Epithelium Depletion in Polarized Human Bronchial Double

Double Mechanism for Apical Tryptophan Depletion in Polarized Human Bronchial Epithelium

This information is current as Olga Zegarra-Moran, Chiara Folli, Benedetta Manzari, of September 25, 2021. Roberto Ravazzolo, Luigi Varesio and Luis J. V. Galietta J Immunol 2004; 173:542-549; ; doi: 10.4049/jimmunol.173.1.542 http://www.jimmunol.org/content/173/1/542 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Double Mechanism for Apical Tryptophan Depletion in Polarized Human Bronchial Epithelium1

Olga Zegarra-Moran,2* Chiara Folli,* Benedetta Manzari,† Roberto Ravazzolo,*‡ Luigi Varesio,† and Luis J. V. Galietta*

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-␥ in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-␥. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-␥ caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-␥ treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a trans- ؉ epithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na -dependent Downloaded from amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, /a removal through a Na؉-dependent amino acid transporter and an IFN-␥-inducible degradation by IDO. The Journal of http://www.jimmunol.org Immunology, 2004, 173: 542–549.

he periciliary fluid (PCF),3 which covers the airway sur- other types of cells is important in the local resistance to infectious face, is very important in the protective role played by the agents. airway epithelium against injuring particles and infectious IFN-␥ is secreted by CD4ϩ Th1 cells, NK cells, and a subset of T ϩ agents in the breathed air. Optimal thickness and fluidity of PCF CD8 T cells (7), and it is a potent modulator of airway epithelium are essential to maintain effective cilia beating and to allow mu- functions. IFN-␥ alone or in combination with other cytokines in- cociliary clearance. PCF volume and ion composition are con- duces several responses, including the expression of ICAM-1 (8), by guest on September 25, 2021 trolled by the balance between fluid/electrolyte secretion and ab- NO synthase 2 (9), cyclooxygenase (10), and chemokines (11, 12). sorption. The airway epithelium may also modify the composition We obtained evidence supporting that IFN-␥ can change PCF of PCF by secreting or removing various organic molecules such properties. We used a differentiated model of airway epithelium in as proteins, peptides, and amino acids, and influence the innate and which bronchial epithelial cells are allowed to polarize on a per- adaptive immune response (1, 2). Indeed, epithelial cells directly meable support, and we found that IFN-␥ changes the activity of secrete antibacterial products in the PCF under resting and stim- the epithelial Naϩ channel (ENaC) and of Ca2ϩ-activated ClϪ ulated conditions (3, 4). During an inflammatory response, airway channels in a way that would favor fluid secretion vs absorption epithelial cells also produce regulatory soluble factors (e.g., PGs, (13). To further investigate the effect of IFN-␥ on airway epithe- NO, cytokines, and chemokines) upon direct interaction with lium functions, we recently analyzed differential gene expression pathogens or following stimulation by cytokines secreted by leu- by using microarrays, and we found that, among others, IDO was kocytes (1, 2, 5, 6). The cross talk between airway epithelium and strongly up-regulated by IFN-␥ (our unpublished results). IDO is a mammalian enzyme that catabolyzes the essential amino acid tryptophan by oxidizing its pyrrole moiety. It represents the rate- *Laboratorio di Genetica Molecolare and †Laboratorio di Biologia Molecolare, Isti- limiting enzyme of the kynurenine pathway, the major tryptophan tuto Giannina Gaslini, Genoa, Italy; and ‡Dipartimento di Pediatria e Centro de Ec- catabolic pathway in mammals (14). IDO is ubiquitous in nonhe- cellenza per la Ricerca Biomedica, Universita`di Genova, Genoa, Italy patic tissue, where it is inducible by inflammatory mediators, in- Received for publication September 25, 2003. Accepted for publication April 21, 2004. cluding IL-12, IL-18 (15), bacterial LPS (16), and IFNs (17). IDO up-regulation by IFN-␥ is associated to expression of antimicrobial The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance response (18, 19) designed to limit the proliferation of invading with 18 U.S.C. Section 1734 solely to indicate this fact. pathogens by depleting the essential amino acid tryptophan from 1 This study was supported by Fondo per gli Investimenti della Ricerca Grant to the intra- and extracellular environments. Furthermore, IDO may R.R. O.Z.-M. was supported by Fondo per gli Investimenti della Ricerca funds to Dipartimento di Pediatria e Centro de Eccellenza per la Ricerca Biomedica, Univer- also contribute to modulation of leukocyte function (20). A role for sita`di Genova. IDO in T cell tolerance has been reported, and the suppressor 2 Address correspondence and reprint requests to Dr. Olga Zegarra-Moran, Labora- activity of macrophages and dendritic cells on T cell proliferation torio di Genetica Molecolare, Istituto Giannina Gaslini, L.go G. Gaslini, 5, Genoa in vitro may involve reduction of tryptophan concentrations in the 16148, Italy. E-mail address: [email protected] medium via IDO-dependent mechanisms (21Ð23). Tryptophan-de- 3 Abbreviations used in this paper: PCF, periciliary fluid; CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; ENaC, epithelial Naϩ channel; Isc, short- rived catabolites, in particular L-kynurenine, seem to be responsi- circuit current. ble at least in part for the inhibition of T and NK cell proliferation

