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Morphological damage induced by Escherichia coli lipopolysaccharide in

cultured hepatocytes: Localization and binding properties

Article  in  British Journal of Experimental Pathology · September 1988

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Br. J. exp. Path. (I988), 69, 537-549

Morphological damage induced by Escherichia colilipopolysaccharide in cultured hepatocytes:

localization and binding properties

Raffaella Pagani, M. Teresa Portoles, Ines Diaz-Laviada andAngel M. Municio

Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense,28040-Madrid, Spain

Received for publication 2 December I987Accepted for publication i March I988

Summary. Lipopolysaccharides (LPS) from Gram-negative bacteria are considered to be theresponsible agents for the induction of endotoxic shock, affecting the liver as a target organ. Inthis study, the cell morphology and some biochemical properties of 24 h-culture-hepatocytemonolayers treated with Escherichia coli o01i B4 lipopolysaccharide, were observed. Cellmorphology was observed by scanning electron microscopy and immunofluorescencemethods. LPS interaction induced an increase in rounded cells with diminished adhesioncapacity. As biochemical parameters, albumin synthesis and 2-deoxyglucose uptake weremeasured. LPS decreased the hexose uptake in a dose-dependent manner. Binding of (14C)LPSto cultured hepatocytes showed that LPS binds to non-specific constituents of the membranebilayer.

Keywords: endotoxins, endotoxic shock, hepatocyte cultures, scanning microscopy, immuno-fluorescence, LPS binding, deoxyglucose transport

The molecular bases for toxicity of Gram-negative bacterial endotoxins are poorlyunderstood. Cytotoxic lesions as well asfunctional and metabolic disturbances occurmainly in the liver. This organ exerts anendotoxin clearance function in which bothKuppfer cells (Mori et al. I973; Nolan 975)and parenchymal cells (Willerson et al.I970) participate. Thus, the complexitiesimposed on the in-vivo studies by factorssuch as cellular heterogeneity ofthe liver andhomeostatic mechanisms might alter thedetermination of specific parenchymal cellalterations.

In-vitro studies on liver functions were

carried out on either perfused liver or liverslices. These procedures represent some dis-advantages in that only a limited number ofsimultaneous experiments can be made onperfused liver. Additionally, many cells inliver slices are damaged and both methodsyield preparations that remain viable for onlya few hours.

Since most of the specific liver functionsare located in hepatocytes (Guguen-Gillouzo& Gillouzo I983), a first attempt to simplifythe experimental model made use of isolatedhepatocytes obtained by the collagenaseperfusion method (Pagani et al. I98I).Although many liver functions are retained

Correspondence: Dr Raffaella Pagani, Departamento de Bioquimica y Biologia Molecular, Facultad deC.C. Quimicas, Universidad Complutense, 28040-Madrid, Spain.

537

R. Pagani et al.by hepatocytes isolated by collagenase (Gers-chenson & Thompson I975; Tager et al.I976), several reports have indicated theimpairment of some functions in freshlyisolated cells which can be restored byculture for a few days (Tanaka et al. 1978).Thus, the activity of 5'-nucleotidase inrecently isolated cell membranes is lowerthan that in intact liver membranes andnormal levels are regained when cells arecultured (Kato et al. I979).

In order to investigate the endotoxic effectat the cellular level, binding of endotoxiclipopolysaccharide (LPS) from Escherichia colito freshly isolated hepatocytes was pre-viously studied (Pagani et al. I98I). Thefindings show that LPS binds to particularingredients of the membrane bilayer of thesecells. The bilayer interaction is accompaniedby an alteration of metabolic pathwaysinvolving altered hormone-receptor binding,gluconeogenic response and second mes-senger production (Pagani et al. I984; Por-toles et al. I987).The aim of this study was to observe the

morphological alterations induced by endo-toxin treatment on cultured hepatocytesusing scanning electron microscopy, and tomonitor the LPS-membrane interaction byimmunofluorescence procedures. Bindingproperties of ('4C)-labelled LPS to the hepato-cyte cultures and LPS effect on membranetransport, were also estimated.

