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Inflammatory response in obstructive jaundice and peritonitis

Sewnath, M.E.

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Citation for published version (APA):Sewnath, M. E. (2003). Inflammatory response in obstructive jaundice and peritonitis.

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Download date: 03 Jan 2020

Endogenouss interferon-gamma protects against cholestaticc liver injury in mice

Miguell E. Sewnath1, Tom van der Poll2,3, Cornells J.F. van Noorden4,, Fiebo J.W. ten Kate\ and Dirk J. Gouma1

Departmentss of Surgery. "Experimental Internal Medicine, ''Infectious Diseases. Tropical Medicinee and AIDS. Cell Biology and Histology, and 'Pathology. Academic Medical

Center.. University of Amsterdam. Amsterdam. The Netherlands.

HKPATOLOGYHKPATOLOGY2002;2002; 36:1466-1477

Interferon--;Interferon--; protects against cholestatic liver injury

ABSTRACT T

Cholestaticc patients stiller from high perioperative morbidity and morlalilv. but the pathophysiology is still unknown.

Inlerferon-«ammaa (II-'N-y) max plav a role during cholestasis. Therefore, bile duel ligation (lull ) was induced in 11 X-y

((-chainn receptor-deficient (IFX-7R1- -) and wild t\pe (II N-/R ) mice. Hdl elicited increased IFN-y mRNA and

proteinn levels in the liver. One week alter bdl. IFN-yR,+/+ mice showed less severe jaundice and li\er injury than UN

y R rr - mice as relleeted bv lower bilirubin and liver en/yme levels. In accordance, livers of I l 'VyR i +/+ mice displaced

smallerr areas of necrosis b\ 2 3 than IFN-7R-/- mice upon histopaihologic examination (/> < (1.1)5). whereas mitotic

activityy and 1'CXA labeling index was more than twice higher in II X-yR,+/+ mice (/> 0.05). l ivers of I I 'X- /R+/ +

micee dispiaved higher rales of apoptosis as indicated bv DNA fragmentation rate, the number of apoptolie bodies, and

PARI'' immunostaining. Bdl was not associated with lethality in II X-yk : mice: II N-yR -/- mice, however, died from

100 daxs onwards and survival after 2 weeks was 02' \. (10 16). In conclusion, these data suggest that Il-'N-y protects

againstt liver injurv during exlrahepalic cholestasis by stimulation o\ apoptosis and subsequent proliferation of

hepatoevtess leading to elegant removal of damaged hepatoevtes thus preventing necrosis and concomitant inflammatory

responses. .

INTRODUCTIO N N

Thee pathophysiology of high perioperative morbidity and mortality of cholestatic patients is

largelyy unknown. Surgical mortality rates are higher than expected independently of the cause

off the biliary obstruction either benign or malignant.1'"" A variety of physiological abnormalities

occurss during obstructive cholestasis, including delayed wound healing with increased rates of

woundd infection, increased propensity to renal failure, increased plasma endotoxin levels, and

cholangitis."'"'1 1

Cholestasiss is a main feature of many human liver diseases, including primary biliary cirrhosis,

primaryy sclerosing cholangitis, and chronic allograft rejection following liver transplantation. In

manyy of these cholestatic diseases, the initial insult is directed towards the bile ducts and olten

mediatedd by T lymphocytes and cytokines.

Il-'N-yy is a dimcric glveoprotein that has mam immune regulator) functions and induces cell

proliferationn and apoptosis. Il-'N-y is mainh synthesized by natural killer cells and activated 1

cells.ss I he cell surface receptor complex for IFN-y is composed of two chains. Il ;N-yR] and

H-'N-yR:.1'""" The IFN-y homodimcr binds two molecules of the II-'N-yR, chain. Although the

IFN-yR:: chain cannot bind II-'N-y by itself.': it is required lor IFN-y signaling and receptor

complexx formation.

Thee technique of targeted gene deletion has been shown useful for the elucidation of functions

off cytokines.14 In particular, generation of mice with a homozygous disruption of the If N-yR,

chainn gene (IFN-yR|-/- mice) enables the evaluation of the tissue protective role oï' IFN-y in

immunee response and host defense during cholestasis. II-N-yR,-/- mice are normal, healthy, and

fertilee without obvious phenotypic differences when compared with wild type mice.

