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Copyright #ERSJournalsLtd 2004 EurRespir J 2004;23: 327– 333 EuropeanRespiratory Journal DOI: 10.1183/09031936.03.00098803 ISSN0903-1936 Printedin UK –allrights reserved

REVIEW

Innateimmunity in the : how epithelialcells Ž ght against respiratorypathogens

R. Bals*,P.S. Hiemstra #

Innateimmunity in the lung: how epithelial cells ® ghtagainst respiratory *Deptof Internal Medicine, Division of R Bals, P S Hiemstra #ERS JournalsLtd 2004 PulmonaryMedicine, Hospital of the Uni- versityof Marburg, Philipps-University, ABSTRACT: Thehuman lung is exposed to a largenumber of airborne pathogens as a # resultof the daily inhalation of 10,000 litres of air Theobservation that respiratory Marburg,Germany; Deptof Pulmonology, LeidenUniversity Medical Center, Leiden, infectionsare nevertheless rare is testimony to the presence of anef®cient host defence TheNetherlands systemat the mucosal surface of the lung Theairway is strategically positioned at the interface with the Correspondence:P S Hiemstra,Dept of environment,and thus plays a keyrole in this host defence system Recognition Pulmonology,Leiden University Medical systemsemployed by airwayepithelial cells to respond to microbialexposure include the Center, P O Box9600, 2300 RC Leiden,The actionof the toll-like receptors Netherlands Theairway epithelium responds to such exposure by increasing its production of Fax:31 715266927 mediatorssuch as , and antimicrobial peptides Recent® ndings E-mail: p s hiemstra@lumc nl indicatethe importance of these peptides as effector molecules of innate by Keywords:Innate immunity, , cathe- killingmicroorganisms, but also as regulators of in¯ ammation, immunity and wound licidins,, epithelium, antimicrobial repair Finally,the clinical relevance of thefunctions of theairway epithelium in innate peptide immunityis discussed EurRespir J 2004;23: 327± 333 Received:August 29 2003 Accepted:October 16 2003

Thestudies were supported by grants from: DeutscheForschungsgemeinschaft the Bun- desministeriumfu ÈrBildungund Forschung, andthe Cystic ® brosis foundation,Dutch Asthmafoundation and Netherlands Organi- zationfor Scienti®c Researchand the EuropeanRespiratory Society

The integrity of the critically depends on a whichcytokines and other mediators secreted from the airway tightly regulated host defence apparatus The innate immune epitheliumare likelyto playa critical role [3] The role of system provides initialprotection against microorganisms antimicrobialpeptides, smallendogenous antibiotics with and stimulates the adaptive immuneresponse [1] Cellular proin¯ammatory, and other functions, is less clear components ofthe innate immunesystem include The aimof this review is to highlightthe role of the airway such as or , natural killercells, epitheliumin host defence and to describe new developments ,mast cells, and others Epitheliaof the inthis rapidlyevolving area of research Both the recognition humanbody form interfaces between the internal milieuand systems used byairwayepithelial cells to © sense the microbial the external environment In the respiratory tract, the world’ and the effector molecules produced inbasal condi- epitheliallining the airways is the ®rst point of contact for tions and inresponse to microbialexposure are described inhaledsubstances such as environmental pollutants, cigarette The focus inthis latter part is on antimicrobialpeptides smoke, airborne , and microorganisms [2] In recent Whereas this review is mainlydevoted to airwayepithelial years it has become clear that airwayepithelial cells not only cells, also research on other epithelia (mainlythose of the provide apassive barrier function, but also actively contribute and the intestine) that is relevant to pulmonaryresearch is to the innate immunesystem [2,3] discussed Important input to the content of this review came The innate immunefunctions of airwayepithelial cells are from the European Respiratory Society research seminar of signi®cance for the pathogenesis of avariety of human "Host defence function of the airwayepithelium " that was diseases Failureof the localhost defence apparatus may held inNoordwijkerhout, The Netherlands, 7± 8November, result inmicrobialcolonisation and subsequently of 2002 the airways and the lungparenchyma Mechanisms of the basal immunityprovided by airwayepithelial cells have a critical role inthese settings Activities of the innate immune Recognitionof