(24). Interestingly, L-kynurenine inhibits proliferation only of ac- to normalize IDO transcript abundance. Data were analyzed by using Se- tivated cells, leaving resting cells able to respond to a subsequent quence Detector Systems version 2.0 software (Applied Biosystems). IDO reverse and forward primers were the same used for RT-PCR. Reverse and stimulus. Evidence that IDO-dependent T cell inhibition may op- ␤ Ј forward primers for 2-microglobulin were 5 -TCCAATCCAAATGCG erate also in vivo has been presented. An important consequence of GCATC and 5Ј-GCGCTACTCTCTCTTTCTGG, respectively. IDO activity has been identified in the placenta, where tryptophan degradation seems to play an important role in the prevention of the allogeneic fetus rejection by maternal T cells (25). Ion-pairing reverse-phase HPLC To our knowledge, there is only one report on IDO gene ex- Apical and basolateral medium bathing bronchial monolayers were har- pression induction by IFN-␥ in the airway epithelium (26). How- vested 0, 24, and 48 h from the beginning of the experiment and stored ever, there is no indication of the consequences of IDO activation frozen. The sample for HPLC analysis was prepared, as described (29). on a polarized epithelium. We aimed at elucidating the effects of Briefly, tissue culture supernatants were adjusted to a final concentration of ␥ 2% perchloric acid, kept on ice for 30 min, and centrifuged 5 min at IFN- on IDO expression and tryptophan metabolism on a polar- 13,000 ϫ g, and the pellet of precipitated proteins was discarded. The ized preparation of human bronchial epithelial cells. Our purpose perchloric acid-soluble fraction was neutralized by adjusting to 0.4 M po- was to determine whether tryptophan is transported through the tassium hydroxide; the clear supernatant was filtered through a 0.45-␮m polarized epithelial barrier and whether IDO up-regulation de- filter (Minispike; Waters, Milford, MA) and analyzed by HPLC. The HPLC separation module was a Waters model 2690 controlled by Mille- pletes tryptophan from apical and/or basolateral sides of epithe- ϩ nium 32 data system equipped with a Symmetry Shield RP18 column (Wa- lium. Our results show that a Na -dependent amino acid trans- ters). Tryptophan was measured with a fluorescence detector (Waters 474 porter is responsible for the unbalanced distribution of tryptophan scanning fluorescence detector) at an excitation wavelength of 285 nm and