116 6

ChapterChapter 7

Inn the present study, we used Ii-'N-yR,-/- and wild type mice to determine the role oflT'N-y in hepaticc responses to acute biliary obstruction induced by bile duct ligation (bdl).

MATERIAL SS AND METHODS

Animal s s

Breedingg pairs o\' 129 Sv K\ wild type (II N-yR ) mice and 129 S\ interferon--.' receptor l(cc-chain)-deficient mice

116-200 g) were kindly provided by Dr. Manfred knpf (Basel Institute lor Immunology. Basel. Switzerland). Male mice

att the age ol S-10 weeks were used. They were all housed in one animal room, maintained under a 1 2-hour light/dark

cyclee with regular mice chow (SRM-A: Hope Farms. Woerden, the Netherlands) and w a t e r s/ libitum.

Surgicall procedures

Thee Institutional Animal Care and Lse Committee of the Academic Medical Center. Amsterdam, the Netherlands

approvedd all experiments.

Bdll and sham surgeiy was performed exactly as described pre\ iously. "" Briefly. after 5 days of acclimatization. surger\

wass performed under sterile conditions (t = 0). Mice were di\idcd randomly into sham-operated control groups and bdl

groups.. In total. 1 S4 Il'N-yR, - - mice and 1 K4 IFN-vR,-/- mice were used. Halt" of the mice in both groups were sham

operatedd and hall Underwent bdl.

Organn and blood sampling

Mortalityy was monitored during the first 14 days after sham operation and bdl (n = 16 in each groupi. In another

experiment,, animals were sacrificed daily during the first week and al 14 days after sham operation and bdl (n = 8 per

groupp per time point). Since ll-'X-vR-/ - mice started to die from hepatic failure from 10 days onwards (see results), data

obtainedd at 14 days were obtained from animals that were still alive. Blood samples were transferred to tubes containing

heparin,, and immediately placed on ice: plasma was prepared by cenlrifugation (3000 x i>. 10 min. 4"C), after which

aliquotss were stored at - 2 0 C. Samples from all liver lobes were removed rapidly after killin g the animals, dissected in

smalll portions and either stored at -20 C for biochemistry and histochemistry or fixed for histologv.

Histologyy and histochemistry

Smalll pieces of liver tissue from left and right liver lobes were fixed in Vi buffered freshly-prepared paraformaldehyde,

embeddedd in paraffin and stained for routine histologv (haemaloxylin and eosin). Histological examination was

performedd on coded samples by 2 independent investigators, blinded for treatment groups and biochemical and

histochemicall parameters. Four histopathologic features were scored semic]uantati\ely in 4 grades of severity (0-3):

portall inflammation, necrosis, ductular proliferation, and fibrosis. The 4 features were equalh weighed and summed.

Thiss scoring system has been described previously.

Too detect proliferating cells, liver specimens were stained lor proliferating cell nuclear antigen (PCNA) usin'j a

monoclonall antibody (Dako. Glostrup. Denmark). endogenous peroxidase activity was blocked with 0.1'/( NuN ,/(J.3'/f

(v/v)) H : 0 : dissolved in 5 ml dimethylformamide and 95 ml acetate buffer (pH 4.9. 50 niM) and 0.03'v (v/v) H (X As

secondaryy antibody, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Dako) was used. Finally, slides

117 7

Interferon--/Interferon--/ protects against cholestatic liver injury

weree rained lor HRP activity hy incubating the sections with 5 my/ml 3-amino-9-eth\l carhazole (Sigma. St.Louis. MO)

andd 0.03' < H O . '10 control tor background staining, incubations vv ilhout primary antibody were performed, from each

mouse-.. 4 sections of different liver lobes were stained and the number of PCN.\-expressing hepaioeytes was determined

inn 20 randomK selected fields. An average number of positive cells per Held was calculated and expressed as PCNA

labelingg index (PCNA-LI).

Periportall and periceniral /ones of li\er lobules were compared. Pericentral /ones were defined as areas of 5 layers ol

hepatocuess surrounding terminal branches ol hepatic \euis. For each liver lobe. I 2 sections were used and 3 periatrial

andd pericentral /.ones were randomK selected in each section.

