Lysophosphatidylcholine increases rat permeability to

Gut, 1985, 26, 369-377

Lysophosphatidylcholine increases rat ileal permeabilityto macromoleculesC TAGESSON, L FRANZEN, G DAHL, AND B WESTROM

From the Departments of Clinical Chemistry, Occupational Medicine, and Pathology II, LinkopingUniversity, and Department ofZoophysiology, University of Lund, Sweden

SUMMARY The influence of lysophosphatidylcholine (LPC) on macromolecular permeability inthe distal ileum has been studied. Using a rat experimental model, we determined the intestinalpermeability to different sized dextrans (3000-70 000 daltons) and bovine serum albumin (BSA)in the absence and presence of LPC. We also examined the morphology of the ileal mucosa afterdeposition of LPC in the gut lumen, and determined N-acetyl-Pi-glucosaminidase, 5'-nucleotidase, and alkaline phosphatase activities in suspensions of isolated mucosal cells anddifferent concentrations of LPC. We found that 20 mM LPC damaged the ileal mucosa and that itincreased its permeability to all the molecules investigated. Moreover, mixtures of mucosal cellsand 0.01-1 mM LPC showed increased N-acetyl-,-glucosaminidase activity: the higher the LPCconcentration, the higher the enzyme activity. These findings indicate that LPC, a naturallyoccurring surfactant in the intestine, might damage mucosal cells and release lysosomal enzyme

activity, and that higher LPC concentrations may impair the mucosal barrier function andincrease the gut permeability to macromolecules such as proteins. This could have relevance tothe development of various disease states, in which increased intestinal absorption ofmacromolecules is of importance.

Intestinal absorption of macromolecules may be ofcritical importance to the pathogenesis of manydiseases, especially those involving allergic reactionsto ingested foreign substances or toxic reactions tobacterial and parasitic products.1 Little is known,however, about the mechanisms by which theabsorption of macromolecules could be facilitated invarious disease states. Chadwick et a!2 recentlyshowed that some of the naturally occurring bileacids in man can alter rabbit colonic structure andfunction, and that bile acid induced fluid secretionwas invariable associated with epitheliolysis andincreased mucosal permeability. Alternatively, wehave found that another naturally occurringdetergent, lysophosphatidylcholine (LPC), canimpair the mucosal barrier in the distal part of ratileum and thus enable larger molecules such asdextran 3000 to traverse the intestinal wall.3 Thedetailed mechanisms behind this effect are notknown, however, and it is therefore unclear whetherLPC may increase also the permeability to largerAddress for correspondence: Christer Tagesson, MD, Department ofOccupational Medicine, Linkoping University, S-581 85 Linkoping, Sweden.

Received for publication 1 June 1984

macromolecules such as proteins.The present study was conducted with a view to

obtain more detailed information concerning howLPC might influence intestinal permeability tolarger molecules. We therefore investigated theinfluence of LPC on rat intestinal permeability tolarger dextrans (3000-70 000 daltons) and to bovineserum albumin, a 70 000 dalton protein. In addition,we examined the morphology of the ileal mucosaafter deposition of LPC in the bowel lumen, anddetermined N-acetyl-a-glucosaminidase, 5'-nucleo-tidase, and alkaline phosphatase activities inmixtures of isolated mucosal cells and LPC. Theresults indicate that LPC may act as a detergent andrelease lysosomal enzymes from mucosal cells invitro, and that higher LPC concentrations increasethe shedding of enterocytes at the villous tips andaugment the mucosal permeability to dextran 70 000and bovine serum albumin.

Methods

CHEMICALSFluorescein-isothiocyanate (FITC) conjugated

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Tagesson, Franzen, Dahl, and Westrom

dextrans with molecular weights of 3000 and 10 000daltons were purchased from Pharmacia FineChemicals, Uppsala, Sweden, whereas FITC-dextran 70 000 was bought from Sigma ChemicalCompany, St Louis, Missouri, USA. Lyso-phosphatidylcholine (1-acyl-sn-glycero-3-phosphocholine) was Sigma type I, whereas bovineserum albumin was Sigma type V.

