UW-preservation of cultured human gallbladder epithelial cells: Phenotypic alterations and differential mucin gene expression in the presence of bile

UW-Preservation of Cultured Human Gallbladder Epithelial Cells: Phenotypic Alterations and Differential Mucin Gene Expression in the Presence of Bile J E A N - P I E R R E CAMPION, 1' 2 NICOLE PORCHET, 3 J E A N - P I E R R E AUBERT, 3 A N N I E L'HELGOUALC'H, 1 AND BRUNO CLI~MENT 1

In orthotopic liver transplantation, extended cold ischemia of the graft may i n d u c e cell damage, particularly in biliary epithelium. We h a v e investigated the effects of a cold University of Wisconsin (UW) solution on cultured h u m a n gallbladder biliary epithelial cells (GBEC) exposed or not exposed to stagnant bile. In UW solution, morphological alterations of cultured GBEC were not p r o m i n e n t u n d e r light microscopy after 16 hours at 4°C, b e i n g more striking after 24 to 48 hours. Ultrastructural examination of GBEC s h o w e d a condensation of chromatin at the periphery of the nuclei after 16 hours in cold UW solution. B o t h protein and DNA syntheses were strikingly reduced in these cells. After rewarming in standard Williams' m e d i u m at 37°C for 24 hours, cultured GBEC exhibited both normal morphology and function. As in both freshly isolated and routinely cultured GBEC, rewarmed cells expressed various m u c i n genes, n a m e l y MUC1, MUC3, MUC4, MUC5AC, and MUC5B genes, w h e r e a s MUC2 mRNAs were barely detectable. A dramatic decline in the steady-state mRNA levels of both MUC3 and MUC5B was found in cultured GBEC versus freshly isolated cells. Addition of bile into UW solution at 4°C h a d n o significant effect o n GBEC m o r p h o l o g y and DNA and protein syntheses. When bile was added during the r e w a r m i n g period, both protein and DNA syntheses were strongly reduced. Addition of bile d u r i n g either storage in UW solution or r e w a r m i n g period induced increased steady-state MUC2, MUC3 and MUC5AC mRNA levels. These results s h o w that UW is a reliable cold storage solution for GBEC and the presence of stagnant bile w i t h i n the culture m e d i u m during the rewarming period leads to deleterious p h e n o t y p i c alter-

Abbreviations: UW, University of Wisconsin; GBEC, gallbladder biliary epithelial cells; cDNA, complementary DNA; mRNA, messenger RNA; HEPES, N-2-hydroxyethylpiperazine-N ' -2-ethanesulfonic acid. From the 1Unit~ de Recherches H~patologique~ Inserm U-49, 2Clinique Chirurgicale, CHRU Pontchaillou, Rennes, and ~Inserm U-377, Place de Verdun, Lille, France. Received May 6, 1994; accepted August 9, 1994. Supported by the Institut National de la Sant~ et de la Recherche M~dicale, the "Association pour la Recherche contre le Cancer" (ARC, Paris, France), and the "Association Fran~aise de Lutte contre la Muscoviscidose" (AFLM, Paris, France; grant no. 94021). Address reprint requests to Bruno Clement, PhD, Inserm U-49, HSpital Pontchaillou, 35033, Rennes Cedex, France. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2101-003453.00/0

ations of these cells. This suggests c h a n g e s in the management of liver graft during orthotopic liver transplantation. (HEPATOLOGY1995;21.'223-231.)