Doublee stammu immunohistochcmisiiA to locali/e UN- / in relation with CD4-positive cells or macrophages was

performedd as described pre\ uuislv with minor modifications.' Between each step, sections were rinsed 3 limes in 0.01

mol/LL phosphate-buffered saline (PBS). pH 7.4. unless specified otherwise. Serial sections (ft LJ.ni thick) were cut on a

crvostat.. mounted on gelatin-coaled glass slides, and fixed in ice-cold acetone for 10 minutes. Sections were then

incubatedd in a moisi dark chamber with 10'r normal rabbit serum for 10 minutes and then incubated overnight at 4 C'

withh the primary monoclonal rat anti-mouse IF\'-v antihodv (IgG . clone XMGI .2; Pharmingen. San Diego. CA) diluted

inn PBS. HRP activitv was visualized bv incubating sections in a medium containing 50 mg 3.3'-diaminobenzidine-

tetrachloridee (Sigma) dissolved in 100 ml Tris-HC'l buffer (50 mM at pH 7,6) containing H : 0 : in a tlnal concentration

off 0 .01 ' ;. Subsequently, sections were incubated with the second primary antibody, either a monoclonal rat anti-mouse

CD44 (clone GKI.5: Pharmingen) antibody or a monoclonal ral anti-mouse pan macrophage (mO) (clone L4-80:

Pharmingen)) for I hour at room temperature. Then, sections were incubated again with a HRP-conjugated secondary

rabbitt anii-motisc lg(i. The second peroxidase activity was visualized with the use of 40 mg 4-ehloro-l -naphtol (Sigma)

dissolvedd in 0.2 ml dimcthylformamide and 0.3 ml ethanol and added to 100 ml of 50 mM Tris HCI buffer at pH 7.6

eontaininuu 0.01'-; H-0-. Linallv. sections were rinsed in distilled water and mounted in glycerin-gelatin. In control

incubations,, one of each oflhe primary antibodies was omitted.

Too evaluate hepatocyte proliferation, mitotic figures in 100 hepatocytes were counted in 12 different fields at x 400

magnificationn in liver sections ol 6 mice per group al each lime point and were expressed as percentage.

Assays s

Totall plasma bilirubin, alkaline phosphatase, /-glutamyl transferase, alanine aminotransferase (ALT) , and aspartate

aminotransferasee (AST) were determined will ) commercially available kils i Sigma) using a Hitachi analyzer

ii Boehrineer. Mannheim, (iermanvt according to the manufacturer's instructions. Cytokines were measured in duplicate

b\\ enzv me linked immunosorbent assay iLLISA i according to the instructions of the manufacturer i with detection limits

inn pmnl): IFV-Ml-Vz) . tumor necrosis factor-re (T\'F-u) (3 1.3) (both from R&D Systems. Abingdon. 1'nited Kingdom)

andd inierleukiii-6 ilL-6 ) OfXl (Pharmingen).

Live rr homogenutt ' preparation s

Forr measurements of cvtokines. liver homogenates were suspended in 2 volumes of lysis buffer containing 300 mM

NaC'1.. 30 mM Iris. 2 mM MgC'l,. \''< Triton X-100. and pepstatin A, leupeptin and aproptinin (all 2(1 ng/mi: pH 7.4:

Sigma)) and incubated at 4 (' for 30 min. Homogenates were centriluged at 3.000 \ ,<„< at 4 C for 10 mm to remove cell

debris,, and supernatanls were stored at 20 C' until assays were performed.

118 8

ChapterChapter 7

RNAA preparation and reverst' transcriptase PCR

Tissuee samples were homogenized in 1 ml of TRIzol Reagent (Gibco, Berlin. Germany > according lo the manutacturer's

instructionss and total RNA was isolated using chloroform extraction and isopropanol precipitation. RNA was dissolved

inn diethvlpyrocarbonate (DFPC)-treated water and quantified by spectrophotometis. cDNA was s\nthesi/ed by mi\in»

22 u.gol RNA with 0.5 ^g oligo(dT) (Gibco). and by incubating the solution (total volume 12 u.1) tor 10 minutes at 72

C.. Subsequently. S pJ of a solution containing 5 x Superscript First Strand Synthesis System for RT-PCR (Gibco). 1.25

niMM of each of dNTPs (Amersham. Amersham. United Kingdomt. 10 inM dithiothreitol (D IT ; Gibco). and the

Superscriptt Pro-amplification system (Gibco) wore added, and the final solution was incubated for fit) min at 37 C. For

RR I-PCR of IFN-y. equivalent amounts of cDNA (5 p.1) were amplified m a solution (20 p.1) containing 4'< DMSO

II Merck. Miinchon. Germany >. 12.? ^g BSA (Biolabs. Carle Place. NY>. 1.2? niM of each ol dNTPs, I0\ PCR buffer

(0.677 M Tris-HCI (pH S.S).67 mM MgCI-.O.I M |Fmercaptoethanol.67 p.M FDTA. 0.166 M < MR )S04) . 0.5 I of

AmpliTaqq DNA polymerase (Perkin Rimer. Branchburg, NJ), and the forward (F) and the reverse (R) primers < 100 mM

each). .