across the polarized epithelium that accumulates in the basolateral an emission wavelength of 360 nm. Kynurenine was detected at a wave- Downloaded from side. IFN-␥, by activating IDO, causes an additional depletion of length of 360 nm using a UV detector. A diode-array detector was used for the identification of peaks. Tryptophan and kynurenine were identified by tryptophan at the apical side to almost undetectable levels. comparing the retention times and spectral data (obtained by diode-array detection) with the standards. The HPLC conditions were previously de- Materials and Methods scribed (29). Briefly, the sample was injected in the column equilibrated in Cell culture a running buffer composed by 2% methanol, 1 mM tetrabutylammonium hydrogen sulfate, and 30 mM phosphate buffer, pH 8.0, and eluted iso- Human bronchial epithelial cells were cultured, as previously described cratically for 15 min. http://www.jimmunol.org/ (13, 27). Briefly, cells were detached from bronchi after overnight incubation with protease XIV. Cells were obtained from lung transplants (cystic fibrosis (CF) patients) or lung resections (non-CF patients). After detach- [3H]Lysine and [3H]tryptophan transport ment, cells were grown on culture flasks for three to six passages in serum- free LHC basal medium/RPMI 1640 medium with 2 mM L-glutamine, 100 For uptake experiment on permeable supports, the apical and basolateral U/ml penicillin, and 100 ␮g/ml streptomycin. Cells used in this work were culture medium was removed and replaced with 0.5 and 2 ml of Krebs ␮ obtained from three non-CF and from two CF donors. We found no sig- solution, respectively. The apical medium contained either 100 M lysine ␮ 3 ␮ ␮ 3 nificant differences among donors; therefore, data from different individu- and 1 Ci/ml [ H]lysine or 50 M tryptophan and 1 Ci/ml [ H]trypto- ␮ als were pooled. CF cells were homozygous for ␦F508 mutation. To obtain phan. Aliquots of 20 and 1000 l were removed every 5 min from apical Ϫ5 and basolateral medium, respectively. After each sampling, the filter was polarized monolayers, cells were plated at high density (5 ϫ 10 cells/ by guest on September 25, 2021 cm2) on permeable supports (Snapwells; Corning-Costar, Cambridge, moved to another well containing fresh basolateral solution. Experiments MA). The medium was DMEM/Ham’s F12 (1:1) and contained 2% FBS, were done at 37¡C. The radioactivity in the samples was determined by 2mML-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and var- liquid scintillation counting to calculate the amount of lysine or tryptophan ious hormones and supplements (27, 28). Cells were maintained at 37¡Cin remaining in the apical medium at each time, and the efflux of radioactivity at the basolateral side. a humidified incubator in an atmosphere containing 5% CO2, and the experiments were done 8Ð11 days after plating. At this time, the epithelial resistance was 1182 Ϯ 27 ⍀/cm2 (n ϭ 52) in control cells, and 3212 Ϯ 108 ⍀/cm2 (n ϭ 31) in IFN-␥-treated cells. Ussing chamber experiments Conventional RT-PCR After 8Ð11 days in culture, permeable supports with human bronchial epithelial monolayers were mounted in a vertical diffusion chamber (Corning- Total RNA was extracted from cultured epithelia with TRIzol. Reverse Costar). The apical and basolateral chambers were filled with Krebs bicar- transcription on 1 ␮g of total RNA was performed with a commercial kit bonate solution that contained (in mM): 126 NaCl, 0.38 KH2PO4, 2.13 (BD Clontech, Palo Alto, CA), following the manufacturer’s instruction. K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose. The solution PCR was done using Amplitaq polymers and a reaction kit (PerkinElmer, was bubbled with 5% CO2-95% air. Experiments were done at 37¡C. The Wellesley, MA). PCR cycles included denaturation at 95¡C (40 s), primer transepithelial potential difference was clamped at the desired value with a annealing at 60¡C (30 s), and extension at 72¡C (30 s). Specific sequences voltage clamp amplifier, connected to the apical and basolateral chambers ␤ Ј for amplification of 2-microglobulin were 5 -GCGCTACTCTCTCTT via Ag-AgCl electrodes and agar bridges. For convention, reported volt- TCTGG-3Ј and 5Ј-TCCAATCCAAATGCGCCATC-3Ј (sense and anti- ages are referred taking the basolateral side as ground. Potential difference sense strand, respectively). PCR using these amplimers yields a 380-bp and fluid resistance between potential-sensing electrodes were product. IDO-specific sequences were amplified by using the following compensated. sequences:5Ј-CAAAGGTCATGGAGATGTCC-3Јand5Ј-CCACCAATAG AGAGACCAGG-3Ј as the sense and the antisense primer, respectively. PCR product was 240 bp length. Number of PCR cycles (reported in Re- Chemicals sults) was chosen to keep the reaction in the linear amplification phase. LHC9 medium was prepared from LHC basal medium (Biofluids, Rock- Quantitative real-time RT-PCR ville, MD) with the addition of supplements, as previously described (27, 28). Triiodothyronine and retinoic acid were from Biofluids. [3H]Trypto- Real-time PCR amplification reaction was done in a total volume of 25 ␮l phan and [3H]lysine were from Amersham Biosciences (Amersham, Buck- containing 5 ␮l of cDNA sample, 12.5 ␮l of SYBR Green PCR Master Mix inghamshire, U.K.). All other chemicals were from Sigma-Aldrich (St. (Applied Biosystems, Foster City, CA), and specific primers. The final Louis, MO), except DMEM, RPMI 1640, Ham’s F12, and serum that were concentration of primers was 7.5 pM. Primers and probes were designed from Euroclone (Paignton, Devon, U.K.). according to manufacturer’s guidelines. An ABI Prism 7700 sequence detection system (Applied Biosystems) with SYBR Green fluorescence was used for the assays. Cycling conditions were: 10-min hot start at 95¡C, Statistics followed by 35 cycles of denaturation step at 95¡C for 40 s, an annealing step at 63¡C for 30 s, and an extension step at 72¡C for 30 s. Each sample Data are presented as representative traces or as means Ϯ SEM. Signifi- ␤ was run in triplicate. 2-microglobulin was used as housekeeping mRNA cance was assessed using Student’s t test for unpaired groups of data. 544 TRYPTOPHAN DEPLETION IN HUMAN BRONCHIAL EPITHELIUM

Results basolateral side, because it is effective only from this side of the IFN-␥ induces the expression of IDO on bronchial epithelial monolayer (our unpublished results). cells We measured tryptophan and kynurenine concentration in the apical and basolateral fluid of cultured bronchial epithelia. Initial To evaluate IDO expression, we used a semiquantitative RT-PCR tryptophan concentration in our culture medium was 36 Ϯ 3 ␮M. on cDNA samples generated from control and IFN-␥-treated hu- When the medium was recovered from untreated cell cultures after man bronchial epithelia grown on permeable supports. Untreated a 24-h incubation, we found an asymmetrical distribution of tryp- epithelia did not show an IDO signal after 25 amplification cycles tophan levels. Taking into consideration the volumes of the culture using either diluted or undiluted cDNA. On the contrary, a clear medium on both sides of epithelium (see Materials and Methods), amplification product was detected from the cDNA of cells incu- the tryptophan changed from 18 Ϯ 1.5 nmol to 6 Ϯ 1 nmol on the bated with IFN-␥ even at very low concentrations of the template apical side, and from 72 Ϯ 6 nmol to 84 Ϯ 4 nmol on the baso- (Fig. 1). The housekeeping gene, ␤ -microglobulin, was similarly 2 lateral side (Fig. 2, A and B). After 48 h, tryptophan levels were expressed on both cell preparations. IDO expression could be dem- even lower on the apical (4 Ϯ 1.5 nmol) and did not change on the onstrated in untreated cells only after 32 PCR cycles using the basolateral side (81 Ϯ 6 nmol). undiluted template. To quantify the IDO response to IFN-␥,we determined the relative amount of mRNA using a real-time RT- PCR (Fig. 1B). We found that IDO mRNA increased at ϳ60- or 40-fold after 24 or 48 h of IFN-␥ stimulation, respectively. These results indicate that IDO mRNA is expressed at very low levels in Downloaded from resting bronchial epithelial cells, and that its expression is strongly up-regulated by treatment with IFN-␥.