Cold storage solutions are required for successful preservation of organs before orthotopic liver transplantation. Despite recent improvement in surgical techniques, the incidence of biliary related complications reaches a rate between 11% and 24%. 1'2 These complications involve various mechanisms including technical defect, hepatic artery thrombosis, viral infection, acute or chronic rejection, and original disease recurrence. However, these etiologies do not account for all pathological situations. Recent reports suggest the deleterious effect of prolonged cold ischemia time, 1-3 others being controversial. 4 Biliary epithelium is undoubtedly involved, but the mechanism(s) of cell injury related to cold preservation is unclear. The structure and functions of the liver have been extensively studied in hypothermically preserved organs depending on the duration and conditions of preservation. Thus, the efficacy of the University of Wisconsin (UW) solution for extended liver preservation, up to 16 hours, has been clearly shown. ~ In addition, it has been shown that both cooling and rewarming of the organs are critical steps for a rapid reestablishment of liver functions. Although hepatocytes were only slightly affected, marked alterations of nonparenchymal cells, namely sinusoidal endothelial cells and biliary cells occurred during cold storage in UW solution and rewarming of the liver. 6'v Biliary epithelial cells are the major cell type forming both bile ducts and the gallbladder, s During its passage into the biliary tree toward the duodenum, the bile is modified by these cells that reabsorb and secrete fluids and inorganic electrolytes. In the gallbladder, they concentrate bile by transferring large volumes of water and electrolytes and maintaining a concentration gradient between the lumen and extracellular spaces. An important additional physiological role of biliary epithelial cells is to secrete mucus that serves as lubricant and protective layer. The physiochemical properties and functions of mucus are attributed to mucins, a large family of heavily O-glycosylated proteins. Biophysical and biochemical properties of mucins purified

223

224 CAMPION ET AL from t h e airway, or from colonic or gastric m u c u s h a v e b e e n extensively described, b u t little is k n o w n a b o u t mucins from t h e biliary tree. T h e first i n f o r m a t i o n on t h e s t r u c t u r e of gallbladder m u c i n s suggested t h a t rep e a t e d amino acid sequences occur in bovine m u c i n proteins. 9 In addition, t h e s e p r o t e i n s can contain at least two distinct domains, including a h y d r o x y - a m i n o a c i d - r i c h d o m a i n c o n t a i n i n g the m a j o r i t y of covalently b o u n d c a r b o h y d r a t e s a n d a nonglycosylated d o m a i n t h a t binds hydrophobic ligands such as bilirubin a n d cholesterol a n d m a y c o n t r i b u t e to the p a t h o g e n e s i s of cholesterol cholelithiasis. 1° P a r t i a l c o m p l e m e n t a r y (c) DNA sequences of t h e core proteins of several mucins from both i n t e s t i n a l a n d t r a c h e o - b r o n c h i a l sources w e r e r e c e n t l y obtained. 11-14 T h e lack of a p p r o p r i a t e tools m a y explain t h a t t h e effects of cold storage on biliary epithelial cells, p a r t i c u l a r l y m u c i n gene expression, has b e e n poorly explored. P r i m a r y c u l t u r e s of h u m a n gallbladder biliary epithelial cells (GBEC) are a n a t t r a c t i v e in vitro model s y s t e m to s t u d y biliary cellular functions. C u l t u r e d G B E C h a v e b e e n morphologically a n d functionally c h a r a c t e r i z e d e l s e w h e r e J 5~s Following dissociation of gallbladder mucosa, epithelial cells can a t t a c h on various s u b s t r a t e s , form colonies, a n d r a p i d l y proliferate. G B E C express specific m a r k e r s , e.g., 7 - g l u t a m y l t r a n s p e p t i d a s e and c y t o k e r a t i n s , a n d m a i n t a i n m u c u s secretion for at least 1 w e e k in culture. 16 To i n v e s t i g a t e t h e efficacy of U W solution on the cold storage of biliary cells, we h a v e studied t h e viability a n d function of cult u r e d G B E C stored at 4°C in U W solution a n d h a v e focused on t h e differential expression of six m u c i n genes. We show t h a t U W solution is suitable for cold p r e s e r v a t i o n of G B E C in vitro a n d t h a t the p r e s e n c e of s t a g n a n t bile in r e w a r m i n g m e d i a r e s u l t s in m a r k e d p h e n o t y p i c a l t e r a t i o n s a n d changes in t h e expression of specific m u c i n m e s s e n g e r (m)RNAs. MATERIALS AND METHODS