PERMEABILITY MEASUREMENTSAll experiments were carried out on 225-250 gfemale Sprague-Dawley rats maintained on astandard laboratory diet. They were anaesthetisedby an intraperitoneal injection of Ketalarg (Parke,Davis, Pontypool, UK; 50 mg/kg body weight) andRompung (Bayer AG, Leverkusen, West Germany;5 mg/kg body weight). A polyethylene catheter (0 =0-96 mm) was placed in one of the carotid arteries.Thereafter, a 10 cm segment of the distal ileumimmediately proximal to the caecum was convertedto a tied loop. Before ligating the proximal end, aflexible cannula with an injection valve was put intothe segment through a separate insertion. Thepermeability markers (FITC-dextran 3000, 10 000,or a mixture of FITC-dextran 70 000 and bovineserum albumin) were dissolved in 1 ml 150 mMNaCl together with (or without) 20 mM LPC or 1%Triton X-100, and instilled into the loop. Bloodsamples (200 ,ul) were withdrawn from the carotidartery before instillation and at intervals thereafter.The samples were analysed for FITC-dextrans and/or bovine serum albumin.To determine the concentration of FITC-dextran,

the sample was mixed with 3*8 ml Tris (50 mM)-NaCl (150 mM), pH 10-3, and centrifuged at 2800 gfor five minutes. The supernatant was then analysedfor FITC-dextran by using fluorescence spectro-metry as described elsewhere.3 Bovine serumalbumin in serum was determined by electro-immunoassay using crystallised bovine serumalbumin (Sigma type A 7638) as standard.4 Specificantiserum to bovine serum albumin was obtainedfrom Miles Laboratories (Freehold, USA).

MORPHOLOGYTied loops prepared as above were either leftunfilled or filled with 1 ml 150 mM phosphatebuffered saline (PBS), 20 mM LPC in 1 ml PBS, or1% Triton X-100 in 1 ml PBS. After 30 minutes, 300ml 2% glutaraldehyde in 0-1 M Na-cacodylate-HClwith 0*1 M sucrose and 2% polyvinylpyrrolidone(PVP, molecular weight 44 000 daltons) wasperfused via the heart.5 6 The perfusion wasconducted during 10 minutes at a constanthydrostatic pressure of 110 mmHg. The pH of thefixative was 7-2, its temperature 37°C and its total

osmolality about 560 mOsm (vehicle osmolalityabout 300 mOsm).For light microscopy, five 2 mm thick segments

were sampled from each loop and immersed in theabove fixative for another three days at 4°C. Thespecimens were then dehydrated, embedded inSorwall JB4 resin, cut into 3 Am thick sections andstained with either haematoxylin eosin,haematoxylin van Gieson, or 0.5% toluidine blue atpH 4.The morphological changes in villous tips were

quantitatively assessed by examining intestinalcircumferences in sections stained withhaematoxylin van Gieson. Tips were thus classifiedinto three categories according to their morpho-logical appearance: (i) normal morphology withregular columnar epithelium, (ii) moderatelychanged morphology with loss of columnarcharacteristics, partial desquamation, karyolysis andkaryorrhexis, and (iii) severely changed morphologywith patchy desquamation of epithelium leavingdenuded areas of basal membrane. A mean of 36villi (range 22-69) were examined in each circum-ferential section.For transmission electron microscopy, two 1 mm

wide segments of each loop were immersed in theabove fixative for another three days at 4°C. Three 1mm cubic pieces were taken from each segment,rinsed in 0-15 M Na-cacodylate-HCI buffer (CAC)and postfixed in 1% OSO4 in 0.15 M CAC. Afteranother rinse in 0-15 M CAC, the specimens were'stained' in 2% uranyl acetate in 50% ethanol overnight. After dehydration in a graded ethanol seriesand embedding in Epon 812, the specimens were cutwith diamond knives on a LKB III ultratome,stained with lead citrate and examined in a Philips300 electron microscope at 60 kV.