Extracellular tryptophan levels under resting and IFN-␥- stimulated conditions The effect of IFN-␥ on extracellular tryptophan concentrations was http://www.jimmunol.org/ investigated on human bronchial epithelial cells grown on permeable supports as a polarized epithelium. IFN-␥ was applied to the by guest on September 25, 2021

FIGURE 1. IDO expression on bronchial epithelial cells. Semiquanti- tative PCR (A) using serial dilutions of cDNA from control and IFN-␥- treated human bronchial epithelial cells. Dilutions 1/2, 1/4, 1/8, and 1/16 FIGURE 2. Time course of apical and basolateral tryptophan and indicate that 0.5, 0.25, 0.125, and 0.0625 ␮g of RNA were used for the kynurenine levels in control and in IFN-␥-treated epithelia. IFN-␥ caused ␥ ␤ PCR. Treated cells were incubated for 48 h with 1000 U/ml IFN- . 2- a reduction of apical (A) and basolateral (B) tryptophan (TRP), and an microglobulin was used as housekeeping gene. In control conditions, ep- increase of apical (C) and basolateral (D) kynurenine (KYN). Each point is ithelia did not show an IDO signal after 25 amplification cycles even using the mean of 9Ð12 experiments Ϯ SEM. The effect of IFN-␥ was partially undiluted cDNA (1 ␮g). In contrast, a stable amplification product was prevented by norharmane (n ϭ 5). The IDO inhibitor norharmane (50 ␮M) found from the cDNA of cells incubated with IFN-␥ still after 16-fold was added to the apical and basolateral medium at the same time as IFN-␥. ,ء .dilution of the template. B, A quantitative determination of IDO mRNA Asterisks indicate statistically significant differences with IFN-␥ alone p Ͻ 0.01. Time course of kynurenine level on both sides of ,ءء ;was conducted using real-time RT-PCR. Calculation of IDO abundance p Ͻ 0.05 ␤ ␥ after normalization to 2-microglobulin showed changes due to IFN- the epithelia. After exogenous application of 15 nmol of apical kynurenine, treatment. Each bar is the mean of six measurements on different cell prep- we found that it decreases on the apical (E) and increases on the basolateral arations. Error bars represent SEM. (F) side of the monolayer (n ϭ 3). The Journal of Immunology 545

Kynurenine was absent from the medium of bronchial epithelial the epithelia at t ϭ 0. The amount of lysine that disappeared from cells under resting conditions (Fig. 2, C and D), thus confirming the apical chamber in 20 min was 10.1 Ϯ 1.4 nmol (n ϭ 10; Fig. that basal IDO expression is very low and suggesting that the de- 3). A total of 100 ␮M tryptophan or 100 ␮M kynurenine added to crease of apical tryptophan in untreated cells is not due to catab- the apical solution at the beginning of the experiment reduced olism. After IFN-␥ treatment, tryptophan decreased on both sides significantly the lysine uptake, suggesting that both compounds of cultured epithelia, disappearing completely from the apical side compete with lysine for the transporter. The uptake in Naϩ-free in 48 h and decreasing to 20 Ϯ 3 nmol on the basolateral side. In solution was almost zero, as expected for a Naϩ-dependent parallel, kynurenine increased dramatically in the basolateral me- transporter. dium to ϳ50 nmol, whereas kynurenine levels on the apical side Ϯ The transport of tryptophan and kynurenine is electrogenic and were modest (2 0.5 nmol). Experiments were done to establish ϩ whether changes in tryptophan and kynurenine levels were indeed Na dependent mediated by IDO activity. As shown in Fig. 2, 50 ␮M norharmane, One characteristic of the apical amino acid transporter in cultured a concentration near the IC50 for IDO inhibition (30), partially bronchial epithelial cells is the ability to generate a transepithelial prevented the tryptophan depletion and the production of kynure- electrical current (28). After clamping epithelia at 0 mV and block- nine induced by IFN-␥. ing ENaC with amiloride, we measured the short-circuit currents We hypothesized that the asymmetrical kynurenine distribution (Isc) in response to amino acids added to the apical chamber. As could be due to the presence of a specific transepithelial transport. previously shown, apical lysine generates an Isc in a dose-depen- According to this hypothesis, kynurenine would be produced by dent way in airway epithelial cells (Fig. 4, A and E). Interestingly,