Cell Isolation a n d Culture All experiments were carried out in accordance with French law and regulations and in agreement with local and national ethics committees. Gallbladders were obtained from organ donors. Immediately after gallbladder removal, bile was stored at 4°C. The gallbladder was incised, rinsed thoroughly with saline solution, and checked for signs of mucosal inflammation. The mucosa was carefully dissected from underlaying muscular layer and adventitia and subsequently washed in cold L-15 Leibovitz medium. The remaining tissue was cut into fragments that were thoroughly stirred for 40 minutes at 37°C in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution containing 137 retool/ L NaC1, 2.7 mmol/L KC1, 0.28 mmol/L Na2HPO4, 12 H20 and 10 mmol/L HEPES, pH 7.65, supplemented with 0.025% collagenase (Boehringer-Mannheim) and 5 mmo]/L CaC12. Dissociated GBEC were collected by low-speed centrifugation and washed twice in HEPES solution and once in serum-free Williams' medium (GIBCO-BRL). Then, 5 x 10~ cells were seeded onto 35-ram plastic dishes (Nunclon, InterMed Nunc, Roskilde, Denmark) in Williams' medium containing 10% fetal calf serum and antibiotics (penicillin 10 IU/mL; strepto-

HEPATOLOGYJanuary 1995 mycin 50 #g/mL). The medium was changed after 24 hours and everyday thereafter, unless otherwise indicated. Hypothermic Preservation of Cultured GBEC One day after cell seeding, media were removed, and cultured GBEC were placed either at 37°C in Williams' medium or at 4°C for 16 hours, 24 hours, or 48 hours in either UW solution or UW solution containing bile from the same donor at the concentration of 1/100 (vol/vol). After storage, media were removed, and cell cultures were placed at 37°C for either 4 hours or 24 hours in fresh Williams' medium containing 10% fetal calf serum, supplemented or not with 1/100 bile (vo]/vol), under a humidified atmosphere composed of 95% air and 5% CO2. Cells were routinely examined under phasecontrast microscopy. To investigate the influence of bile on the preservation of GBEC, various storage and culture conditions were established as follows: condition I, cells stored for 16 hours at 4°C in UW solution and then cultured for 6 hours at 37°C in Williams' medium: condition II, cells stored for 16 hours at 4°C in UW solution containing 1/100 bile (vol/vol) and then cultured for 6 hours at 37°C in Williams' medium; condition III, cells stored for 16 hours at 4°C in UW solution and then cultured for 6 hours at 37°C in Williams' medium containing 1/100 bile (vol/vol); condition IV, cells stored for 16 hours at 4°C in UW solution containing 1/100 bile (vol/vol) and then cultured for 6 hours at 37°C in Williams' medium containing 1/100 bile (vol/vol). Electron Microscopy For electron microscopic analysis, cultured GBEC were fixed in 2.5% glutaraldehyde buffered with 0.1 mol/L sodium cacodylate pH 7.4 for 5 minutes at 4°C, postfixed in a 1% tetroxide solution in cacodylate buffer for 30 minutes, dehydrated in alcohols and embedded in epoxy resin. U]trathin sections were stained with uranyl acetate and lead citrate before examination. DNA and Protein Synthesis Protein content was estimated by the Bradford's method using Biorad protein reagent (Bio-Rad Laboratories, Ivry Sur Seine, France). For protein synthesis determination, cell cultures were incubated for 4 hours in serum-free, leucine-free Williams' medium, containing 2 #Ci [14C]leucine per mL of medium (340 mCi/mmol, Amersham, Arlington Heights, IL). Cell layers were extensively washed with cold media, and intracellular proteins were subsequently precipitated, washed, dissolved, and counted. DNA synthesis was measured following incubation of biliary cell cultures with [3H]thymidine in serum-free Williams' medium for 4 hours (5 Ci/mmol, Amersham). Then, cell layers were extensively washed with cold media and sonicated before counting. Each assay was carried out in triplicate in three independent experiments. The Mann-Whitney test was used for significance assesment. RNA Extraction and Northern-Blot Analysis Total RNAs were extracted from both freshly isolated and cultured GBEC using the guanidium-thiocyanate/cesium chloride method. 19 RNAs (5 ~g per lane) were resolved by electrophoresis in a denaturing 1.1 mol/L formaldehyde-agarose gel and transferred onto Hybond-N sheets (Amersham). Hybridizations were performed at 42°C in 6x standard sodium phosphate ethylenediaminetetraacetic acid, 5x Den-

