazidime, and 2 g/ml fluconazole. Basolateral culture medium was changed every 2 4 days. All epithelia were studied at least 14 days after seeding when they had differentiated. All epithelia were evaluated with scanning electron microscopy for the development of a ciliated apical surface. Figure 5. Effect of Rinse Duration on 3 H 2 O Movement Each data point indicates n 18 from 3 different experiments. Lines are linear least-squares fit of the data. 36 Cl, and 3 H 2 O, and for measurement of [Na] and [Cl] by flame photometry and chloridometry, respectively. From these measure- ments, we determined the 3 H 2 O activity per l of water, the cpm of 22 Na per mole of Na, and the cpm of 36 Cl per mole of Cl in the basolateral medium. ASL volume was calculated from the ASL 3 H 2 O collected divided by the ratio of 3 H 2 O activity per l of basolateral medium. Na content was calculated from the ASL 22 Na collected divided by the ratio of 22 Na per mole of Na in the basolateral medium. ASL [Na] was calculated from the Na content divided by the volume of ASL. [Cl] was determined in a similar manner. Incomplete collection of ASL would not alter the calculated [Na] and [Cl] because concentrations are calculated from the ratio of 22 Na or 36 Cl to 3 H 2 O in the single sample of ASL removed by rinsing. However, incomplete collection could cause an underestimate of ASL volume. ASL was removed with a 1 sec rinse of the apical surface. Movement of 3 H 2 O from basolateral to apical solution through the water-permeable epithelium during the rinse could cause an underestimate of ion concentrations. To examine the extent of 3 H 2 O movement during the rinse, we varied the rinse time (Figure 5). Linear extrapolation to time zero indicates that we could have underestimated [Na] and [Cl] by at most 10%. Measurement of Liquid Absorption and Cl Flux Liquid absorption was measured using methods similar to those previously described (Mangoo Karim et al., 1989) and modified for Measurement of Transepithelial Electrical Properties airway epithelia (Smithet al., 1994). Onlyepithelia that had a transep- For measurement of transepithelial electrical properties, epithelia ithelial electrical resistance 800 cm2 (EVOM; World Precision were mounted in Ussing chambers and studied as previously de- Instruments, Sarasota, FL) were used. At the start of the experiment, scribed (Smith et al., 1994; Zabner etal., 1996). Epithelia were bathed basolateral solution was replaced with 500 l fresh medium conin symmetrical solutions containing (in mm): NaCl 135, K 2 HPO 4 2.4, taining 10 M forskolin and 100 M IBMX; all epithelia were treated KH 2 PO 4 0.6, CaCl 2 1.2, MgCl 2 1.2, dextrose 10, and HEPES 5 (at ph with camp agonists during measurement of liquid absorption to 7.2, 37 C), and gassed with 100% O 2. Isc (Amil) is the decrease in maximally activate CFTR Cl channels. To the apical surface we current after apical addition of 10 M amiloride or benzamil. Bumeta- applied 60 l of saline containing (in mm): NaCl 137.8, KCl 4, NaHCO 3 nide-sensitive Isc (Isc (Bumet) ) is the decrease in current after basolat- 29, CaCl 2 1.2, MgCl2 0.6, and NaH 2 PO 4 1; osmolality of the submucoeral addition of 100 M bumetanide to epithelia studied in the pres- sal solution was adjusted to equal that of mucosal solution using a ence of apical 10 M amiloride and basolateral camp agonists vapor pressure osmometer (Wescor Inc., Logan, UT). After incuba- (10 M forskolin plus 100 M 3-isobutyl 1-methylxanthine, IBMX); tion for 4 hr, apical solutions were collected under mineral oil and Isc (Bumet) is a measure of the transepithelial Cl transport pathway their volume measured as previously described (Smith et al., 1994). that includes CFTR. Methods to measure Cl flux were similar to those used to assay liquid absorption. Saline (250 l as described above) was applied Measurement of ASL [NaCl] The basolateral medium (500 l) of epithelia was spiked with cpm of 3 H 2 O and 22 Na or 36 Cl and then placed in a sealed chamber containing a water-saturated atmosphere of 5% CO 2 in air. Water used for chamber humidification was labeled with the same specific activity of 3 H 2 O to ensure that at equilibrium the ratio of labeled to unlabeled water would be identical in the water vapor, culture medium, and ASL. Non-CF and CF epithelia were always studied at the same time in the same chamber. Preliminary studies indicated that the tracer content of ASL had reached equilibrium by 24 hr. After incubation at 37 C for 48 hr, ASL was collected by rapidly rinsing the apical surface with 100 l medium. The ratio of 22 Na or 36 Cl to 3 H 2 O was determined by liquid scintillation. Aliquots of submucosal solution were also collected for measurement of 22 Na, to the apical surface. The basolateral surface was bathed in 750 l of isosmolar medium. To measure basolateral to apical and apical to basolateral flux, 36 Cl at cpm/ml was added to the appropriate solution. Ten l samples were collected from apical or basolateral solutions at indicated times. Radioactivity was measured by liquid scintillation. Interventions Serum-Free Medium Epithelia were cultured in the absence of serum for 4 days. Incubation in serum-free medium inhibits Isc (Amil) without altering CFTR function (see Figure 2A and unpublished data). For studies of liquid absorption, incubation in serum-free medium began 2 days before the start of the 48 hr study period.