IDO-mediated tryptophan degradation and released by diffusion Downloaded from through apical and basolateral membrane. Subsequently, apical kynurenine would be removed by an apical membrane transporter. To test this hypothesis, we added 15 nmol of kynurenine to the apical culture medium. Kynurenine was rapidly removed from the mucosal surface and transported to the basolateral side of the monolayers, thus

producing an inverted asymmetric distribution (Fig. 2, E and F). http://www.jimmunol.org/

Tryptophan and kynurenine are actively removed from the apical surface Our data indicated that tryptophan and kynurenine asymmetrical distributions under resting and stimulated conditions could be due to a transepithelial transport. We reported previously that the apical membrane of cultured bronchial epithelia has an electrogenic amino acid transporter that couples Naϩ inﬂux to the uptake of neutral and cationic amino acids (28). Given the broad selectivity, by guest on September 25, 2021 we hypothesized that this transporter could be also responsible for the apical removal of kynurenine as well as of tryptophan. First, we tested this possibility indirectly by measuring lysine uptake. A total of 100 ␮M lysine (50 nmol) was applied to the apical side of

FIGURE 4. Ussing chamber experiments. The transepithelial potential difference was short circuited at 0 mV. Isc elicited by increasing concentrations of lysine (A), kynurenine (B), and tryptophan (C). The effect of FIGURE 3. Time course of lysine uptake. Lysine uptake measured lysine and tryptophan/kynurenine was not additive, suggesting that they alone (F) or in the presence of 100 ␮M tryptophan (‚) or kynurenine (Ⅺ). compete for the same amino acid transporter. As expected, in Naϩ-free In Ⅺ, the lysine uptake in a Naϩ-free solution. Cells were grown for 8Ð11 solution, no Isc increase was observed after kynurenine or lysine addition days on permeable supports. Before experiments, the apical and basolateral (D). E and F, Dose-response relationships of tryptophan, kynurenine, and culture medium was removed and replaced with 0.5 and 2 ml of Krebs lysine. The points were ﬁtted to a Michaelis-Menten function. The maxi- solution. A total of 100 ␮M lysine (1 ␮Ci/ml [3H]lysine) was applied to the mal current obtained with tryptophan was 1.3 Ϯ 0.3 ␮A/cm2, about one- apical side of the epithelia at t ϭ 0, and every 5 min thereafter, aliquots of half that of kynurenine (2.6 Ϯ 0.7 ␮A/cm2), and one-third that of lysine 20 and 1000 ␮l were removed from apical and basolateral medium, re- (3.9 Ϯ 0.6 ␮A/cm2). The half-effective concentration was instead 12.1 Ϯ spectively. The radioactivity in the samples was determined by liquid scin- 2.9, 13 Ϯ 3.4, and 79.3 Ϯ 5.8 ␮M for tryptophan, kynurenine, and lysine, tillation counting to calculate the lysine uptake. Each point is the mean of respectively. No differences on these parameters were observed on IFN- 4Ð10 experiments Ϯ SEM. ␥-treated epithelia (F, open symbols). 546 TRYPTOPHAN DEPLETION IN HUMAN BRONCHIAL EPITHELIUM apical kynurenine also generates a current, and the same effect was induced by tryptophan (Fig. 4, B and C). In agreement with the hypothesis of a common transporter for these three amino acids, saturating concentrations of tryptophan or kynurenine largely prevented lysine induced current. In agreement with uptake experiments and with the assumption of a common Naϩ-dependent transporter, kynurenine, tryptophan, and lysine were ineffective under Naϩ-free conditions (Fig. 4D). Dose-response relationships for tryptophan and kynurenine in control and IFN-␥-treated epithelia were similar (Fig. 4F and Table I), thus suggesting that IFN-␥ treatment does not modify transporter expression or activity.

The amino acid transport depends on transepithelial potential Given the electrogenic activity of the apical amino acid transporter, we anticipated that the response to amino acids had to be affected by transepithelial electrical potential. We measured lysine- and tryptophan-induced Isc at ϩ30, 0, and Ϫ30 mV. The ﬁt of dose-response relationships to a Michaelis-Menten function Downloaded from (Fig. 5) yielded similar half-maximal activations at the three voltages (77.8 Ϯ 15.9, 79.8 Ϯ 11.1, and 96.3 Ϯ 21.9 ␮M for lysine, and 6.72 Ϯ 3.15, 14.24 Ϯ 4.01, and 10.06 Ϯ 2.11 ␮M for tryptophan; n ϭ 4Ð5 at each condition). In contrast, the maximal activity was statistically different: 13.5 Ϯ 1.8, 6.1 Ϯ 0.8, and 3.5 Ϯ

0.5 ␮A for lysine, and 2.18 Ϯ 0.25, 1.76 Ϯ 0.2, and 1.11 Ϯ 0.11 http://www.jimmunol.org/ ␮A for tryptophan at ϩ30, 0, and Ϫ30 mV, respectively.