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FIG. 1. Phasecontrast microscopy.GBECwere cultured for 24 hours in Williams' medium at 37°C (A) and subsequently preserved at 4°C in UW solution for 16 hours (B), 24 hours (C), and 48 hours (D). After 48 hours, UW solution was replaced by Williams' medium for a further 24 hours conventional culture at 37°C (E) (original magnification x150).

hardt's solution, 0.5% sodium dodecyl sulfate, 50% formamide, 100 #g/mL calf thymus DNA, overnight. After washes in 1x standard sodium phosphate ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate at 65°C, filters were exposed to Hyperfilm MP (Amersham) at -80°C 1 day for/~-actin, MUC1, MUC3, MUC5B, and MUC5AC or 3 days for MUC2, MUC4 and MUC6. cDNA probes were pMUC7 (450 bp) for MUC1, 2° SMUC41 (800 bp) for MUC2,11 SIB124 (400 bp) for MUC3, TM JER64 (1830 bp) for MUC4,la JER58 (836 bp) for MUC5AC, 14 and JER57 (1831 bp) for MUC5B. 21 RESULTS

Effects of UW Solution on Cold Storage of Cultured GBEC Phase Contrast Microscopy. GBEC were isolated and seeded onto 35-mm plastic dishes in Williams' medium. They rapidly attached, formed colonies, and proliferated. Confluency was reached in less t h a n 24 hours (Fig. 1A). After this period of time, media were removed and cells were preserved at 4°C in nonsupplemented

UW solution. Light microscopic examination of biliary cells did not show any obvious morphological change after a 16-hour hypothermic preservation, when compared with unpreserved cells, i.e., cultured at 37°C in Williams' medium (Fig. 1B). After 24 hours in UW solution at 4°C, cells became rounded (Fig. 1C). After 48 hours in UW solution at 4°C, numerous biliary cells detached from the plastic surface (Fig. 1D). Colonies of remaining 24-hour and 48-hour UW stored GBEC recovered a normal morphological appearance and proliferated when Williams' medium replaced UW for a 24-hour conventional culture at 37°C (Fig. 1E). Electron Microscopy. Unpreserved and hypothermically preserved GBEC for 16 hours and 24 hours were examined under electron microscopy. Biliary cells in conventional culture conditions exhibited a typical ultrastructure, with a b u n d a n t microvilli and tight junctions between cells (Fig. 2A). Organelles in the cytoplasm and one nucleus with one or two nucleoli were identified. Numerous membrane-limited vesicles con-

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FIG. 2. Electron microscopy. GBEC were cultured for 24 hours in Williams' medium at 37°C (A) and subsequently preserved at 4°C in UW solution for 16 hours (B) and 24 hours (C). After 16 hours in UW solution at 4°C, GBEC were cultured for a further 24 hours in Williams' medium at 37°C (D). In 16-hour UW-preserved GBEC, chromatin is condensed at the i n n e r periphery of the nucleus (arrows); in both control (A) and rewarmed GBEC (D), numerous membrane-limited vesicles t h a t probably contain mucins are visible (stars). (A: original magnification x7,900; B: original magnification x18,600; C: original magnification x10,700; D: original magnification x6,400.)

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TABLE 1. Protein and DNA Syntheses in Cultured GBEC UWPreserved Cultures

Rewarmed C u l t u r e s After UW P r e s e r v a t i o n (16 h at 4°C a n d

(16 h at 4°C)

12 h at 37°C)

95

1695 ± 75

3842 ± 101

5970 ± 140

1075 ± 85

6153 ± 106

Standard Cultures

[14C]leucine incorporation (dpm]#g proteins) [3H]thymidine incorporation (dpm/~g proteins)

3530 ±

NOTE. D a t a are the m e a n s ± SD in triplicate f r o m a typical experiment.