Materials and methods

Materials

(14C)Lipopolysaccharide was obtained fromE. coli oiiia, oiIIb: K58: H21 (ATCC)grown in a medium using (1 4C)glucose as theonly carbon source. The extraction of(14C)LPS (S.A. 2.23 jCi/mg) was carried outaccording to Romanowska's modification(Romanowska 19 70) ofthe method by West-phal et al. (I952). Unlabelled LPS from E. coliO0 I IiB4, obtained according to the above-mentioned method, was supplied by Difco(Detroit, MI, USA); A260/A240 was deter-

mined for purity verification (values higherthan I.2 would implicate a RNA contamina-tion, Romanowska I970). Collagenase,Type I, was provided by Boehringer (Mann-heim, FRG), William's E medium wasobtained from Flow Laboratories (Irvine,Ayrshire, UK), penicillin and streptomycinfrom Antibioticos S.A. (Le6n, Spain) andfoetal calf serum from GIsco Europe (Paisley,Renfrewshire, UK). ('4C)2-Deoxyglucose(S.A. 3I8 mCi/mmol) was from AmershamInternational Ltd. (UK). Unlabelled 2-deox-yglucose was obtained from Sigma ChemicalCo. (St Louis, MO, USA). Rat albumin andanti-albumin serum were kindly donated byDr F. Lampreave (Universidad de Zaragoza,Spain). Other chemicals were purchasedfrom Merck (Darmstadt/FRG). Tissue culturebottles and plates were from Costar (Cam-bridge, MA, USA).

Animals

Male Wistar rats (I50-200 g body weight),fasted over 20 h with water adr;iinistered adlibitum, were used in all experiments.

Isolation and culture of hepatocytes

Hepatocytes were isolated by the perfusiontechnique, using collagenase in Krebs-Ringer-bicarbonate solution (KRB-medium)according to the general method ofBerry andFriend (I969) as described previously(Pagani et al. I98I). Cell suspensions werepurified by repeated centrifugations in Wil-liam's E sterile medium, with 500 U penicil-lin and o.i mg streptomycin per ml, in arefrigerated RC-5 Sorvall centrifuge with anangular rotor, at i65 g for 20 seconds. Cellviability was 8 5 to 95% as determined by thetrypan blue exclusion test.Maintenance of cell structure was checked

by electron microscopy of thin sections ofEpon-embedded cell suspensions cut on anLKB Ultratome III ultramicrotome, andexamined in a Jeol IOO electron microscope.Yield of viable hepatocytes was (IOO± 20)Io6per rat liver.

538

Morphological damage induced by E. coli LPSThe isolated hepatocytes were cultured

in the presence of IO% foetal calf serum.Cultures were grown at 3 70C in 25 cm2bottles (6 x io6 cells/bottle), under C02/02(5%: 95%) atmosphere during 24 h until theformation of a continuous monolayer. Cellculture was performed under sterile condi-tions in a laminar flow chamber, TelstarCAM 700I.

Albumin measurement

Albumin levels in the medium culture weremeasured according to the immunoelectro-phoresis method of Laurell (I966) as modi-fied by Grieninger et al. (I979).

2-Deoxyglucose transport

Hepatocytes cultured in tissue culture plates(5 x IO' cells per well) were preincubatedunder C02/02 (5%: 95%) atmosphere for20 min at 3 70C in either the presence orabsence of different concentrations of LPSfrom E. coli oIII:B4 (io-ioo ,ug/well) inHanks' medium. Preincubation volume was0.5 ml/well. Afterwards, the medium wasaspirated and 0.5 ml/well of (14C)2-deoxy-glucose at different concentrations in Hanks'medium (0.05-I mM) were added and thecells were incubated during 5 min underC02/02 (5%:95%) atmosphere.

After the incubation was stopped by aspir-ating the medium, cells were sonicated dur-ing 5 min in an Ultrasons bath S-5 I 3(Selecta, Spain) in order to remove themonolayer. Then, 50 p1 ofH20, 50 pl of6o%perchloric acid and 50 P1 of 12 M NaOH wereadded per well. Cells were scraped andincorporation of radioactivity was measuredin a Packard Tri-Carb 3255 scintillationcounter using toluene-triton X-ioo-H20(2: I:0.2, V:V:V), PPO 5 g/l, POPOP 0.3 g/lafter adding I.5 ml to the sample.

("4C)Lipopolysaccharide binding assayThe standard binding assay was based on themethod of Rodbell et al. (I9 7I). Cultured

hepatocytes were incubated in plates at3 71C, during 20 min, in Hanks' mediumcontaining (14C)LPS (S.A. 4.5 pCi/mg). Theincubation volume was 500 1I; 5 x IO5 cellsand (14C)LPS (io to IOO Mg) per well.The generated data which employed

labelled endotoxin alone were considered astotal binding (Kahn I976). The non-specifi-cally bound LPS was determined by themethod of McQuarri et al. (I976), preincu-bating the cells with a high concentration ofunlabelled LPS (i mg per well) oiii: B4from E. coli, during 20 min at 3 70C. After-wards, the medium was removed and a freshmedium with (14C)LPS was added and in-cubated for 20 min. Subtraction of non-specifically bound LPS from the total radioac-tive uptake generated specific bindingcurves.