6 Molecular Cell 402 Chronic camp Agonists Goldman, M.J., Anderson, G.M., Stolzenberg, E.D., Kari, U.P., Zasloff, camp agonists (10 M forskolin and 100 M IBMX) were added to M., and Wilson, J.M. (1997). Human -defensin-1 is a saltcamp the medium for 20 hr before study. Chronic treatment with camp sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell agonists increases Isc (Amil) (Figure 2A; unpublished data) and is similar 88, 2 9. to addition of cholera toxin to the culture medium (Yankaskas Hull, J., Skinner, W., Robertson, C., and Phelan, P. (1998). Elemental et al., 1985). During all studies of liquid absorption, camp agonists content of airway surface liquid from infants with cystic fibrosis. were present during the study to maximally activate CFTR. Am. J. Respir. Crit. Care. Med. 157, Benzamil Hyde, S.C., Gill, D.R., Higgins, C.F., Trezise, A.E., MacVinish, L.J., Benzamil (10 M, Research Biochemicals International, Natick, MA) Cuthbert, A.W., Ratcliff, R., Evans, M.J., and Colledge, W.H. (1993). was added to the basolateral solution rather than the apical surface Correction of the ion transport defect in cystic fibrosis transgenic to avoid adding liquid to or disturbing the ASL. Benzamil is more mice by gene therapy. Nature 362, potent thanamiloride at inhibiting thena channel in airway epithelia Jiang, C., Finkbeiner, W.E., Widdicombe, J.H., McCray, P.B., Jr., and (Kleyman and Cragoe, 1988; Stutts et al., 1995). When benzamil is Miller, S.S. (1993). Altered fluid transport across airway epithelium in added to the basolateral (or apical) solution of epithelia studied in cystic fibrosis. Science 262, Ussing chambers, it rapidly blocks amiloride-sensitive current (data Johnson, L.G., Dickman, K.G., Moore, K.L., Mandel, L.J., and Bounot shown). cher, R.C. (1993). Enhanced Na transport in an air-liquid interface Adenovirus culture system. Am. J. Physiol. 264, L560-L565. CF epithelia were treated with a recombinant adenovirus expressing CFTR (Ad2/CFTR-16; Genzyme) or -galactosidase Ad2/ Gal-16) Joris, L., Dab, I., and Quinton, P.M. (1993). Elemental composition as previously described (Zabner et al., 1996). Epithelia were incu- of human airway surface fluid in healthy and diseased airways. Am. bated with 50 MOI for 12 hr on days 3, 7, and 10 after seeding. Rev. Respir. Dis. 148, Kleyman, T.R., and Cragoe, E.J., Jr. (1988). Amiloride and its analogs Acknowledgments as tools in the study of ion transport. J. Membr. Biol. 105, Knowles, M.R., Clarke, L.L., and Boucher, R.C. (1991). Activation by We thank Pary Weber, Jan Launspach, Tom Moninger, Tony Thompof extracellular nucleotides of chloride secretion in the airway epithelia son, and Theresa Mayhew for excellent assistance. We especially patients with cystic fibrosis. New Engl. J. Med. 325, appreciate the help of James Abbenhaus, James Flynn, E. L. Knowles, M.R., Robinson, J.M., Wood, R.E., Pue, C.A., Mentz, W.M., Grandon, Denzel Hartshorn, Mike McCubbin, Thomas Pasic, William Wager, G.C., Gatzy, J.T., and Boucher, R.C. (1997). Ion composition Portilla, Mary Prudinsky, Warren Regelman, Mary Schroth, and their of airway surface liquid of patients with cystic fibrosis as compared associates, and the Iowa Statewide Organ Procurement Organiza- with normal and disease-control subjects. J. Clin. Invest. 100, 2588 tion. We thank Genzyme and the University of Iowa Gene Transfer Vector Core (supported in part by the Carver Charitable Trust) for Mangoo Karim, R., Uchic, M.E., Grant, M., Shumate, W.A., Calvet, adenovirus vectors. This work was supported by the National Insti- J.P., Park, C.H., and Grantham, J.J. (1989). Renal epithelial fluid tutes of Health (HL42385, M. J. W., andhl42368, J. H. W.), the Cystic secretion and cyst growth: the role of cyclic AMP. FASEB J. 3, Fibrosis Foundation, and the Howard Hughes Medical Institute. J. Z is supported by the Carver Charitable Trust. M. J. W. is an Investiga- McCray, P.B., and Bentley, L. (1997). Human airway epithelia extor of the Howard Hughes Medical Institute. press a -defensin. Am. J. Respir. Cell Mol. Biol. 16, Received May 13, 1998; revised July 2, Quinton, P.M. (1990). Cystic fibrosis: a disease in electrolyte transport. FASEB J. 4, References Quinton, P.M. (1994). Viscosity versus composition in airway pathology. Am. J. Respir. Crit. Care Med. 149, 6 7. Basbaum, C.B., and Finkbeiner, W.E. (1989). Mucus-producing cells Rubenstein, R.C., and Zeitlin, P.L. (1998). A pilot clinical trial of of the airways. In Lung Cell Biology, D. Massaro, ed. (New York: oral sodium 4-phenylbutyrate (Buphenyl) in deltaf508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR Marcel Dekker, Inc.), pp function. Am. J. Respir. Crit. Care Med. 157, Boucher, R.C., Stutts, M.J., Knowles, M.R., Cantley, L., and Gatzy, Schneeberger, W.E., and Lynch, R.D. (1992). Structure, function, J.T. (1986). Na transport in cystic fibrosis respiratory epithelia. and regulation of cellular tight junctions. Am. J. Physiol. 