taining amorphous or dense material were observed in the cytoplasm. When cell cultures were preserved for 16 hours at 4°C in UW solution, the most striking effect was on chromatin structure, with disappearance of well formed nucleoli and condensation of chromatin at the inner periphery of the nucleus in the majority of cells (Fig. 2B). Ultrastructural alterations of intracytoplasmic organeUes were different from one cell to another. In numerous GBEC, but not all, the cytoplasm-to-nucleus ratio was reduced. After 24 hours in UW solution at 4°C, numerous cells exhibited typical features of apoptotic cells, with shrinkage of cells and alteration of chromatin integrity (Fig. 2C), others being less altered (not shown). A normal ultrastructure of GBEC preserved for 16 hours at 4°C in UW solution was observed in the majority of cells after a further 24 hours in fresh Williams' medium at 37°C (Fig. 2D). Protein and DNA Synthesis. When compared with unpreserved GBEC, a 52% decline in protein synthesis was found in leucine incorporation in UW-preserved cultures, in three independent experiments (P < .009). Results obtained in a typical experiment are shown in Table 1. Similarly, hypothermic preservation had a striking effect on DNA synthesis, with an 82% decrease in thymidine incorporation in UW-preserved cells compared with unpreserved cells in three independent experiments (P < .008). After UW removal and 12-hour conventional culture in Williams' medium at 37°C, no significant change was found in both leucine and thymidine incorporation in UW preserved cells, compared with control cells (Table 1). Influence of Bile on UW-preserved GBEC Morphology. Examination of biliary cell cultures under phase contrast microscopy indicated that addition of bile in UW solution during hypothermical preservation had no obvious effect on cell morphology (condition II), compared with control (condition I) after a 6-hour rewarming period in Williams' medium at 37°C (Fig. 3A and B). This was confirmed under electron microscopic examination (data not shown). Conversely, when bile was added to Williams' medium at 37°C during the 6hour rewarming period (conditions III and IV), numerous cells detached from the plastic surface (Fig. 3C and D). The surviving cells started to proliferate during the

following 24 hours in the absence of bile. These cells did not exhibit particular ultrastructural alterations compared with GBEC cultured in the absence of bile (Fig. 2). DNA and Protein Synthesis. When bile was present in UW solution during 4°C storage (condition II), no significant change was found in both [14C]leucine and [3H]thymidine incorporation when compared with control cultures (condition I) (Fig. 4). When bile was added during the 4-hour rewarming period at 37°C in Williams' medium, a dramatic decrease in both protein and DNA synthesis was found in biliary cell cultures preserved in UW solution either in the absence (condition III) or presence of bile (condition IV). The decline reached, respectively, 65% (P < .0009) and 56% control values (P < .0006) for protein neosynthesis and 81% (P < .008) and 63% control values (P < .0006) for DNA synthesis in three independent experiments. No significant change was found in both [3H]thymidine and [14C]leucine incorporation in cultured GBEC after an additional 24 hours in fresh bile-free Williams' medium at 37°C, regardless of storage conditions. Mucin Gene Expression

Total RNA was prepared from freshly isolated GBEC, standard GBEC cultures in Williams' medium, and UW-preserved GBEC under conditions I, II, III, and IV, as described above. The expression of six different mucin genes, namely MUC1, MUC2, MUC3, MUC4, MUC5AC, and MUC5B was analyzed by Northern blotting (Fig. 5 and Table 2). MUC1. Freshly isolated GBEC contained a major MUC1 transcript, whereas an additional mRNA species was detectable in primary culture. In UW-preserved cultures, only one MUC1 mRNA species was found. No obvious change in the steady-state MUC1 mRNA level was detected regardless of culture conditions, i.e., I, II, III, and IV. MUC2. MUC2 mRNA species was barely detectable in both freshly isolated and cultured GBEC. A weak signal was observed after a longer exposure (data not shown). The steady-state MUC2 mRNA level was increased in UW-preserved cultures (condition I), with an additional increase in that steady-state level in the presence of bile during preservation (condition II). The effect of bile on MUC2 mRNA content was even much higher when bile was added during the rewarming of UW-preserved biliary cell cultures in Williams' medium at 37°C (condition IV). MUC3. A polydisperse signal was found by Northernblotting using the MUC3 cDNA probe. This signal was markedly decreased in cultured GBEC compared with freshly isolated cells. The presence of bile during either hypothermic preservation (condition III) and/or rewarming of cells in Williams' medium at 37°C (conditions II and IV) resulted in an increased steady-state MUC3 mRNA level. MUC4. A polydisperse signal was shown in freshly isolated GBEC using the MUC4 cDNA probe. In cul-