Thee PCR reactions were carried out in the thermocycler Gene AMPQ-0 PCR System 9700 < Perk in Rimer. Branchburg.

NJ).. Cycling conditions for PCR amplification of IFN-y and |}-actin wore: at 94 C for 5 minutes i I eyele) followed b\

333 (IFN-y) or 24 <|ï-actim cycles at 95 C for 60 seconds, at 5S C lor 60 seconds and 72 C tor 60 seconds, followed

byy a final extension phase at 72 C for 10 min. The primers used were: IFN-y (F): 5 -

GAAGTGAAGTGGAAGGGXXXAGAAX-.V .. (R) 5-AGGGAAACTGGGAGAGGAGAAATAT-3'; and |3-actin (F):

5-GTCAGAAGGACTCCTATGTG-3'.. <RI: 5--GCTCGTTGCCAATAGTGATG-3'. The PCR products were

separatedd on 1.5'.i agarose gel containing 0.5 x TBF. (50 mM Tris, 45 mM boric acid. 0.5 mM FDTA. pH X.3) with 0.5

H-gmll ethidium bromide. For IFN-y and |i-actin. the number of cycles that resulted in linear amplification was

determinedd (data not shown) Single band intensities of IFN-y and iFactin were analyzed using the Ragle Rve II \ ideo

systemm (Eaglet) Software System: Strulagenc. Fa Jol la. CAi using the negative image as described pre\ IOUSK . '

Apoptosis s

MorphologicMorphologic determination of apoptotic bodies

Apoptoticc bodies were scored as defined by Kerr and co-workers.' The number of apoptotic bodies per I 00 hepatoevtes

wass counted in I 2 different fields at \ 400 magnification in liver sections of 6 mice per group at each time point and

expressedd as percentage.

DNADNA fragmentation assay

Too quantify apoptosis as characterized by DNA fragmentation, cytoplasmic oligonucleosome-bound DNA was

determinedd using an RLISA kit according to the manufacturer's instructions (Bochringer). In brief, cytoplasmic fractions

off homogenized liver tissues after centri (ligation (angle rotor. 4 C. 15 min, 13.000 x g supernatant) were used as antitzen

sourcess in ERISA's. Percentages of fragmentation in comparison to controls wore calculated from the absorbance values

ass previously described.

ImmunodetectionImmunodetection of poly (ADP-ribose) polymerase (PARH) p<S5 fragment -positive cells

Too rule out fnlse-positivitv in the determination of apoptosis based on DNA fragmentation rates due to apoptosis of non

parenchymall cells and/or hepalocy te necrosis. PARP immunohistochemistry was performed as additional methodology.

119 9

Interferon--;Interferon--; protects against cholestatic liver injury

Sectionss ot paraftin-emhedded li\cr were deparalllni/ed in \ \ lunc. rehydraled in a descending series ot alcohols, and

endogenouss peroxidase was blocked by incubaiion with 0 .3 ', H : ( ) : in niclhanol tor 20 min. Liver sections were then

incubatedd in a microwave oven at 100 C lor Hl min in citrate butler (0.0 I M. pH 6). Sections were washed in PBS and

incubatedd with 10', normal goat serum tor 15 min. The primary anti- PARP pK5 fragment polyclonal antibody

iPromega.. Leiden. The Netherlands! was diluted in PBS and sections were incubated overnight at 4 C. Altera briet rinse

inn PBS. sections were incubated lor 30 mm at room temp with hiotinylated swine anti-rabbit antibody (Dakoi in PBS

containine.. 5'< normal mouse serum and subsequently with sireptavidin-bioiin peroxidase complex (Dakoi tor 30

minutess at mom temp. Peroxidase acli\ii \ was detected by incubation in a solution ot' I mg/ml 3.3-diammoben/idine

tetralndrochloridee iSigmai and 0 0 1 ', H.-().- in 50 niM Tris-HCI (pH 7.6>. Sections were counterstained with

l iaemaumlin.. In control incubations, the primary antibodv was replaced by PBS. Apoptosis was estimated hy counting

thee number of PAKP-positi\e cells per unit area.