Incubation was stopped by aspirating themedium. Cells were then sonicated, themonolayer was removed and the radioacti-vity was measured as in the (14C)2-deoxy-glucose transport assay.

Statistics

Each experimental point represents themean± SD of triplicate determinations.

Scanning microscopy studies

Scanning microscopy studies were carriedout on cultured hepatocytes on circularmicrocoverslips, some of which were pre-coated with collagen Type I, according to themethod of Neupert and Vogel (I984).

Collagen-coated dishes were prepared byaddition of a collagen solution (Olmo et al.I985). Dishes, prepared in this manner,were dried in a laminar flow chamber andthen sterilized by exposure to ultraviolet lightduring 4 to 6 h immediately before use(Grinnell I982).The attached cells on glass or collagen-

coated cover-slips, were rinsed four times inphosphate-buffered saline, pH 7.4, during 45min at room temperature.

Dehydration was performed by slow water

539

R. Pagani et al.

replacement using series of acetone solutionswith final dehydration in absolute dryingacetone, allowing samples to dry at roomtemperature and under vacuum.The coverslips were mounted on stubs and

coated in vacuum with gold-palladium.Control and treated-LPS (I g/l) cells were

examined with a Jeol JSM-35C scanningelectron microscope.

Immunofluorescence studies

Immunofluorescence studies on binding ofLPS to hepatocytes were carried out on 24 hcultured cells in 25 cm2 bottles (8 x06cells/bottle) or in microcoverslips (Silber-mann et al. I983). Anti-LPS serum, obtainedby Dr. F. Diaz de Espada (Clinica Puerta deHierro, Madrid, Spain) according to Morri-son and Leive method (I975), was purifiedby saline precipitation before use. LPS anti-serum reactivity was assayed by the Ouch-terlony method. A positive and high specificreactivity was observed using i: i and I: 2dilutions of the antiserum.

Control and treated cells were examinedwith a Leitz Ortholux 2 (Ploemopack 2.5).

Results

Morphological observations

The hepatocyte monolayers, observed byphase-contrast microscopy, after 24 h ofculture in standard conditions, show anoptimal cell viability.

In order to estimate the morphologicalpreservation, cells were observed by scan-ning electron microscopy. Monolayers ofhepatocytes, grown on glass coverslips dur-ing 24 h, showed a mosaic-like morphology,very similar to the in-vivo structure, withpolygonal-shaped cells having intact micro-villi. Only a small percentage ofrounded cellswere attached to the monolayer (Fig. i).

Treatment with LPS from E. coli O I I : B4

(i g/l) increased the number ofrounded cellsin the monolayer in comparison with that incontrol cells, even when the hepatocyteswere grown on microcoverslips precoatedwith collagen that assures both a higheradhesion to the substrate and the polygonalshape of the cells (Fig. 2A,B). These roundedcells had a diminished adhesion althoughthey remained attached to either the sub-strate or to other cells. When cells weregrown without collagen, the cytoplasmicretraction, due to LPS, was more evident(Fig. 3).

Biochemical parameters. Albumin synthesis and2-deoxyglucose uptake

In order to test cell viability and function ofthe hepatocyte cultures, the albumin syn-thesis during the culture was measured.Figure 4 shows that the levels of albumin inthe medium increased gradually with pro-gressing culture time, reaching a maximumtowards 24 h.

Since glucose uptake by hepatocytes canbe related to a well-preserved mechanism oftransport through the membrane, the glu-cose uptake by cultured hepatocytes wasassayed by the ('4C)2-deoxyglucose incor-poration method either in the absence orpresence of LPS. The 24 h-cultured hepato-cytes maintained a normal rate of uptake of("4C)2-deoxyglucose (Fig. 5), whereas the E.coli OIII : B4 LPS treatment decreased thisuptake in a dose-dependent manner (Fig. 6).

Lipopolysaccharide binding studies

Immunofluorescence studies of LPS (i g/l)bound to hepatocyte monolayers, in theabsence of collagen, show that LPS wasbound mainly to the plasma membrane ofrounded cells (Fig. 7A,B), coating the surfaceof these cells (Fig. 7C,D).A very high non-specific binding was

observed with hepatocyte cultures and totalbound ('4C)LPS followed a linear saturationcurve up to 100 jg of (14C)LPS (Fig. 8).