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FIG. 3. Phase contrast microscopy. GBEC were cultured for 24 hours in Williams' medium, then cells were (A): stored for 16 hours at 4°C in UW solution and then cultured for 6 hours at 37°C in Williams' medium (condition I); (B): stored for 16 hours at 4°C in UW solution containing 11100 bile (vol/vol)and then cultured for 6 hours at 37°C in Williams' medium (condition II); (C): stored for 16 hours at 4°C in UW solution and then cultured for 6 hours at 37°C in Williams' medium containing 11100 bile (vol/vol)(condition III); (D): cells stored for 16 hours at 4°C in UW solution containing 11100bile (vol/vol)and then cultured for 6 hours at 37°C in Williams' medium containing 11100 bile (vol/vol)(condition IV) (original magnification ×370).

t u r e d GBEC, this signal was b a r e l y detectable, regardless of the c u l t u r e conditions. MUC5AC. A polydisperse signal was obtained in both freshly isolated and cultured GBEC using a MUC5AC cDNA probe. A decline in the steady-state MUC5AC mRNA level was noted after UW-preservation (condition I). Addition of bile during the r e w a r m i n g period of cells at 37°C in Williams' medium, resulted in a m a r k e d increase in MUC5AC mRNA content (conditions II and IV). MUC5B. I n t e n s e signal c o r r e s p o n d i n g to M U C 5 B m R N A was found in freshly isolated GBEC. M U C 5 B m R N A was b a r e l y d e t e c t a b l e in c u l t u r e d biliary cells, i n d e p e n d e n t of c u l t u r e conditions. fl-Actin. I n t e g r i t y control of RNA was achieved by probing/~-actin on t h e blots first h y b r i d i z e d w i t h m u c i n cDNA probes. No c h a n g e in t h e s t e a d y s t a t e fl-actin m R N A level was found, except in condition IV. In this c u l t u r e condition, the low level of/~-actin e x p r e s s i o n

was c o r r e l a t e d w i t h s t r i k i n g morphological d a m a g e s (Fig. 3). DISCUSSION

The effects of h y p o t h e r m i c a l p r e s e r v a t i o n in U W solution on liver cells, p a r t i c u l a r l y h e p a t o c y t e s , h a v e b e e n i n v e s t i g a t e d both in vivo and in vitro. 6'7'22"23 Little is known, however, c o n c e r n i n g the effects of l o n g - t e r m storage in U W solution on t h e viability a n d functions of biliary epithelial cells e i t h e r in situ or in vitro. We chose to i n v e s t i g a t e the effects of h y p o t h e r m i c preserv a t i o n on biliary cells isolated from the gallbladder. G B E C can be easily p r e p a r e d in h i g h yields a n d h a v e b e e n c h a r a c t e r i z e d elsewhere. 151s I n t e r e s t i n g l y , epithelial cells from b o t h gallbladder a n d bile ducts exhibit similar functions. Thus, both cell types r e a b s o r b a n d secrete fluids and inorganic electrolytes in bile,

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cated that cold storage in UW did not result in equal injury of all the cells, thus suggesting that a subpopulation of GBEC was indeed selected. Interestingly, a good functional preservation of rat hepatocytes has been reported after cold preservation of parenchymal cells in L-15 medium, including intracellular glutathione content, albumin secretion, and several phase I and phase II drug metabolic reactionsy whereas these cells exhibited altered chromatin structure and lower protein synthesis levels after 48 hours of cold preservation in UW solution. 25 These results indicate that the effects of cold storage in UW solution on cultured GBEC are not specific for these cells and that there is a need of appropriate storage media to improve the functional preservation of cells during storage of liver graft before transplantation.

1200

•~

1000

el II

o

~" t-

800

O

600 -~ t

400

"=

200

|

229

2O00 FI

.o

culture conditions C I H H I IV

el L

oe~ t. o °m

MUC 1

1000

e~ o~

MUC 2 ,q.