Statisticall analysis

Statisticss were performed using the SPSS Base 10.0 for Windows Statistical Software Package (SPSS. Chicago. IL) .

Al ll results are gi\enas mean SLM. Sur\i\al curves were compared with the log-rank test. Differences in biochemical,

immunological,, immunohistochemical and histological data were analyzed using the Mann-Whitney U test. Changes

inn time were anaK/ed h\ one-wav analysis of variance. Differences at indicated time points were assessed by

Bonlerroni'ss test for multiple comparisons where appropriate. A two-tailed p value < 0.05 was considered to indicate

significanll differences.

RESULTS S

Biochemicall evidence for increased cholestasis and liver injur y in IFN-yRi-/ - mice Plasmaa lewis of total bilirubin, alkaline phosphatase, and y-glutamyl transferase (data not shown

lorr y-glutamvl transferase) were eomparable in all 4 experimental groups before surgery (t = ()).

andd signitieant ehanges did not oeeur thereafter in both sham groups. In bdl II'N-yR;+/+ and

Il-'N-yR,-/-- mice, these buxhemical cholestatic parameters increased comparably during the first

77 davs (li g 1 ). At 14 davs after bdl. when the sunning Il-'N-yR,-/- mice were critically ill . these

cholestaticc parameters were higher in bdl Il-'N-yR,-- mice than in Il-'N-yR]+/+ mice.

Shamm and bdl mice exhibited comparable levels of plasma ALT and AST at t = 0. Whereas in

shamm mice plasma levels of these liver enzymes did not change as compared to preoperative

values,, bdl was associated with progressive rises in ALT and AST plasma levels with

significantt higher concentrations m 11'N-yR,-/- mice from day 2 onwards (/J < 0.05). These data

suggestt that although bdl induced the same extent of cholestasis. Il-'N-yR,-/- mice were more

pronee forcholestasis-induced liver injury.

120 0

ChapterChapter 7

JJ 400n o o || 300

-B-IFN N --

---#-IFN--

yR,+/+,, bdl ïR,-/-,, bdl

/RT+/+.. sham /Ri-/- ,, sham

700i i

Dayss after operation 77 8 14

Dayss after operation Figuree 1. Mean ( :SE) plasma levels of total bilirubin, alkaline phosphatase (alk. phosph.). AST and ALT in 1FN-yR|-- and IFN-yR,-/- alter bile duet ligation (bdl) or sham operation (n = 8-10 per group al each time point).

Mortalit yy in cholestatic IFN-yR,-/- mice

Shamm surgery was not associated with morbidity or mortality during the 2-week follow-up. Bdl IFN-yR,+/++ mice became progressively ill from day 14 onwards, but mortality did not occur up too 4 weeks after bdl.(Fig. 2) In contrast, bdl IFN-yR,-/- mice showed severe signs of illness from dayy 7 onwards, as reflected by lethargy, anorexia, ruffled fur. and huddling behavior. Mortality mm cholestatic D-'N-yR,-/- mice occurred from day 10 onwards. Al 14 days after bile duct ligation, survivall in IFN-yRr/- mice was 62% (10/16) as compared to 100% in IFN-yR,+/+ mice (p <

0.01). .

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75 5

50 0

25 5

04 4

PP < 0.01

,, bdl O lFN-yR r / - ,, bdl

-

0 0 - r --9 9 To" "

i i

11 1 77 8 9 10 11 12 13 14

Dayss after bile duct l igation Figuree 2. Mortality in cholestatic IFN-yR, shownn in this figure; n - 16 per group).

andd IFN-yR,-/- mice. Mortality did not occur in the sham groups (no

121 1

Interferon-yprotectsInterferon-yprotects against cholestatic liver injury

Productionn of IFN-y durin « cholestasis Afterr bdl, plasma and liver levels of IFN-y showed a profound increase in time, reaching a

plateauu after 3 days (Fig. 3A, B;/?<0.01), but were 4 to 8-fold lower in IFN-yR,-/- mice than

mm IFN-yR,+/+ mice. In addition. RF-PCR confirmed expression of IFN-y mRNA in livers of

bothh IFN-yR,+/+ and IFN-yRr/- mice after bdl (Fig. 3C).