Amino acid uptake in CF epithelia FIGURE 5. Dose-response relationships at different transmembrane potentials. The transepithelial potential difference was short circuited at dif- Airway epithelia from CF patients have altered transepithelial po- ferent clamping potentials, ϩ30, 0, and Ϫ30 mV. The points were fitted to tential. Given the voltage sensitivity of amino acid transport, we a Michaelis-Menten function. Although half-maximal activations at the decided to measure the lysine uptake on cells obtained from CF three voltages were very similar for each amino acid (ϳ85 ␮M for lysine subjects. We found that the amount of lysine that disappeared from and 10 ␮M for tryptophan), the maximal activity was clearly dependent on the apical chamber in 20 min was 6.6 Ϯ 0.3 nmol (n ϭ 4; Fig. 6A). the transepithelial potential. Each point is the mean of four to five different We measured also lysine uptake on non-CF epithelia in the pres- experiments, and vertical bars are SEM. by guest on September 25, 2021 ence of 400 ␮M glibenclamide, a known inhibitor of CF transmembrane conductance regulator (CFTR), to mimic the CF phenotype. We found that in this condition the uptake of lysine was 1.5 nmol on CF epithelia, while it was without effect on non-CF also reduced to ϳ3.6 Ϯ 0.6 nmol (n ϭ 4). Lysine uptake in CF epithelia. epithelia and in glibenclamide-treated non-CF epithelia was significantly lower than in untreated non-CF epithelia ( p Ͻ 0.05 and Discussion p Ͻ 0.01, respectively). To have a direct evidence of the move- IFN-␥ induces a variety of antimicrobial mechanisms, including ment of tryptophan, we measured also the uptake of this amino induction of IDO and catabolism of tryptophan. The oxidative acid on CF and non-CF epithelia. As shown on Fig. 6B also, the cleavage of the essential amino acid tryptophan by IDO (14) may tryptophan uptake was significantly lower in CF than in non-CF be associated to antimicrobial defense (18, 19). In fact, IDO is epithelia (2.5 Ϯ 1.3 and 6.6 Ϯ 0.3 nmol, respectively). The pres- expressed in cells infected with a variety of intracellular pathogens ence throughout the experiment of either glibenclamide or of the such as Toxoplasma, Chlamydia, and viruses, and it has also been found to inhibit the growth of extracellular bacteria such as group recently identified CFTR-specific inhibitor CFTRInh-172 (31, 32) caused a significant reduction of tryptophan uptake on non-CF B streptococci (19). IDO effectively restricts the growth of tryp- cells to 4.5 Ϯ 0.6 and 3.4 Ϯ 1.5 nmol. We modified the apical tophan-dependent pathogens by starving them for an amino acid. membrane potential also by blocking the ENaC with amiloride. We have studied the functional effects of IFN-␥-dependent IDO This caused a significant increase of tryptophan uptake to 8.6 Ϯ induction in human bronchial epithelia. We hypothesized that IDO up-regulation in airway epithelium could be particularly important to deplete tryptophan in the PCF. Our results first demonstrate that IDO is poorly expressed in resting epithelia. This result is sup- Table I. Kinetic parameters of amino acid-dependent currenta ported by the finding that kynurenine is absent in the culture medium despite relatively high concentrations of tryptophan. Our data Amino Acid nK(␮M) Imaxb (␮A/cm2) m also show that tryptophan is removed from the mucosal surface in L-Lysine 10 79.3 Ϯ 5.8 3.9 Ϯ 0.6 resting conditions and transported to the basolateral side of the L-Tryptophan 5 12.1 Ϯ 2.9 1.3 Ϯ 0.3 epithelium. IFN-␥ treatment strongly induces IDO expression. In ␥ Ϯ Ϯ L-Tryptophan (IFN- ) 4 9.6 2.3 1.1 0.4 this condition, we found IDO responsible for a further decrease in L-Kynurenine 8 13 Ϯ 3.4 2.6 Ϯ 0.7 L-Kynurenine (IFN-␥) 4 11.1 Ϯ 6.1 2.3 Ϯ 0.8 tryptophan concentration and for kynurenine production. Surpris- ingly, kynurenine increased only on the serosal side of the mono- aParameters obtained fitting dose-response relationships to Michaelis-Menten equa- tion. n is the number of experiments. Values obtained on epithelia clamped at 0 mV. layer. We hypothesized that kynurenine produced by IDO-medi- bImax, maximal current. ated tryptophan degradation diffuses through the cell membrane to The Journal of Immunology 547

pressed in the lung, in particular in ciliated epithelial cells of the trachea and bronchioles (34). The involvement of this amino acid transporter in the asymmetric distribution of tryptophan and of kynurenine was established in uptake experiments using radiolabeled amino acids. We found that tryptophan and kynurenine in- hibited the uptake of lysine, a well-known substrate of this transporter. We interpreted this effect as competition between tryptophan/kynurenine and lysine for the same transporter. This behavior was then conﬁrmed by measuring the Isc elicited by direct application of these amino acids to the epithelium apical surface. Both substrates elicited a Naϩ-dependent current, and this effect was not additive with that of lysine. Comparison of the dose- response relationships showed that the maximal current obtained with tryptophan was about one-half that of kynurenine and one- third that of lysine (see Fig. 4, E and F). Interestingly, the apparent