MUC 3

0 condition bile in cold UW solution bile in rewarming medium

I 0 0

II + 0

HI 0 +

IV + +

FIG. 4. DNA and protein synthesis. 3H-thymidine and 14C-leucine incorporation was evaluated in GBEC monolayers cultured in conditions I, II, III, and IV as in FIG. 3. Data are the means _+ SD in triplicate from a typical experiment (dpnd#g total protein from cell layers).

express T-glutamyl transpeptidase and specific cytokeratins and synthesize mucins. 16'24 Our data indicate that UW solution induces reversible cell alterations in cold-stored GBEC. A dramatic decline in both DNA and protein synthesis was found in hypothermically stored cells when compared with routinely cultured GBEC. In addition, alteration of chromatin ultrastructure and cell shrinkage, that are typical features of apoptotic cells, were observed after a 16-hour UW preservation at 4°C. Thereafter, cells became rounded and began to detach after 24 hours. Interestingly, morphological and functionnal alterations were apparently reversible after rewarming of biliary epithelial cell cultures in bile-free Williams' medium. However, electron microscopic examination indi-

MUC 4

MUC 5AC

M U C 5B

~-Actin FIG. 5. Northern-blots. The expression ofMUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and /~-actin genes was analyzed by Northern-blotting using total RNA prepared from freshly isolated GBEC (FI), control GBEC cultured for 24 hours in Williams' medium at 37°C (C) and GBEC cultured in conditions I, II, III, and IV as in FIG. 3.

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TABLE 2. M u c i n m R N A s i n C u l t u r e d G B E C

MUC1 MUC2 MUC3 MUC4 MUC5AC MUC5B /~ actin

Control

Condition I

Condition H

Condition III

Condition IV

111 86 18 76 335 9 108

98 248 45 112 116 8 113

102 922 75 103 816 10 112

106 660 56 89 397 7 106

96 1355 97 97 850 9 47

N O T E . T h e e x p r e s s i o n of six different m u c i n g e n e s a n d ~ - a c t i n w a s a n a l y z e d in f r e s h l y isolated a n d c u l t u r e d G B E C b y N o r t h e r n blot a n d q u a n t i t a t e d by d e n s i t o m e t r i c a n a l y s i s of t h e a u t o r a d i o g r a m (see Fig. 5). R e s u l t s a r e e x p r e s s e d as t h e p e r c e n t a g e of t h e initial v a l u e f o u n d in f r e s h l y isolated GBEC. C u l t u r e conditions (I to IV) w e r e a s described in t h e text.

Although only slight alterations were noted when bile was added to UW solution during cold preservation, addition of bile during the rewarming period had dramatic effects on survival and both DNA and protein synthesis. The toxic effects of stagnant bile during this period seemed to be reversible after removing bile and addition of fresh medium. Thus, it is likely that these effects were only transient. Bile acids are known to promote hepatic injuries. Hepatocellular damages have been reported following acute or chronic administration of bile derivatives in animals, i.e., cholestasis and/ or cirrhosis. 26'27Experimental liver injuries seem to be influenced by bile composition, particularly the bile acid pool. On the other hand, ursodeoxycholic acid is known to improve biochemical indices of liver injury in patients with cholestatic liver diseases such as primary biliary cirrhosis or chronic hepatitis. 2s'29 Interestingly, it has been shown in the dog that biliary lipids and pigments protected gallbladder epithelium from damage by bile salts. 3° Because crude bile was used in our study, we cannot conclude whether a specific bile compound(s) might be responsible for bile-induced effects in gallbladder epithelial cell cultures. It is noteworthy that only trace amounts of lysolecithins were found in bile, even after a 48-hour storage at 4°C, whereas unconjugated bilirubin remained undetectable (data not shown). Although some variations might exist in the degree of toxicity from one experiment to another, the effects of stagnant bile on rewarmed GBEC was reproducible and did not depend on the donor. It must be pointed out that these experiments were performed on proliferating epithelial cell cultures and that addition of bile had a striking effect on DNA synthesis during the rewarming period. The expression of mucin genes in GBEC has never been studied. We found that these cells express several mucin species, derived from at least six distinct genes mapped to different chromosomes. MUC1 is a transmembrane O-glycoprotein expressed by many epithelial cells and all other MUC genes encode mucins norreally secreted by human mucosae. MUC2 gene product is primarily located in colon and respiratory tracts, MUC3 in small intestine, MUC5AC in stomach and