33 P< 0.05 II 1 1 1 1 1 1 1—' I r

0 11 2 3 4 5 6 7 8 14 Dayss after surgery

Dayy 4 Day 7 Day 4 Day 7

ï\ ï\ II FN-/

B B 800 0

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00 1 2 3 4 5 6 7 8 14 Dayss after surgery

•• IFN-yR, OO IFN-yR, -•- IFN-yR! !

• QQ IFN-yR,

+/+.. sham -/-.. sham

+/+,, bdl

-/-,, bdl

Figuree 3. Mean I SEM) levels of IFN-y protein inn plasma (A) and livers (B) of IFN-yR +/+ and IFN-yRr/-- mice after bdl or sham operation (;i = 88 per group at each time point, except for bdl IFN-yR,-/-- al 14 days. // 5) and IFN-y and [5-actin mRNAA levels m livers of IFN-yR +/+ mice al 4 andd 7 days alter bdl or -.ham (/i = S per group) (C). .

^^ gp ^ j? «*

IFN-yR,+/+ + IFN-yy R,-/-

Cytokincc response to bdl in IFN-yR,-/-mice e

Plasmaa TNF and lL-6 levels in both bdl IFN-

yR,+/++ and IFN-yR i-/- mice increased

significantlyy from da\ 2 after surgery compared to sham mice ( Fig. 41. From day 3. plasma TNF

andd II.-o levels were sigmiicanth higher in bdl IFN-yR,-/- mice than in bdl ll ;N-yR,+/+ mice.

Plasmaa TNF and lL-6 levels remained undetectable in all sham mice at all time points.

125 5

ff 100H "& & BB 75-U. .

ZZ 50-

25--

0J J

BB 90Ch

II 600-01 1

dd 300-

•• IFN--..R,H

OO IFN-yR,

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DD IFN-yR,

fr,fr, Sham

/-.. sham

/ • .bd l l

/-.. bdl

00 2 3 4 5 6 7 8 14 0 11 2 3 4 5 6 7 8 14 Dayss after surgery

Figuree 4. Mean ( SEM) TNF (A) and IL-6 (B) plasma levels in II N-yR or IFN-yR -/- mice alter bdl or sham operationn [n = S-l 2 per group al each time point,.

122 2

ChapterChapter 7

Increasedd liver pathology in cholestatic IFN-yR,-/- mice LiverLiver histopatlwlogy

Inn both sham Il-'N-yR,+/+ and II ;N-yRr/- mice, liver tissue appeared to he normal at all time

points.. IFN-yR,-/- mice sacrificed from 3 days alter bdl onwards showed overt lesions in the

liverr that were macroseopically visible. In all bdl Il-'N-yR,-/- mice from 4 days after bdl onwards,

extensivee necrosis of hepatocytes was observed {Fig. 5B). whereas at this time point only slight

degenerativee changes were observed in hepatocytes without necrosis in sections of bdl IFN-

yR,+/++ mice (Fig. 5A). The size of these necrotic lesions in bdl Il-'N-yR,-/- mice increased

dramaticallyy in time, occupying up to MV/t of the total area of the liver sections at 14 days after

bdll in mice that were still alive (Fig. 5I-') Furthermore, these Il-'N-yR,-/- mice showed

significantlyy more inflammatory cells in portal tracts, predominantly consisting of

polymorphonuclearr cells (PMN). than bdl Il-'N-yR]+/+ mice. Moreover, infiltrations of PMN

weree prominent in liver parenchyma, often causing occlusion of sinusoidal lumens (data not

shown).. In contrast. IFN-yR,+/+ mice that underwent bdl only showed mild forms of hepatic-

lesionss (Fig. 5A. C. E). Parenchymal necrosis and portal inflammation were observed in livers

off these bdl mice from day 3 onwards (Fig. 5A. C), and becoming more severe only after 2

weekss (Fig. 5F).

Liverr tissue of bdl IFN-yR,-/-mice showed significantly more damage than liver tissue of bdl

IFN-yR,+/++ mice at 7 days after bdl (Table 1). Scores for fibrosis and necrosis were significantly

higher,, but not for portal inflammation and ductular proliferation. The total score of liver damage

afterr bdl as plotted on a time-scale, shows that liver damage was larger in bdl IFN-yR,-/- mice

thann in bdl IFN-yR, +/+ mice (Fig. 6).