Km for tryptophan and kynurenine were lower than that for lysine (see Table I) and for other good substrates of the transporter such as arginine and alanine (28), thus indicating that tryptophan and kynurenine are also physiological substrates. In IFN-␥-treated Downloaded from cells, the maximal current and the half-effective concentration of tryptophan and kynurenine were similar to those of control cells, indicating that IFN-␥ does not modify the transporter expression or activity. An important ﬁnding was that the rate of amino acid uptake by the apical transporter strongly depended on transepithelial poten- http://www.jimmunol.org/ tial, as expected from its electrogenic nature. Indeed, the maximal Isc for lysine and tryptophan at ϩ30 mV was 13.5 and 2.2 ␮A, while it was 3.5 and 1.1 ␮A, signiﬁcantly lower, at Ϫ30 mV. This is not surprising because more positive potentials impose a higher driving force for apical-to-basolateral Naϩ movement through the epithelium that might drive faster amino acid uptake. Conversely, at more negative transepithelial potential, this driving force is re-

duced. Given these results, we speculate that apical membrane by guest on September 25, 2021 potential changes might affect the rate of amino acid uptake. CF is a disease in which the mutation of a gene encoding an apical ClϪ channel, the CFTR, causes the transepithelial potential difference to be significantly more negative than in non-CF subjects (35). There are indications that in CF the sodium absorption through FIGURE 6. Amino acid uptake in CF and non-CF bronchial epithelial ENaC is up-regulated (36), and this could be the main reason for cells. A, Time course of lysine uptake in CF (E) and non-CF (F) cells, or the more negative value of the transmembrane potential. In a pre- in non-CF cells in the presence of 400 ␮M glibenclamide (Ⅺ). Each point vious work, we measured the activity of the apical amino acid Ϯ is the mean of 4Ð10 experiments SEM. B, Tryptophan uptake in CF and transporter in CF and non-CF epithelia in short-circuit conditions, non-CF (WT) epithelia measured at 20 min in the absence or presence of ␮ ␮ ␮ i.e., with transepithelial voltage clamped at 0 mV (28). We found 10 M amiloride (amil), 20 M CFTRInh-172 (Inh-172), 400 M glibenclamide (glib), or in a Krebs solution without Naϩ (Na-free). Each bar is that the half-effective concentration and the maximal current the mean of 4Ð6 experiments Ϯ SEM. Asterisks indicate that values are evoked by arginine on epithelia obtained from CF patients were -p Ͻ similar to those of non-CF cells, indicating that CFTR is not di ,ءء ;p Ͻ 0.05 ,ء :statistically different from the respective control value 0.01. As for experiments on Fig. 3, cells were grown for 8Ð11 days on rectly involved in the modulation of the transporter. However, we permeable supports. Before experiments, the apical and basolateral culture could not exclude an indirect effect mediated by electrochemical medium was replaced with Krebs solution, and 100 ␮M lysine or 50 ␮M coupling. Therefore, in this study, we measured lysine and tryp- 3 3 tryptophan (1 ␮Ci/ml [ H]lysine or [ H]tryptophan) was applied to the tophan uptake on CF cells in conditions in which the transepithe- ␮ apical side of the epithelia. Aliquots of 20 and 1000 l were removed from lial potential is not clamped. Our results demonstrate that, in open apical and basolateral medium every 5 min, and the radioactivity in the circuit conditions, CF epithelia exhibit a reduced lysine and tryp- samples was determined by liquid scintillation counting to calculate the lysine/tryptophan uptake. tophan uptake compared with non-CF cells. We hypothesized that this result is due to an altered transepithelial voltage in CF. Actu- ally, the lack of functional CFTR could make the voltage more both apical and basolateral compartments. Then apical kynurenine negative by two mechanisms that could happen at the same time. is rapidly removed by the same amino acid transporter that is re- In the absence of CFTR protein, the ENaC would be hyperactive, Ϫ sponsible for the clearance of tryptophan from the apical surface in as reported previously (35Ð37). Furthermore, reduced Cl perme- ϩ resting conditions. ability will result in the lack of a shunt conductance for Na trans- A few years ago, we described the characteristics of a broad port. In both cases, the outcome would be hyperpolarization of the specificity Naϩ-dependent amino acid transport in the apical mem- epithelium that could decrease voltage-dependent amino acid brane of bronchial epithelial cells (28). This activity might be me- transport. We tested this hypothesis using selective inhibitors of diated by the recently cloned hATB0,ϩ (33), which is highly ex- ENaC and CFTR. The block of ENaC should increase amino acid 548 TRYPTOPHAN DEPLETION IN HUMAN BRONCHIAL EPITHELIUM transport by making the transepithelial voltage less negative. In- References terestingly, the treatment with the ENaC blocker amiloride restores 1. Martin, L. D., L. G. Rochelle, B. M. Fischer, T. M. Krunkosky, and K. B. Adler. the amino acid uptake on CF to levels seen in non-CF epithelia. In 1997. Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators. Eur. Respir. J. 10:2139. contrast, we treated non-CF epithelia with two CFTR inhibitors to 2. Thompson, A. B., R. A. Robbins, D. J. Romberger, J. H. Sisson, J. R. Spurzem, mimic the CF phenotype. We found that in these conditions the H. Teschler, and S. I. Rennard. 