INFLUENCE ON MUCOSAL CELLS IN VITROThe distal ileum (about 20 cm) was excised and thefaecal contents removed. The mucosa was thenscraped off with a curette. Special precaution wastaken to scrape only the superficial layers of themucosa. The cells were suspended in Krebs-Ringerphosphate buffer containing 10 mM glucose (KRG)and 0-5% (w/v) trypsin to a concentration of 15 mgtissue/ml. This suspension was subjected tomagnetic stirring for 30 minutes, Vortex-mixed for 1minute, and filtered through gauze. The filtrate wascentrifuged (800 g, 10 min), the supernatantdiscarded, and the pellet resuspended in KRG. Onehundred microlitres of this cell suspension was thenmixed with 100 ,u LPC (final concentration 0*01-1mM), and the mixture incubated at roomtemperature for 30 minutes. The activities ofalkaline phosphatase,7 5'-nucleotidase8 9 and N-

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Lysophosphatidylcholine and ileal permeability

acetyl-fS-glucosaminidase8 10 were then determined.To study the influence of LPC on non-lysosomalN-acetyl-13-glycosaminidase, the mucosal cells weredisintegrated in a glass homogenizer, subjected tothree freeze-thawing cycles, and centrifuged at30 000 g for 30 minutes. The supernatant was thenwithdrawn and used for further experimentation.The material was incubated with varying concen-tration (0.001-10 mM) LPC as described above, andthe activity of N-acetyl-,3-glucosaminidasedetermined.

STATISTICAL DETERMINATIONSStatistical analysis were carried out with the use ofStudent's t test.

Results

INFLUENCE OF LPC ON INTESTINAL PERMEABILITYThe intestinal absorption into blood of differentsized dextrans in the absence and presence of LPC isillustrated in Figure 1. After deposition in theabsence of LPC, only minute amounts of dextran3000 were found in the circulation, whereas in thepresence of LPC considerable amounts were found.Lysophosphatidylcholine also enhanced the transferof dextran 10 000, although as shown in Figure 1,the transfer of dextran 10 000 was less than that of

81

FITC - Dextran(nmol/ml blood)44

dextran 3000. For comparison, the influence ofTriton X-100, a non-ionic detergent, on thepermeability to dextran 3000 and 10 000 is alsoshown in Figure 1. Thus, like LPC, Triton X-100significantly augmented the permeability both todextran 3000 and 10 000 (Fig. 1, right).The influence of LPC on the transfer of dextran

70 000 and bovine serum albumin is illustrated inFigure 2. We thus found that the presence of LPCsignificantly increased the absorption also of thesetwo compounds.

INFLUENCE ON INTESTINAL MORPHOLOGYThe operative procedure by itself had no apparentinfluence on the morphology of the intestine.Instillation of PBS (with or without LPC or TritonX-100) caused a slight distention of the loop and aflattening of the mucosa with broadening of the villi.No apparent infiltration of inflammatory cells wasfound in the mucosa after either treatment.Phosphate buffered saline alone had no evidenteffect on epithelial morphology as seen by lightmicroscopy (Fig. 3) or by transmission electronmicroscopy. On the other hand, instillation of LPCor Triton X-100 caused profound changes of theepithelium along most of the villous tips (Fig. 3).These changes included loss of columnar character-istics and varying degrees of karyorrhexis and

Gex-.-.a- :A: nE---o8 12 0

Time (min)4 8 12

Fig. 1 Influence of20mM LPC (left) and l %Triton X-100 (right) on intestinal permeability to FITC-dextran 3000 ()and 10 000 (O). Permeability in the absence ofLPC and Triton X-100, respectively, is indicated by open symbols ((O)FITC-dextran 3000, (O) FITC-dextran 10 000). The ordinate shows the blood concentration at different times afterdeposition of3-3 mM FITC-dextran in the distal ileum. Mean offive experiments, vertical bars indicate standard error of themean.

0 4=- } s @ -m~19i'Ar-ir-m --l -=Tl

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Tagesson, Franzen, Dahl, and Westrom

FITC Dextmn 70000(,umol /ml blood )

0

Time (hours)

BSA(,umol /ml blood)

Fig. 2 Intestinal permeability to FITC-dextran 70 000 (left) and bovine serum albumin (right) in the absence (O) andpresence (0) of20mM LPC. The ordinate shows the serum concentration at different times after deposition ofa mixture ofFITC-dextran 70 000 (50 mglml) and bovine serum albumin (50 mglml) in the distal ileum. Mean of6-10 experiments;vertical bars indicate standard error ofthe mean.

karyolysis. Furthermore, both partial desquamationwith cells still sticking to the remaining coveringepithelium and total desquamation were seen.When the degree of morphological changes in thevillous tips was quantitatively assessed, theepithelial damage after instillation of Triton X-100was found to be more pronounced than that afterinstillation of LPC (Table).

