Permeability and Modulation of the Intestinal Epithelial Barrier
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Permeability andmodulatio n ofth e intestinal epithelial barrier invitr o ErwinDuize r Promotores Dr.J.H .Koema n Hoogleraar in de Toxicologic Landbouwuniversiteit Wageningen Dr.P.J . van Bladeren Bijzonder hoogleraar ind eToxicokinetie k en Biotransformatie Landbouwuniversiteit Wageningen enTN OVoeding ,Zeis t Co-promotor Dr.ir .J.P .Grote n Hoofd Afdeling Verklarende Toxicologic TNOVoeding ,Zeis t ^6SS pMo^1 \ Permeability andmodulatio n ofth e intestinal epithelial barrier invitr o Erwin Duizer Proefschrift ter verkrijging van de graad van doctor op gezag van derecto r magnificus, van de Landbouwuniversiteit Wageningen, dr. C. M. Karssen in het openbaar te verdedigen opwoensdag 16juni 1999 desnammidag s te vier uur in de Aula. Permeability and modulation of the intestinal epithelial barrier in vitro Erwin Duizer ThesisWageninge n Agricultural University, The Netherlands - Withreferences , -Wit h summary in Dutch ISBN 90-5808-067-6 Subject headings: intestinal epithelium / Caco-2/ IEC-18/ paracellular Cover: Confocal laser scanning microscopy image of filter-grown IEC-18 cells labeled witha polyclonal anti-ZO-1antibod y and visualized with aTRITC-conjugate d streptavidin- biotin system. Print: ADDIX,Wij k bij Duurstede The investigations described in this thesis were carried out at theToxicolog y Division of the TNONutritio n and Food Research Institute (Zeist, Netherlands). Financial support for the publication of this thesis waskindl y provided by TNONutritio n and Food Research Institute andth e Dutch Alternatives to Animal Experiments Platform. ^MO^'i^55 STELLINGEN behorende bij het proefschrift "Permeability and modulation of the intestinal epithelial barrier invitro " vanErwi n Duizer, te verdedigen op woensdag 16jun i 1999,o m 16.00uu r teWageningen . 1 Voor het modeleren van de paracellulaire permeabiliteit van de dunne darm voor hydrofiele macromoleculen voldoet het twee-compartimenten systeem beter als het wordt uitgerust met dunne darm cellen (IEC-18) dan met, van oorsprong, dikke darm cellen (Caco-2) (ditproefschrift). 2 Veranderingen in de transepitheliale elektrische weerstand (TER) zijn geen goede kwantitatieve indicatie voor veranderingen in de transepitheliale permeabiliteit voor macromoleculen (ditproefschrift). 3 Het voorspellen van de orale bio-beschikbaarheid van een stof, ongeacht de absorptie route, aan de hand van een enkel in vitro model is nu nog niet mogelijk (dit proefschrift). 4 Cadmium heeft enkeleeigenschappe n gemeenme td e ideale "drug absorption enhancer" (ditproefschrift). 5 Het gebruik van "permeation enhancers" kan gemakkelijk leiden tot een verhoging van hetaanta l mensenme t voedselallergie. (Pena and Crusius. Food allergy, coeliac disease and chronic inflammatory bowel disease in man. Vet. Q. 1998;20Suppl3:S49-52. Ahmed and Fuchs.Gastrointestina l allergy to food: areview .J . Diarrhoeal Dis.Res . 1997;15:211-23). 6 Dat verminderde gluthation peroxidase activiteit door Selenium-deficientie kan leiden tot enterovirale myocarditis, is eerder toe te schrijven aan een verhoging van de virulentie van de enterovirussen dan aan een verminderd functioneren van het immuunsysteem. (Beck etal. Increased virulence of a human enterovirus (Coxsackievirus B3) in selenium-deficient mice. J. Inf. Dis. 1994;170:351-357. Beck etal. Glutathione peroxidase protects mice from viral-induced myocaritis. FASEB J. 1998;12:1143- 1149). 7 Inee n celkweek laboratorium ise ree npositiev ecorrelati etusse n de reproduceerbaarheid van deexperimente n enhe talcoho l gebruik. (Ojeh etal. Evaluation of the effects of disinfectants on rotavirus RNA and infectivity by the polymerase chain reaction and cell-culture methods.Mol .Cel l Probes 1995;9:341-346). 8 De angst dat chimpansees een reservoir zullen vormen voor levend poliovirus en daarmee de eradicatie van polio in gevaar brengen is ongegrond, het is waarschijnlijker datd echimpansee s eerderzij n ge-eradiceerd danhe tpoliovirus . (Dowdle and Birmingham. The biological principles of poliovirus eradication. J. Infect. Dis. 1997;175:S286-S292). 9 Eenturne r staathe tliefs t nietme tbeid ebene no pd egrond . 