540

Morphological damage induced by E. coli LPS

Fig. i. Scanning electron micrograph of 24 h cultured hepatocytes from adult Wistar rats. Cells weregrown on glass coverslips in William's E medium supplemented with io% foetal calfserum (bar= iO gm).

Discussion

The precise mechanisms involved in endo-toxin-induced shock are not yet clearlyunderstood. A biphasic behaviour has beenobserved in endotoxin-induced shock attri-buted to a direct interaction of bacteriallipopolysaccharide with the membrane oftarget cells and an interaction of multiplehomeostatic regulatory mechanisms (Brad-ley I979).The liver is the decisive target organ that

controls the metabolic changes during shock(Hardaway 1979), exhibiting some distur-bances in its morphology and function.

In order to simplify the in-vivo studies andto evaluate the primary steps of the directaction of LPS on parenchymal liver cells, theLPS binding to freshly isolated hepatocyteshas been previously studied, demonstratingthe non-existence of functional specific

receptors for the LPS in the cell membrane.However, the endotoxin does bind to someconstituent of the membrane bilayer, induc-ing a destabilization of the membrane(Pagani et al. I98I; Portoles et al. I987) andproducing metabolic disorders (Pagani et al.I984).The non-replicating 24 h-cultures used in

this work provide a useful experimentalmodel for studying the primary steps of directaction of bacterial endotoxins on parenchy-mal liver cells because of the reestablishmentof various functions altered by the collage-nase treatment during cell isolation. It hasbeen shown that protein synthesis, which isvery low in fresh hepatocytes (Tager et al.1976), is restored when cells are cultured.The glycocalyx is also repaired during thefirst hours of culture (Kato et al. I979), thusindicating the recovery of the normal metab-olism and structure of the cells.

54I

R. Pagani et al.

...'

i

Fig. 2. Scanning electron micrographs of cultured hepatocytes in presence or absence ofLPS from E. coliO II0 : B4. Hepatocytes from adult Wistar rats were grown on glass coverslips precoated with collagen, inWilliam's E medium supplemented with io% foetal calf serum during 24 h. A, Control monolayermaintained during IO min in serum-free medium (bar= 50 pm); B, Hepatocytes after a IO min treatmentwith i g/l LPS in serum free medium (bar= 50 Pm).

542

* y

Morphological damage induced by E. coli LPS

Fig. 3. Magnification of the cytoplasmic retraction in cultured hepatocytes treated with LPS from E. coliO I I : B4, observed by scanning electron microscopy. Hepatocytes from adult Wistar rats were grown onglass coverslips non-precoated with collagen, in William's E medium supplemented with io% foetal calfserum during 24 h. Monolayers were treated with LPS (i g/l) during IO min in serum-free medium(bar= 5 pm).

543

Hours

Fig. 4. Albumin levels in the medium of hepatocyte cultures at different times. Cultures were grown at370C in 25 cm2 bottles (8 x io6 cell/bottle), under C02/02 (5%: 95%) atmosphere during 24 h. Albuminlevels in the medium culture were measured at different times by Laurell immunoelectrophoresismodified by Grieninger.

2

Ec

0.1 0.5 1

mM

Fig. 5. 2-Deoxyglucose uptake by cultured hepatocytes. Twenty-four hour cultures in tissue cultureplates (24 flatbottom wells) (5 x Io5 cells/well) were incubated under C02/02 (5%:95%) atmosphereduring 5 min at 37°C with 0.5 mI/well of (14C) 2-deoxyglucose (S.A. 3I8 mCi/mmol) at differentconcentrations (0.05-I mM) in Hanks' medium. After aspirating medium, the monolayer was removedand incorporation of radioactivity was measured.

I - * I _

1

Morphological damage induced by E. coli LPS

100 4

90

Ca1)0

a)0-

80

70

60

50

K

20 40 60 80 100jg

Fig. 6. Effect of different concentrations of LPS on the 2-deoxyglucose uptake by cultured hepatocytes.Twenty-four hour cultures in tissue culture plates (24 flatbottom wells) (5 x I05 cells/well) werepreincubated during 20 min at 3 70C in either the presence or the absence of different concentrations ofLPS (IO-IOO 4g/o.5 ml/well) in Hanks' medium. After aspirating medium, 0.5 ml/well of 0.5 mM('4C)2-deoxyglucose (45 mCi/o.s ml) were added and the cells were incubated at 37TC during 5 niinunder C02/02 (5%:95%) atmosphere. Then, medium was aspirated, the monolayer was removed andincorporation of radioactivity was measured. Percentages are referred to the incorporated (14C)2-deoxyglucose by control cultured hepatocytes in the absence of LPS.