Ultrastructurally, the above findings wereverified. A reduction in number of microvilli,

Table Quantitative assessment of the influence ofLPCand Triton X-100 on intestinal morphology. The Tableshows the percentage of villous tips showing differentdegrees ofmorphological changes after exposure to thevarious agents. Moderate changes include loss ofcolumnarcharacteristics, karyorrhexis, karyolysis orpartialdesquamation, whereas severe changes represent totaldesquamation ofepithelium leaving some areas of the basalmembrane denuded. A mean of36 villi (range 22-69) wereexamined in each intestinal circumference. Values are mean± SEM

Circum- Morphological changesferences

Agent counted (no) None Moderate Severe

None 10 95-0±1-5 5-0±1-5PBS 10 91-0±4-4 9 0±4 4LPC (20mM) 10 11-7±3-0 80-9±2-8 7 5±3 5TritonX-100(1%) 9 1.2±0.9* 59.6+7.4* 39-2±7-7t* p<001 vs LPC treatment. t p<0-001 vs LPC treatment.

swelling of mitochondria and cytoplasmicvesiculation were noted after treatment with eitherLPC (Fig. 4a) or Triton X-100. Furthermore, TritonX-100 caused more pronounced damage with totalepithelial desquamation of many villous tips (Fig.4b). Tight junctions were sometimes opened upbetween clearly degenerating cells still sticking tothe basal membrane, but not between normal oronly slightly damaged cells.

INFLUENCE ON MUCOSAL CELLS IN VITROThe influence of LPC on activities of alkalinephosphatase, 5'-nucleotidase, and N-acetyl-13-glucosaminidase from isolated mucosal cells in vitrois illustrated in Figure 5. Thus, mixtures of mucosalcells and increasing LPC concentrations showedincreased enzyme activity: the higher the LPCconcentration, the higher the enzyme activity. In thepresence of 1 mM LPC, the N-acetyl-i-glucosaminidase was increased 4.8 times over thatattained in the absence of LPC, whereascorresponding values for alkaline phosphatase and5'-nucleotidase were 1*7 and 1.5, respectively. Bycontrast, there was no significant influence of 1,uM-10 mM LPC on the N-acetyl-,3-glucosaminidasein a 30 000 g supernatant fraction of disintegratedmucosal cells.

Discussion

Altered intestinal permeability to food constituents

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C _Fig. 3 Photomicrographs of intestinal villi exposed to (a) PBS, (b) 20mM LPC, and (c) 1% Triton X-100. (Weigert'shaematoxylin-eosin; 570). In (a) the villus is covered with regular columnar epithelium whereas in (b) the epithelium isdegenerated and partially desquamated - that is, some cells are still sticking to the remaining epithelium. A totaldesquamation ofepithelium with denuded basal membrane is seen in (c) (arrows). (The dilated capillaries under the basalmembrane of villous tips in (a) and (b) are due to the perfusion fixation).

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Fig. 4 TEM micrographsfrom villous tips exposed to (a)20mM LPC, and (b) 1%Triton X-100. Note in (a)severely damaged enterocyteswith distorted microvilli (M),numerous cytoplasmicvacuoles, and dilatedmitochondria with disruptedcristae. At the bottom of (a) areseen a nucleus (N) and thebasal membrane (BM). In (b),denuded basal membrane(BM) is seen. Note also in (b)dilated capillary (C) beneathbasal membrane (effect ofperfusion fixation) .

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Relativeactivity 3

0 0.01 01 1

Lysophosphatidylcholine (mM)

Fig. 5 Activities ofalkaline phosphatase (a),5'nucleotidase (0), and N-acetyl-3-glucosaminidase (A) inmixtures of rat intestinal mucosal cells and different LPCconcentrations. The ordinate shows enzyme activity relativeto the activity in the absence ofLPC. Mean ofthreeexperiments; vertical bars indicate standard error of themean.