Inummohistochemistrx Inummohistochemistrx

Cellss invading portal areas included both PMN and mononuclear ceils. To define the phenotype

off these mononuclear ceils, serial liver sections at 7 days after operation were stained for CD4+

TT cells and (resident) m<J)s (Table 2).

Cholestasiss caused a significant rise in the number of infiltrating CD4+ T cells in portal tracts

off bdl mice as compared to sham mice, but diil'erenees were not found between IFN-yR,+/+ and

IFN-yR,-/-- mice. In cholestatic livers, the number of CD4+ T cells that were positive for IF'N-y

wass similar in livers of bdl Il-'N-yR, +/+ (Fig 7A) and bdl IFN-yR,-/- (Fig. 7B) mice. The

numberr of (resident) m<t>s was significantly increased in livers of bdl mice as compared to sham

mice.. The number ofmOs in livers of Il-'N-yR 1+/+ and IFN-yR 1-/- sham mice was similar:

however,, the number of m<t>s was significantly higher in livers of bdl IFN-yR I-/- mice as

comparedd with IFN-yR 1+/+ (p < (>.(>5). These cells were further characterized in a double

stainingg analysis using the IFN-y-specifie antibodv XMCi 1.2.

Interferon-';Interferon-'; prolecis against cholestatic liver injury

Dayy 4

I F N - T R ^ / * * IFN-yR.,-/--

PVV p-

Dayy 7 LQ.

Dayy 14

Figur ee 5. Representative H&E-stained liver sections from cholestatic mice (n = 8-10) at 4 (A. B), 7 (C, I)), and 14 (E.. F) days after bdl. Note the larger size and number of necrotic lesions (arrowheads) in cholestatic Il'N-vR,-/- mice (B.. 1). and F) as compared to cholestatic [FN-yR,+/+ mice (A. C'. El. PV = portal vein. CV = central vein. Bar = 1000 p.m.

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Figur ee 6. Mean ) totall histological scores of liverr damage in IFN-',/Rii • - and IFN-yR,-/-micee during 14 days after hdll or sham operation (// == 6-8 per group at each timee point). The total histologicall score is composedd as in Table l.

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Interferon-yInterferon-y protects against cholestatic liver injury

Inn sham mice, no mOs were found that were positive for IFN-y. In contrast, mOs positive for

IFN-yy were found both in bdl [FN-yR,+/+ (Fig. 7C) and in bdl IFN-yR,-/- (Fig. 7D) mice.

indicatingg that in mice IFN-y is produced b\ activated mOs under cholestatic circumstances.

Notee that the number of mOs positive for IFN-y were significantly lower in IFN-yRr/- mice as

comparedd to [1- N-yR,+/+ mice.

Figuree 7. Representative liver sections from IFN-yR, • (A. C i and IFN-yR -/- 1B, D) mice al 7 days after bdl (n = 88 mice per group) thai were double stained lor CD4 T cells (arrows) and IFN-y (A. 1?) or m<t>s (arrow heads) and IFN-yy <c- D). The number of CD4 • T cells thai were positive for IFN-y «ere comparable in bdl ll'N-yR +/+ (A) and bdll IFN-yR -/- |B) mice. However, the number of mOs that «ere positive for UN-, was larger in livers oi bdl IFN-yR,, • - mice (C) than in bdl IFN-yR -/- mice (Di. Bar = 100 uni.

Decreasedd liver regeneration capacity in cholestatic IFN-yR r /- mice Liverr damage induces increased mitotic activity as assessed by PCNA expression. PCNA

stainingg was virtually absent in sham mice, with an average PCNA-LI ol 2 1 at 7 days alter

operationn (Fig. 8A. B). In contrast, proliferative activity was high al 7 days after induction of

cholestasis,, particularl) in periportal /ones (Fig. 8C, D). PCNA expression in hepatoeytes was

significantlyy higher m bdl IFN-yR,+/+ I big. 8C) mice than in bdl IFN-yR,-/- (Fig. 8D) mice. The

averagee PCNA-LI in liver sections of bdl IFN-yR,+/+ vs. IFN-yR,-/- mice was 42 9 and 22

7.. respectively, at 7 days after bdl (p < 0.051. Thee number of mitoses in sham mice did not exceed baseline levels (0.43 0.13% ) (Fig. 9A). Thee number of mitoses in bdl U;N-yR|+/+ mice was higher at each time point than in bdl 1FN-

120 0

ChapterChapter 7

yR,-/-- mice, reaching peak levels after 4 days and slowly decreasing afterwards. At 7 days of

cholestasis,, mitotic activity in bdl II-N-yR,+/+ mice had decreased from a peak level of 3.6

0.6%% (data not shown) to 2.4 0.4'i: this was still significantly higher than 1.6 0.3 '?i in bdl

IFN-yR,-/-- mice (Fig.9A;/? = 0.01 ).