10 Voorzowe lcabare tprogramma' sal swetenschappelijk e artikelengeld t "gebruik altijd de teksten van een ander tenzij die van jezelf beter zijn" en "schrijven isschrappen" . (Herman Finkerse ndit proefschrift). In dankbare herinnering, voor mijn vader Agood place tobegin is at the beginning, because otherwise things canget confuzzled. Winnie-the-Pooh Contents SectionI :Introductio n andMethodolog y Chapter1 GeneralIntroductio n 3 § 1.1 Introduction 4 §1.2 Transepithelial permeation routes inth e intestine 5 § 1.3 Thetranscellula r barriers ofth eintestina l epithelium 8 § 1.4 Theparacellula r barrier of the intestinal epithelium 10 § 1.5 Tight Junctions: The gate-keepers of theparacellula r barrier 11 § 1.6 Modulation of the intestinal epithelial barrier: Therelationshi p between increased permeability andtoxicit y 13 § 1.7 Intestinal epithelial cell lines asmode l for the intestinal epithelium: Paracellular permeation prognosis beyond Caco-2 15 §1.8 Objectives andApproac h 17 Chapter2 Methodology 19 Application of epithelial cell culture systems to investigate intestinal permeability andpermeabilit y modulation §2.1 Thetwo-compartmen t cell culture system 19 § 2.2 Theintestina l epithelial cellline sCaco- 2an d IEC-18 22 § 2.3 Methods to assess the functionality ofth e intestinal epithelium in vitro §2.3. 1 Measurement andmeanin g of the transepithelial electricalresistanc e (TER) of filter grown cells 23 §2.3.2 Assessment of transepithelial permeation rates 23 § 2.3.3 Qualitative Immunofluorescence localization ofjunctiona l proteins 24 § 2.3,4 Quantification ofjunction-relate d ZO-1usin g confocal scanning lasermicroscop y and imageprocessin g 25 § 2.3.5 Assessment of adverse effects on the intestinal epithelium in vitro in studies toth e modulation of transepithelial permeability 26 SectionII :Permeabilit y ofth eIntestina l Epithelial BarrierIn Vitro Chapter 3 Comparison ofpermeabilit y characteristics of thehuma n colonic Caco-2 and thera t small intestinal IEC-18cel l lines 31 Chapter4 Carrier-mediated transport inra t small intestinal IEC-18cells .Compariso n withth ehuma n colon carcinoma Caco-2 cell line 45 Chapter5 Dexamethasone inhibits cell proliferation andenhance s theparacellula r barrier function ofra t ilealIEC-1 8cell s 59 SectionIII : Modulation ofth eIntestina l Epithelial BarrierIn Vitro Chapter6 Absorption enhancement, structural changes in tightjunction s and cytotoxicity caused bypalmitoy l carnitine inCaco- 2 andIEC-1 8 77 Chapter7 Effects of cadmium chloride on the paracellular barrier function of intestinal cell lines 95 SectionIV :Summarizin g Discussionan d Conclusion Chapter8 Summary andConcludin g remarks 115 §8.1 Transepithelial permeability of the filter grown Caco-2 andIEC-1 8cell s 116 § 8.2 Modulation of the intestinal epithelial barrier: increased permeability and toxicity in cell culture systems 121 §8.3 Conclusions andPerspective s 126 References 129 Samenvatting 139 Curriculum Vitae 149 List ofpublication s 150 Dankwoord 151 SectionI Introduction and Methodology Chapter 1 Introduction Chapter 1 General Introduction Manuscript based on the chapters 1,2 an d 8i n preparation; tob e submitted toATLA . Ina challengin g effort togai na bette r insight inora lbioavailabilit y offoo d components, toxicants, anddrugs , we studied the absorption andbarrie r characteristics of two intestinal cell lines,Caco- 2an dIEC-18 ,culture d ina two-compartmen t system.W ewondere d whetherth era t small intestinal cell line IEC-18 could provide a better model than the Caco-2 cell line, in obtaining in vitro permeation rates to predict oral bioavailability in vivo, for non-nutrient hydrophilic molecules. Secondly,i norde rt ogai ninsigh ti nth emechanism sb ywhic hth eintegrit yo fth eepithelia l barrier may be compromised, thefilter-grown cell s are exposed to a drug absorption enhancer (palmitoyl carnitine) and to apossibl e food contaminant (cadmium). In these experiments the relationbetwee n increased permeability for hydrophilic macromolecules, analtere d paracellular barrier structure, andth e viability of theepithelia l cells was assessed. Chapter 1 Introduction § 1.1 Introduction Thebioavailabilit y ofal lingeste dcompound si st oa grea texten ddetermine db yth eabilit y ofthes ecompound st opas sth eintestina lepithelium .A hig hbioavailabilit y isguarantee dfo rmos t nutrientsan delectrolyte s sincethe yar eactivel y absorbed byth eepithelium .Th esam eepithelium , however,present sa nalmos timpermeabl ebarrie rt onon-nutrien t (potentiallyharmful ) hydrophilic (macro-) molecules,viruse s and bacteria, rendering their entrance into the systemic circulation very low.T operfor m itsfunctio n asa selectiv e absorber (or a selective barrier) onth e interface between the intestinal lumen and the internal host milieu, the intestinal epithelium has evolved intoa highl y specialized structure.Th esingl ecel llaye rformin g theintestina lepitheliu m consists of several cell types,eac h with a specific morphology and function. Forexample ,th e intestinal epithelial absorptivecells ,th eenterocytes ,ar epolarize d toallo wvectoria l transport,the y posses microvilli toenlarg eth eabsorptiv e area,an dth ecell sar eseale dtogethe rb ya junctiona l complex tomaintai na barrie ri norde rt oavoi dabsorptio n ofpotentiall y harmful compoundsan dt opreven t back-flow of theactivel y absorbed nutrients. Thecomplexit yo fth egastrointestina l tract,an