The culture conditions were testedthrough morphological studies such asphase-contrast microscopy and scanningelectron microscopy (Fig. i), as well as byevaluation of some biochemical parameterssuch as albumin synthesis (Fig. 4) and 2-deoxyglucose transport (Fig. 5). These dataindicate that hepatocyte cultures exhibit agood viability with a restored protein syn-thesis and an unaltered membrane permea-bility. For these reasons, 24 h-cultures wereused in all experiments to evaluate the effectof LPS treatment.The study of morphological alterations

induced by LPS treatment evidences that

endotoxin produces an hepatocyte cytoplas-mic retraction, diminishing their adhesioncapacity. Hepatocytes remain attached tothe monolayer by retraction fibres and pre-sent a rounded aspect. This effect can beobserved by phase-contrast microscopy and,in more detail, by electron microscopy (Figs 2& 3), even under good adhesion conditions(i.e., when coverslips are precoated withcollagen).We have previously observed that the LPS-

membrane interaction induces an increase ofthe cell membrane microviscosity (Portoleset al. I98 7), indicating that the endotoxinbinds to hepatocytes altering the physical

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R. Pagani et al.

Fig. 7. Immunofluorescence study of the LPS from E. coli oiii:B4 bound to 24 hours culturedhepatocytes. Cells were grown under standard conditions on tissue culture bottles (8 x io6 cells/bottle).A, Control ( x 450); B, Hepatocytes after a io min treatment with i g/l LPS ( x 450); C,D, Magnification ofthe binding to the cytoplasmic membrane (x goo).

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Morphological damage induced by E. coli LPS

0

I

-

m

I9

Fig. 8. Association of (14C)LPS with cultured hepatocytes. The total binding curve (0) was determinedusing 24 h cultures in tissue culture plates (24 flatbottom wells) (5 x IO' cells/well) and I0-I00i ug(14C)LPS/well (S.A. 4.5 pCi/mg). The non-specific points (-) were determined by preincubation with img LPS/well for 20 min at 370C. The specific binding curve (A) was calculated by difference.

properties of the cytoplasmic membrane.This effect could be related to the diminutionof the adhesion capacity and to changes inthe cell morphology.The LPS treatment decreases the (14C)2-

deoxyglucose uptake in a dose-dependentway (Fig. 5). This fact agrees with theendotoxin effect described in isolated adipo-cytes (Leach & Spitzer I98I), suggesting thatthe LPS-membrane interaction can alter,even at low doses, the normal mechanism ofglucose transport. This effect is related to thealterations induced by the LPS on the physi-cal properties of the cell membrane. With theaim ofknowing localization of the LPS in thecells, immunofluorescence studies were car-ried out showing that LPS was preferentiallybound to the cytoplasmic membrane of

rounded cells, coating them superficially(Fig. 7).The LPS binding properties were studied

by saturation assays of ("4C)-LPS in hepato-cyte cultures. A non-hyperbolic behaviour ofthe specific binding curve (Fig. 8) suggeststhat LPS interaction with membranes can beattributed to a non-specific binding to hydro-phobic regions, in agreement with previousresults obtained using hepatocyte suspen-sions (Pagani et al. I98I). Therefore, it canbe concluded that liver collagenase perfusionduring hepatocyte isolation does not provokeany damage to the LPS binding domains.Furthermore, the direct action of LPSinduces a morphological damage in hepato-cytes and some functional alterations as 2-deoxyglucose transport and cytochrome

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548 R. Pagani et al.

P450 levels (Pagani et al. I987). In conclu-sion, although further studies are needed fora precise understanding of the mechanismsinvolved in the whole endotoxin action, thehepatocyte cultures provide a useful modelfor the study of primary steps of the endo-toxin action.

Acknowledgements

This study was supported by research grant84/IOOI from 'Fondo de InvestigacionesSanitarias de la Seguridad Social' (Spain) andby research grant PR84-o5o6-Co2-02 from'Comision Asesora de Investigacion Cienti-fica y Tecnica del Ministerio de Educacion yCiencia' (Spain). We are particularly gratefulto Dr M.A. Lizarbe for her help in immuno-fluorescence studies, Dr J.M. Gomez de Sala-zar for technical scanning microscopy assist-ance and M.J. Ainaga for her help inelaborating this manuscript. I.D.L. is greatlyindebted to 'Fondo de Investigaciones Sani-tarias de la Seguridad Social' (Spain) for afellowship on biomedical research (85/533).

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