pathogensby airway epithelial cells system are closely linkedto in¯ammatory processes All majordiseases of the lunginvolve mechanisms of the innate The airwayepithelium senses bacterial exposure and or adaptive immunesystem and chronic obstructive responds accordingly byincreasing its defences Thisresponse lungdisease (COPD)are chronic in¯ammatory diseases, in consists of an increase inthe release of e g antimicrobial 328 R BALS peptides into the lumenof the airways,and the release of Table 1. –Pattern recognition receptorsinvolved in the chemokines and cytokines into the submucosa that initiate an recognition of microorganismsby airway epithelial cells in¯ammatory reaction Thisin¯ ammatory reaction includes the recruitment of phagocytes, that serve to remove micro- Receptor Ligand organisms that are not cleared by the epitheliumitself, and dendritic cells and that mayaid to mount an TLR1 Tri-acyllipopeptides adaptive immuneresponse TLR2 Lipoteichoicacid, peptidoglycan, zymosan, Mechanisms to recognise pathogens by the airwayepithe- microbiallipoproteins and lipopeptides, liumare therefore considered essential to mount aprotective HSP70 (host) response ofthe innate immunesystem It has been knownfor TLR3 double-strandedRNA TLR4 LPS,HSP60 and70 (host),hyaluronic acid alongtime that cells can respond to microbialproducts such fragments (host) as lipopolysaccharide and lipoteichoic acid However, the TLR5 ¯ agellin exact mechanisms and molecules involvedin this response TLR6 di-acyllipopeptides were incompletely understood In the last decade much has TLR7 syntheticcompounds been learnt about the mechanisms that mediate this © adaptive’ TLR8 arm of the innate immunesystem Cellsof the innate immune TLR9 CpG DNA system, includingphagocytes, dendritic cells and epithelial TLR10 cells, use so-called pattern recognition molecules to bindto CD14 LPS conserved molecular patterns that are present on microorganisms CFTR LPS Pattern-recognition molecules can be present insecretions and the circulation insoluble form, such as mannan-binding TLR:Toll-like receptor; HSP: heat shock ; CpG: bacterial lectin (MBL), or they can be transmembrane molecules that deoxyribonucleicacid (DNA) containing unmethylated CpG dinulceo- tides;LPS; lipopolysaccharide; CFTR: cystic ® brosis transmembrane mediate direct cellular responses to microbialexposure The conductanceregulator; Refer to text and references 5± 8; TLR10 toll-likereceptors (TLR)constitute anintensely studied family expression:R Bals,unpublished observation of pattern recognition receptors (10 members of the human familyhave been recognised to date) that are represented detects certain structural variants of LPSsuch as leptospiral by various members inalmost allcells of the body Their LPS [15] The function of TLR3was revealed by studies of expression has been intensely studied on dendritic cells, and it TLR3knockout mice,showing that TLR3is essential inthe is nowrecognised that these, TLRs,help to shape the response to double stranded (ds) ribonucleic acid (RNA)that adaptive immuneresponse by directing the waythat dendritic is produced during viralinfections [16] TLR3has been cells instruct T-cells Airwayepithelial cells also express a implicatedin the response of epithelialcells to e g rhino- variety of TLRsthat mayhelp them to mount an adequate , rhinovirus dsRNAand synthetic dsRNA(polyinosinic- response to microbialexposure Activation of TLRon epithe- polycytidylicacid; poly(I;C)) Thisresponse consists of an lialcells has nowbeen shown to be involvedin the regulation increased expression not onlyof chemokines [6,17], but also of expression of avariety of , includingthose encoding of the human b-defensins (hBD)-2 and -3 [6] TLR9mediates cytokines, chemokines and antimicrobialpeptides the response to bacterial deoxyribonucleic acid (DNA) A Followingthe identi® cation oftollreceptors inDrosophila recent study showed that TLR9mediates the response of ¯ies as receptors involvedin the ¯y ’sresponse to microbial exposure [4],the search for mammalianhomologs led to the colonic epithelialcell lines to bacterial DNA,resulting in discovery of TLRinmammals Avariety of bacterial, fungal expression of interleukin (IL)-8 [18] and viralproducts have nowbeen identi® ed as ligandsfor It is important to note that TLRsmay also mediate the various TLRsand other pattern recognition receptors response to endogenous ligands These ligandsinclude heat- expressed by airwayepithelial cells (summarised intable 1) shock and extracellular matrix components, such as Amongthe members of the TLRfamily,TLR4 has been most fragments of hyaluronic acid that are generated during intensively studied and its role inthe response to lipopoly- in¯ammation Alsoeffector molecules of the innate immune saccharide (LPS) has been subject of alarge number of system mayserve as ligandsfor TLRs The sur- studies Studies inLPS-hyporesponsive mice were essential in factant protein A(SP-A) employs TLR4to activate murine delineating the role of TLR4in the response to LPS,and macrophages [19] Mouse b--2 is another example of demonstrated that LPS-hyporesponsiveness inmice is asso- an effector molecule of innate immunitythat activates cells ciated witha inthe signallingdomain of TLR4 via TLRs Thisantimicrobial peptide was found to activate [9± 11] Subsequent studies inhumans revealed an association dendritic cells viaTLR4, resulting inan increase inco- of selected polymorphisms inthe humanTLR4 witha stimulatory molecules and maturation [20] variety of diseases, includingGram-negative bacterial infec- Whether this is the result of aclassical receptor-ligand tions inpatients inan intensive care unit [12] TLR4 is a interaction remains to be determined [21] central component inthe response of cells to LPS LPS is Activation of TLRsmay be involvedin the regulation of bound by LPS-bindingprotein (LBP),an acute phase protein avariety of genes involvedin host defence, of whichthe that is produced not onlyby cells but also by epithelial cytokines and chemokines have been best characterised It is cells inthe lung[13] LBPserves to transfer LPSto CD14,a nowclear that TLRalso regulate the expression of anti- molecule that together withthe extracellular protein MD-2 is microbialpeptides CD14,a part of the TLR4receptor part of the TLR4complex Thiscomplex is involvedin complex,was found to be essential inthe LPS-induced recognition of LPS,and this is followedby activation of a induction of hBD-2on tracheobronchial epithelialcells [7] signallingcomplex that is associated withthe intracellular Subsequently, TLR2was found to regulate the expression of domainof TLR4and that includes the adaptor molecule hBD-2in response to bacterial lipoprotein inA549 lung MyD88 and related adaptors [5,14] (discussed inmore detail epithelialcells [22],and hBD-2and IL-8, inresponse to elsewhere inthis section) peptidoglycan and yeast cell wallparticles inhumankeratino- TLR2recognises awidearray of microbialproducts from cytes [23] TLRactivation mayalso result inan increase inthe gram-positive and negative and from fungi Like expression of TLRthemselves, whichare normallynot TLR4it is present on airwayepithelial cells Whereas TLR4 present inlarge amounts on epithelialcells Thisis illustrated appears to be the principle receptor for LPS,TLR2 also by the ®ndingthat nontypeable Haemophilusin¯ uenzae INNATE IMMUNIT IN THE LUNG 329 employs TLR2to increase its expression on bronchial epithelialcells [24] In addition,cytokines such as interferon Inflammatory Chemotactic Antimicrobial substances (INF)-c have been found to increase the epithelialexpression mediators mediators of selected TLRs[25] Cytokines LL-37/CAP-18 -defensins Elafin Chemokines -defensins LL-37/CAP-18 Calprotectin Aspeci® cresponse to microbialproducts appears to result, Leukotrienes Chemokines Lysozyme Phospholipase A2 inpart, from individualTLRs possibly havingtheir own Calprotectin Leukotrienes Lactoferrin SP-A, SP-D signallingpathways, resulting inspeci® cresponses Since SLPI Anionic peptides different cell types express distinct subsets of TLRs,the simultaneous activation of different TLRscreates aunique signalto the cells that is characteristic, both for the cell type and the micro-organism involved[5, 14] Recent studies have partly elucidated the intracellular signallingpathways acti- vated by TLRsfollowing binding of TLR-ligands The Fig 1 ±Therole of the airway epithelium in host ’sdefenceagainst commontoll-interleukin-1 receptor (TIR)-domain of TLRs infection Overviewof secreted molecules that play a rolein plays acentral role insignalling TLRsand IL-1R willbind in¯ammation and host defence Someof the depicted molecules the adaptor molecule MyD88 through the TIR-domainthat is appearto be secreted primary to the basolateral side (chemokines), also present inMyD88 MyD88 recruits the serine/threonine whereasothers are secreted to the apical side (antimicrobial peptides) kinase