and microbial products may underlie the patho-genesis of a variety of diseases. Accordingly,increased absorption of macromolecules has beenclaimed to occur in coeliac disease, chronic inflam-matory bowel disease, IgA deficiency, food allergy,various liver diseases, and others. 11-5 Little isknown, however, about the mechanisms by whichintestinal uptake of macromolecules might befacilitated in these disease states. Although certainbile acids,2 16 17 synthetic detergents,'8 andtraumatic injury19 all have been shown to increasegut permeability to macromolecules in variousexperimental systems, their role in the developmentof gastrointestinal disease has not been established.Analogously, we recently found that LPC increasedthe permeability to dextran 3000,3 but the biologicalsignificance of this finding is as yet unclear.The present study shows that LPC may increase

the intestinal permeability not only to dextran 3000,but also to dextran 10 000 and, indeed, 70 000. Italso shows that LPC may augment the intestinalabsorption into blood of bovine serum albumin, a70 000 dalton protein (Fig. 2). Moreover, LPCapparently released considerable amounts oflysosomal enzyme from mucosal cells in vitro (Fig.5), and increased the shedding of enterocytes fromvillous tips in vivo (Fig. 3). These findings indicatethat LPC might damage mucosal cells and impair theintestinal barrier function in the distal part of the

ileum. As a consequence, the intestinal mucosa mayallow permeation of potentially antigenic, toxic orcarcinogenic material.The mechanism by which LPC may damage the

intestinal mucosa has not been elucidated. Wefound, however, that much the same or even greaterinfluence than LPC on mucosal structure andpermeability was obtained with Triton X-100, anon-ionic detergent. It can be hypothesised,therefore, that the observed effects of LPC can beattributed to its detergent properties. Detergentscan alter put permeability20 and damage cellmembranes although the exact mechanism bywhich they interact with membranes is only incom-pletely understood. If LPC-protein interactions aredisregarded, increasing LPC concentrations in thephospholipid bilayer will lead to either a generallyenhanced membrane permeability by increasedmembrane 'fluidity', or to the formation of actualpores.22 Also partial micellisation and solubilisationof membrane lipids have to be considered. Bothreactions will finally lead to free ion permeation andsubsequent osomotic rupture of the cell.Analogously, if the binding affinity of LPC to someintegral membrane proteins are high, the accumula-tion of lysolipid around proteins might result in anextraction of proteins into micelles and, again,formation of 'pores' in the membranes.22 This couldexplain why LPC 'released' N-acetyl-fi-glucosaminidase, a membrane-bounded lysosomalenzyme from the mucosal cells, but had littleinfluence on brush border enzymes such as alkalinephosphatase and 5'-nucleotidase (Fig. 5).We have thus obtained some evidence to indicate

that LPC, a naturally occurring detergent, mayimpair the mucosal barrier in the rat ileum and sofacilitate the intestinal transmission of macro-molecules. The relevance of this finding to thedevelopment of human disease states remains to beestablished. Ammon et a123 recently showed thatLPC affects rat intestinal transport in the same wayas bile acids, fatty acids and synthetic cationic ornonionic detergents, and argued, by comparisonwith the response of the human jejunum to tauro-deoxycholate, that it is likely that LPC generatedduring the normal process of digestion has asignificant influence on intestinal transport in thepostprandial phase under physiological conditions.The postprandial concentrations of LPC in thehuman jejunum range from 2 mM to 4 mM24 25whereas little is known about LPC concentrations inthe distal part of the small intestine. It cannot beruled out, however, that higher concentrations ofLPC might occur, particularly if the mucosa is lesscapable than normal to remove LPC. We recentlyfound that the activity of lysophospholipase was

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376 Tagesson, Franze'n, Dahl, and Westrom

decreased in ileal mucosa of patients with Crohn'sdisease and suggested that the mucosa could be lesswell equipped to handle LPC and, therefore,predisposed for mucosal injury induced by LPC.26This possibility should not be entirely overlookedbecause LPC is generated in inflammatory foci,27has a range of cytotoxic and non-cytotoxic effects ondiverse cells2832 and, as shown in this investigation,may enhance the mucosal permeability to poten-tially antigenic and toxic molecules.

We acknowledge gratefully the excellent technicalassistance of Berit Johansson and Rita Grander.This work is supported by grants from the NationalSwedish Association against Rheumatism, theSwedish Natural Science Research Council, and theSwedish Medical Research Council (B 83-17X-05983-03B).

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3 Bolin T, Sjodahl R, Sundquist T, Tagesson C. Passageof molecules through the wall of the gastrointestinaltract. Increased passive permeability in rat ileum afterexposure to lysolecithin. Scand J Gastroenterol 1981;16: 897-901.

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Lysophosphatidylcholine increases rat permeability to