1995. Immunological functions of the pulmonary non-CF epithelium do behave as a CF tissue displaying a reduced epithelium. Eur. Respir. J. 8:127. 3. Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and amino acid uptake (see Fig. 6). Our conclusion is that membrane J. M. Wilson. 1997. Human ␤-defensin-1 is a salt-sensitive antibiotic in lung that voltage changes really influenced the apical amino acid uptake in is inactivated in cystic fibrosis. Cell 88:553. airway epithelium, and that reduced uptake may occur in CF in 4. McCray, P. B. J., and L. Bentley. 1997. Human airway epithelia express a ␤-defensin. Am. J. Respir. Cell Mol. Biol. 16:343. vivo as a result of CFTR-impaired activity. 5. Black, H. R., J. R. Yankaskas, L. G. Johnson, and T. L. Noah. 1998. Interleukin-8 The degradation of basolateral tryptophan after IFN-␥-depen- production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-␣ dent IDO induction requires tryptophan uptake into the cell. We and respiratory syncytial virus stimulation. Am. J. Respir. Cell Mol. Biol. 19:210. 6. DiMango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas have not studied the basolateral transport system(s) responsible for aeruginosa gene products stimulate respiratory epithelial cells to produce inter- this uptake. However, we can exclude that tryptophan moves leukin-8. J. Clin. Invest. 96:2204. through the same type of Naϩ-dependent transporter found in the 7. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to interferon-␥. Annu. Rev. Immunol. 15:749. apical membrane, because we previously showed that application 8. Look, D. C., S. R. Rapp, B. T. Keller, and M. J. Holtzman. 1992. Selective of basic amino acids on the basolateral side did not cause a change induction of intercellular adhesion molecule-1 by interferon-␥ in human airway epithelial cells. Am. J. Physiol. 263:L79. of the Isc (28), indicating that this transporter is not expressed in 9. Asano, K., C. B. Chee, B. Gaston, C. M. Lilly, C. Gerard, J. M. Drazen, and the basolateral membrane. Tryptophan is normally transported into J. S. Stamler. 1994. Constitutive and inducible nitric oxide synthase gene ex- Downloaded from the cells by the aromatic amino acid system T (38), by branched- pression, regulation, and activity in human lung epithelial cells. Proc. Natl. Acad. Sci. USA 91:10089. chain and aromatic amino acid transporter system L (39, 40), and ϩ 10. Watkins, D. N., M. J. Garlepp, and P. J. Thompson. 1997. Regulation of the by system y L (39). We previously found that amino acid efflux inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) from the basolateral membrane is sensitive to transstimulation or cells by nitric oxide. Br. J. Pharmacol. 121:1482. 11. Sauty, A., M. Dziejman, R. A. Taha, A. S. Iarossi, K. Neote, amino acid exchange (28), a phenomenon characteristic of systems E. A. Garcia-Zepeda, Q. Hamid, and A. D. Luster. 1999. The T cell-specific CXC ϩ y and system L; therefore, it is possible to speculate that one or chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial http://www.jimmunol.org/ both of these systems might be responsible for tryptophan uptake epithelial cells. J. Immunol. 162:3549. 12. Fujimoto, K., T. Imaizumi, H. Yoshida, S. Takanashi, K. Okumura, and K. Satoh. through the basolateral membrane. The same transporters could be 2001. Interferon-␥ stimulates fractalkine expression in human bronchial epithelial responsible for IFN-␥-dependent kynurenine secretion across the cells and regulates mononuclear cell adherence. Am. J. Respir. Cell Mol. Biol. basolateral membrane, which would be facilitated by the exchange 25:233. 13. Galietta, L. J. V., C. Folli, C. Marchetti, L. Romano, D. Carpani, M. Conese, and with tryptophan from the culture medium. O. Zegarra-Moran. 2000. Modification of transepithelial ion transport in human Amino acids are critical for cell survival. In most cases, bacteria cultured bronchial epithelial cells by interferon-␥. Am. J. Physiol. 278:L1186. 14. Shimizu, T., S. Nomiyama, F. Hirata, and O. Hayaishi. 1978. Indoleamine 2,3- would rather use amino acids from their environment than make dioxygenase. J. Biol. Chem. 253:4700. them de novo. Indeed, in nutrient-rich conditions, as is the case of 15. Liebau, C., A. W. Baltzer, S. Schmidt, C. Roesel, C. Karreman, J. B. 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Picolinic acid, a catabolite of L-tryptophan, is a costimulus for the induction of reactive nitrogen In conclusion, our work provides the first evidence that human intermediate production in murine macrophages. J. Immunol. 150:4031. airway epithelial cells maintain low apical tryptophan and kynure- 21. Munn, D. H., E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, and nine concentrations by two important mechanisms: First, under A. L. Mellor. 1999. Inhibition of T cell proliferation by macrophage tryptophan ϩ catabolism. J. Exp. Med. 189:1363. resting conditions, there is a dramatic removal through a Na - 22. Munn, D. H., M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, dependent amino acid transporter. This transporter seems to be B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, et al. 2002. Potential regu- able to remove also kynurenine, and this could be important be- latory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. 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