IL-1R-associated kinase (IRAK) that becomes phos- oftheepithelium phorylated, allowingit to associate withTRAF6 TRAF6 will mediate signallingto downstream molecules such mitogen activity or inhibitgrowth of inhaledmicroorganisms until activated protein kinases and transcription factors such as they are eliminatedby the mucociliaryapparatus, recruited nuclear factor kB Recent studies have also demonstrated the phagocytes and/or the development of an adaptive immune existence of MyD88-independent TLRsignallingpathways response Thisled to the discovery of four other adaptor molecules in Antimicrobialpeptides are effector molecules of the innate additionto MyD88:MyD88 adaptor-like (Mal; also known immunesystem of the lung Recently it was recognised that as Tirdomain-containing adaptor protein [TIRAP]);TIR- they have multipleadditional activities besides their anti- domaincontaining adaptor inducingIFN- b (TRIF; also microbialfunction The term antimicrobialpeptide refers to knownas TIR-containing adaptor molecule-1 [TICAM-1]), ribosomallysynthesised, gene-encoded peptides, meaningthat Trif-related adaptor molecule (TRAM) and sterile a- and one gene inthe codes for one peptide Different HEAT-Armadillomotifs [26] Whereas these adaptors, like groups of antimicrobialpeptides are de® ned, based on MyD88,contain aTIRdomain, they also show marked structural characteristics The defensins and cathelicidins are structural difference withMyD88 These mayallow them to the principalfamilies that are expressed inthe respiratory recruit different transducers resulting inspeci® cdown-stream tract The primary translational product is aprepropeptide signalling Althoughthe speci® cfunction of allthese newly consisting of an N-terminal signalsequence for targeting to recognised adaptors remains to be fullyclari® ed, the obvious the endoplasmic reticulum, apro segment, and aC-terminal advantage of the use of different adaptors by TLRsis to cationic peptide that has antimicrobialactivity after cleavage provide response speci® city Because components from The pro-peptide is cleaved during later stages of intracellular different pathogens willengage different TLRs,an optimal processing orafter secretion response can be generated that results inelimination of a Antimicrobialpeptides inthe humanlung are mainly speci® cpathogen In addition,it is tempting to speculate that produced and secreted by epithelialand phagocytic cells The these adaptor molecules maybe future therapeutic targets in expression and secretion of antimicrobialpeptide genes is e g sepsis therapy aimedto block pro-in¯ammatory cascades tightly regulated Some peptides are produced constitutively, initiated through TLRswhile maintaining TLR-mediated such as hBD-1or mouse b-defensin 1(mBD-1) The expres- protective responses [27] However, whether blockingadap- sion of others is increased by contact of cells withmicrobial tors that mediate pro-in¯ammatory responses is compatible products or proin¯ammatory mediators It has been shown witheffective clearance of microorganisms, and whether that expression of hBD-2,hBD-3, hBD-4, LL-37 and several selective inhibitors of the different adaptors can begenerated, other antimicrobialpeptides is induced in vitro by bacterial is not clear products and in¯ammatory mediators [31±36] These cell culture studies are con® rmed by several patient studies, showingthat the concentration of antimicrobialpeptides such Antimicrobialpeptides and proteins produced by airway as b-defensins is increased invarious body ¯uids during epithelialcells in¯ammatory or infectious diseases, such as pneumonia[37] orcystic ®brosis (38; summarised intable 2) Fromstudies in Airwayepithelial cells secrete alarge array of molecules keratinocytes it is knownthat the epithelialresponse to LPS that are involvedin in¯ ammatory and immuneprocesses [2,3, is greatly enhanced by macrophages through the production 28] Thisvariety of substances produced by airwayepithelial of IL-1 [50]and similarmechanisms mayoperate to activate cells are summarised in® gure 1 Bysecreting these mediators, hBD-2expression inpulmonary epithelial cells [51] This the airwayepithelium is capable to chemoattract and activate maybe an important mechanism for the ampli®cation of cells of the innate and adaptive immunesystem, to immobilise the response to microbialproducts, since epithelialcells and killmicroorganisms, to induce woundhealing and are markedly less sensitive to products such