262, L647- Abnormal basal rate and response to adenylate cyclase activation. L661. J. Clin. Invest. 78, Smith, J.J., Karp, P.H., and Welsh, M.J. (1994). Defective fluid trans- Bradley, S.E., and Purcell, E.F. (1982). The Paracellular Pathway port by cystic fibrosis airway epithelia. J. Clin. Invest. 93, (New York: Josiah Macy, Jr., Foundation). Smith, J.J., Travis, S.M., Greenberg, E.P., and Welsh, M.J. (1996). Crystal, R.G. (1995). Transfer of genes to humans: early lessons and Cystic fibrosis airway epithelia fail to kill bacteria because of abnorobstacles to success. Science 270, mal airway surface fluid. Cell 85, ; and erratum 87(2). El-Etri, M., and Cuppoletti, J. (1996). Metalloporphyrin chloride iono- Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., phores: induction of increased anion permeability in lung epithelial Rossier, B.C., and Boucher, R.C. (1995). CFTR as a camp-depencells. Am. J. Physiol. 270, L386-L392. dent regulator of sodium channels. Science 269, Erjefält, I., and Persson, C.G.A. (1990). On the use of absorbing discs Welsh, M.J., Tsui, L.-C., Boat, T.F., and Beaudet, A.L. (1995). Cystic to sample mucosal surface liquids. Clin. Exp. Allergy 20, Fibrosis. In The Metabolic and Molecular Basis of Inherited Disease. Fleming, A. (1922). On a remarkable bacteriolytic element found in C. R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds. (New York: tissues and secretions. Proc. R. Soc. Lond. Biol. 93, McGraw-Hill, Inc.), pp Flotte, T.R., Afione, S.A., Conrad, C., McGrath, S.A., Solow, R., Oka, Widdicombe, J.H., and Widdicombe, J.G. (1995). Frontiers review: H., Zeitlin, P.L., Guggino, W.B., and Carter, B.J. (1993). Stable in regulation of human airway surface liquid. Respir. Physiol. 99, vivo expression of the cystic fibrosis transmembrane conductance Willumsen, N.J., Davis, C.W., and Boucher, R.C. (1989). Intracellular regulator with an adeno-associated virus vector. Proc. Natl. Acad. Cl activity and cellular Cl pathways in cultured human airway Sci. USA 90, epithelium. Am. J. Physiol. 256, C1033-C1044. Folkesson, H.G., Matthay, M.A., Frigeri, A., and Verkman, A.S. (1996). Wu, D.X-Y., Lee, C.Y.C., Ukekubo, S.N., Choi, H.K., Bastacky, S.J., Transepithelial water permeability in microperfused distal airways. and Widdicombe, J.H. (1998). Regulation of the depth of surface J. Clin. Invest. 97, liquid in bovine trachea. Am. J. Physiol. 274, L388-L395. Gilljam, H., Ellin, A., and Strandvik, B. (1989). Increased bronchial Yamaya, M., Finkbeiner, W.E., Chun, S.Y., and Widdicombe, J.H. chloride concentration in cystic fibrosis. Scand. J. Clin. Lab. Invest. 49, (1992). Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. 262, L713-L724.

7 Control of Airway Surface Liquid in CF 403 Yankaskas, J.R., Cotton, C.U., Knowles, M.R., Gatzy, J.T., and Boucher, R.C. (1985). Culture of human nasal epithelial cells on collagen matrix supports. A comparison of bioelectric properties of normal and cystic fibrosis epithelia. Am. Rev. Respir. Dis. 132, Zabner, J., Zeiher, B.G., Friedman, E., and Welsh, M.J. (1996). Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J. Virol. 70, Zabner, J., Launspach, J., Karp, P., Chenard, C., Stokes, J.B., and Welsh, M.J. (1997). Airway epithelial amiloride-sensitive sodium transport can be regulated by aldosterone in vitro and in vivo. Pediatr. Pulmonol. 14, 233. Zhang, Y., Yankaskas, J., Wilson, J., and Engelhardt, J.F. (1996). In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts. Am. J. Physiol. 270, C1326-C1335. Zhao, C., Wang, I., and Lehrer, R.I. (1996). Widespread expression of beta-defensin hbd-1 in human secretory glands and epithelial cells. FEBS Lett. 396,