bronchus, MUC5B in submaxillary and bronchus glands, and MUC4 in all m u c o s a e . 31 Little is known about MUC6 and MUC7 expression. All these mucins exhibit a repetitive peptide core involving a simple organization with almost identical tandemly repeated peptide domains 11-14'32'33 or a more complex organization involving alternation of heavily or poorly glycosylated domains. 21 Another common and intriguing feature of mucin genes is the polydispersed expression pattern of mRNA in Northen-blot analysis. In freshly isolated GBEC, MUC3, MUC5B, and MUC5AC were expressed at high level. GBEC expressed MUC5B at similar level as airway mucosa from which this gene was previously isolated. Results obtained for MUC2 and MUC3 are in good agreement with those previously obtained by immunohistochemistry.34 Surprisingly, the steady-state MUC3 and MUC5B mRNA levels were dramatically reduced in conventional GBEC primary cultures, thus indicating changes in mucin gene expression in vitro. Whether the expression of both genes are related to a differentiated state of quiescent GBEC in vivo remains to be elucidated. It must be pointed out that the expression of both MUC3 and MUC5B genes remained at high levels when GBEC were cultured on matrigel, a reconstituted basement membrane purified from the Engelbreth-Holm-Swarm tumor that promotes both survival and maintenance of specific functions in a variety of differentiated cells in vitro (Clament et al, unpublished). Cold storage of cultured GBEC in UW solution did not significantly modify the pattern of mucin gene expression. However, striking effects on mucin gene expression were found in rewarmed GBEC in the presence of bile. The steady-state levels of MUC2, MUC3 and MUC5AC mRNA were dramatically increased, whereas those of MUC1, MUC4, and MUC5B were not significantly modified compared with control cell cultures. Thus, the overexpression of at least three distinct mucin genes in cultured GBEC is specifically related to the presence of bile in media and precede the restoration of normal protein and DNA syntheses in rewarmed cells. Further work is needed to investigate whether increased expression of MUC2, MUC3, and MUC5AC corresponds to an adaptation of gallbladder cells to stagnant bile and involves change in mRNA synthesis and/or degradation. Indeed, it can be hypothesized that the overexpression of these mucin genes represent an attempt for GBEC to protect against the toxic effects of bile. In conclusion, our study gives new insights in both cold preservation and functions of gallbladder epithelial cells, particularly differential mucin gene expression. The striking effects of bile during the rewarming of GBEC preserved in UW solution suggest the need of an improvement in the management of liver graft during preservation and rewarming. Our results would suggest that cold ischemia times up to between 16 hours and 24 hours in UW solution are not deleterious to biliary epithelium, provided that the toxic effects of bile is decreased. Clinical studies with extensive washes of the biliary tree or infusion of hydrophilic

HEPATOLOGYVol. 21, No. 1, 1995

bile acid, e.g., ursodeoxycholic acid, are mandatory to evaluate their ability to decrease the rate of biliary complications after liver transplantation, independently of cold ischemia time. A c k n o w l e d g m e n t : W e a r e g r a t e f u l t o D r A. G u i l l o u z o for valuable discussions and helpful criticisms of the m a n u s c r i p t a n d D r O. L o r 6 a l for s t a t i s t i c a l a n a l y s i s . W e t h a n k P r o f e s s o r s J . Y. L e G a l l ( L a b o r a t o i r e d e B i o c h i m i e M 4 d i c a l e , H S p i t a l P o n t c h a i l l o u , R e n n e s ) a n d B. Bihain (Inserm U-391, Rennes) for analyses of bile comp o u n d s , M. P. D e l e s c a u t a n d P. M a t h o n f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e , a n d M. R i s s e l for p r e p a r i n g the illustrations. REFERENCES

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