as LPSwhen angiogenesis inresponse to injuryand to orchestrate the compared to mononuclear phagocytes Mechanisms involved initiationof an adaptive immuneresponse Some of the inthe epithelialregulation of human b-defensin expression secreted products have direct antimicrobialactivity and likely by microbialproducts involves LPSdetection by CD14[7] act as endogenous antibiotics (® g 1) These molecules include and or lipoprotein recognition by toll-likereceptor-2 [22] smallcationic antimicrobialpeptides such as the b-defensins Furthermore, growth factors involvedin wound healing were and LL-37,but also larger antimicrobialproteins such as found to increase expression of hBD-3,human cationic lysozyme, lactoferrin and secretory leukocyte proteinase antimicrobialprotein-18 (hCAP-18)/LL-37and SLPI in inhibitor(SLPI; 29,30) These molecules displaymicrobicidal humankeratinocytes [52] Regulationof LL-37expression 330 R BALS

Table 2. –Presence of antimicrobial peptides produced by associated withCOPD suggests that these peptides are airway epithelial cellsand airway host defence cellsin human important in vivo [66] lung disease Antimicrobialpeptides have avariety of other biological effects besides their antimicrobialactivity Based on their Component Source Increasedlevels in membrane activity, antimicrobialpeptides have aconcentra- lungdisease (references) tion-dependent toxicity towards eukaryotic cells High con- centrations of a-defensins have been described insecretions of a-defensins Epithelialcells Pneumonia (39, 40) patients withcystic ®brosis [41]and chronic bronchitis [44], In¯ammatory Cystic® brosis(41) where these substances likelycontribute to the overwhelming cells in¯ammation Thismay in part be explained by the abilityof Panbronchiololitis(42) ARDS (43) a-defensins to cause of lungepithelial cells and induction Chronicbronchitis (44) of IL-8 production inthese cells [67] Furthermore, whereas Idiopathicpulmonary binding of a-defensins to proteinase inhibitors of the serpin ®brosis(45) familysuch as a1-antitrypsin ( a1-AT) mayrestrict defensin- b-defensin(BD) Epithelialcells Pneumonia (37, 46) induced cytotoxicity, it also decreases the elastase-inhibitory hBD-1 / Cystic® brosis(38, 47) activity of a1-AT [44] macrophages Besides this rather nonspeci® ctoxicity caused by high hBD-2 Dendriticclls Panbronchiolitis (48) concentrations, antimicrobialpeptides bindto cellular recep- hBD-3 tors at lowconcentrations and activate intracellular signalling hBD-4 pathways a-defensins are able to stimulate avariety of cells Cathelicidin Epithelialcells, Pneumonia (46) by mechanisms not yet identi® ed They attract humanCD4/ LL-37/hCAP-18 Neutrophils Sarcoidosis(49) CD45RA+ or CD8+ Tcells [68,69], immature dendritic cells [69],and monocytes [70] They also induce the release of IFN- ARDS:acute respiratory distress syndrome c,IL-6, and IL-10 from T-cells [71] hBD-1and hBD-2were found to bindto achemokine receptor knownas CCR-6[72] inepithelial cells depends furthermore on the differentiation Thisreceptor is found on immature dendritic and memory status ofthe cells [53,54] T cells (CD4+/CD45RO+ )and consequently these ®ndings Antimicrobialpeptides have abroad spectrum activity are interpreted as alinkbetween innate and adaptive immune against Gram-positive and Gram-negative bacteria as wellas mechanisms mediated bydefensins hBD-3and hBD-4chemo- against fungi and enveloped [55] Antimicrobial attract monocytes by mechanisms that have not yet been peptides show synergistic activity withother host defence clari® ed [36,73] Cathelicidins maydisplay similar activities molecules, such as lysosyme and lactoferrin The antimicro- as defensins, since LL-37binds to formyl peptide receptor like bialactivity is based oninteractions between the peptide and 1[74]and attracts neutrophils, monocytes, and CD4T cells surface membranes of the target organisms Functionalstudies and activates mast cells [75] It is interesting to note that on antimicrobialactivity have primarilybeen restricted to whereas antimicrobialpeptides mayact as chemokines, in vitro experiments using puri® ed components Recently, the reverse has also been observed since several intact or several groups published results that provided proof of the truncated forms of chemokines displayantimicrobial activity host defence function of antimicrobialpeptides inliving [76] In additionto these roles of defensins and LL-37in organisms Indirect in vivo evidence for the host defence in¯ammation and immunity,defensins and LL-37have function of antimicrobialpeptides came from astudy on mice also been implicatedin wound repair processes witha disrupted gene for matrilysin,also called matrix metallo- a-defensins were found to cause proliferation of airway protease 7 Matrilysin-de® cient mice were more susceptible to epithelialcells and mediate epithelialwound repair [77], infections withenteropathogens [56] Mice de® cient inan whereas b-defensins maypromote differentiation of kerati- antimicrobialpeptide, mBD-1,revealed delayed clearance of nocytes [78] In linewith this, LL-37is involvedin re- Haemophilusin¯ uenzae from lung[57] Mice withdeleted epithelialisation of cutaneous wounds [79],and induces cathelin-related antimicrobialprotein-18, the murine homo- angiogenesis in vitro and in vivo [80] logue of LL-37,showed more prominent infection after Despite the progress that has been made inthe under- cutaneous inoculationof bacteria [58] Conversely, the standing ofthe basic biologyof antimicrobialpeptides, their overexpression of LL-37by viralgene transfer resulted in role inhuman disease is stillincompletely understood Based augmentation of innate host defence ina bronchial xenograft on their direct host defence and various receptor-mediated modelof cystic ®brosis and inmurine animalmodels of functions, antimicrobialpeptides could have acentral role in pneumoniaand septic shock [59,60] In addition,over- infectious and in¯ammatory diseases Table2 summarises expression of adefensin ina transgenic murine model disease conditions that are characterized by altered concen- provided protection against enteric salmonellosis [61] trations ofantimicrobialpeptides Several lines of evidence indicate the importance of Antimicrobialpeptides have emerged as effector substances antimicrobialpeptides inthe humanlung Usingculture of the innate immunesystem involvingnot onlyactivities as systems of airwayepithelial cells from patients withcystic endogenous antibiotics but also as mediators of in¯amma- ®brosis, it was demonstrated that epithelial-cell derived tion Further analysis ofthe biologicallyrelevant functions of antimicrobialpeptides maybe inactivated by the highsalt antimicrobialpeptides willreveal the role that these molecules concentration inthe epitheliallining ¯ uid[62, 63] Studies in a have indiseases of the lung Here, antimicrobialpeptides humanbronchial xenograft modelrevealed decreased anti- mightcontribute to the development of diseases not onlyas microbialactivity of airwaysurface ¯uidafter inhibitionof endogenous host defence substance but also as pro- or anti- hBD-1synthesis by antisense oligonucleotides [63] Other in¯ammatory mediators Development of antimicrobialpep- evidence for the importance of antimicrobialpeptides comes tides as drugs involves optimized strategies for candidate from studies such as those showingthat their levels are identi® cation, for modi®cation of pharmacodynamic and changed inin¯ ammatory lungdisease (table 2;64), and the pharmacokinetic pro® les and for production Studying the ®ndingthat nasal Staphlococcus aureus carriage is associated biologyof antimicrobialpeptides should allowthe develop- withdecreased antibacterial activity of nasal ¯uid[65] ment of novel therapeutics for infectious or in¯ammatory Finally,also the observation that hBD-1polymorphisms are diseases INNATE IMMUNIT IN THE LUNG 331

Conclusions lipopolysaccharide:evidence for TLR4 as theLps gene product J Immunol 1999; 162: 3749 It is evident that there isarenewed and increased interest in 12 AgneseDM, Calvano JE,Hahm SJ, et al Human toll-like innate immunitythat has intensi® ed studies on the role ofthe receptor4 mutationsbut not CD14 polymorphismsare airwayepithelium that forms the primary interface between associatedwith an increasedrisk of gram-negative infections the environment and the host The epitheliumcontributes to J Infect Dis 2002; 186: 1522± 1525 host defence ina number of ways,including ciliary beat 13 DentenerMA, Vreugdenhil ACE, Hoet PHM, et al activity and mucus production, but also production of Productionof theacute-phase protein lipopolysaccharide- bindingprotein by respiratorytype IIepithelialcells chemokines, cytokines, antimicrobialpeptides, proteinase Implicationsfor local defense to bacterialendotoxins Am inhibitors and surfactant proteins The recognition systems JRespirCell Mol Biol 2000; 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