Apoptoticc body counts at 7 days after operation are shown in Fig. 9B. At this time point, there

weree hardly any apoptotic bodies in both sham groups, whereas liver sections trom bdl IFN-

yR,, +/+ mice showed significantly more apoptotic bodies than bdl IFN-yRr/- mice.

Quantificationn of fragmented DNA revealed that the rate of apoptosis in liver homogenates after

77 days of bdl m CFN-yRr/-mice was significantly lower than that in IFN-yR,+/+ mice (Fig. 9C).

II he apoptotic rate had decreased more strongly m IFN-yR]+/+ mice than m !FN-yRr/- mice at

144 days after bdl but the difference between both bdl groups was still significant (/> < 0.05).

IFN-yR1+/++ IFN-7Rr /-

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Figuree 8. PCX A staining of liver sections from sham (A, B) and bdl iC'. D) animals (n = S mice per group) at 7 days alterr surgerj. Arrows indicate PCNA-positive hepatocj tes, arrow heads necrotic areas. The number of PCNA-positivee cells was decreased and the number of necrotic areas was increased in cholestatic IFN-yR,-/- mice. PV = portall vein, CV = central vein. Bar= 100 u,m.

Liverr sections were stained with the anti-PARP-p85 fragment antibody that binds to the p85-kDa

127 7

Interferon-yInterferon-y protects against cholestatic liver injury

caspasee 3-cleaved fragment of PARP. BDL mice exhibited significantly more PARP-positive

cellss as compared with sham mice. This difference was more overt in bdl IFN-yR,-/- mice (Fig.

10).. confirming both the results of the DNA fragmentation rate analysis and the morphological

determinationn of apoptotic bodies.

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Figuree 9. (A) Mitotic index is presented as mean number I SEM) of mitotic bodies/100 hepatocytes in livers of shamm and bdl animals ai 7 days alter surgery (H 8 mice per group). Mitotic index is decreased in IFN-yR,-/- mice. (B)) Apoptotic activifs in livers of bdl IFN-yR,- - mice is decreased as compared to bdl IFN-yR,+/+ mice at 7 days afterr operation. (C) Mean i SEM) absorbance values as a measure of DNA fragmentation in liver homogenates fromm bdl IFN-yR, - ' and bdl IFN-yR,-/- mice at 7 and 14 days of cholestasis (» = 8 per group, except for bdl IFN-

DISCUSSION N

Inn the present study, we show that murine cholestasis induced by bdl is associated with enhanced

productionn of IFN-y 111 the liver, confirming and extending previous reports. " " More

importantly,, this endogenous IFN-y appeared to play a key role 111 controlling earh cholestatic

liverr injury. The increased liver injury 111 bdl IFN-yR,-/- mice, as reflected by both clinical

chemistryy and histopathology, was associated with mortality within 2 weeks after induction ol

cholestasis,, whereas bdl IFN-yR,+/+ mice survived easily up to 4 weeks of cholestasis.

Furthermore,, li \ er damage 111 bdl IFN-yR,+/+ mice was associated with increased apoptosis and

liverr regeneration, whereas liver damage 111 bdl IFN-yR,-/- mice was characterized by necrosis.

largee inflammatory infiltrates, and a diminished hepatic regenerative capacity. In addition, we

showw that not only CD4+ T cells but also mOs produce IFN-y during cholestasis 111 mice It has

beenn previously reported that murine mOs can be induced to secrete IFN-y after combined

stimulationn with 1L-12 and 11.-18 or in a TNBS (2,4,6-trinitrobenzene sulfonic acid)- induced

128 8

ChapterChapter 7

colitiss model.~''" However, as far as we know this is the first study that reports in vivo [FN-y

productionn by mouse liver mOs The fact that ll-'N-y concentrations were higher in bdl IFN-

yRi+/++ mice than in bdl IFN-yR,-/- mice is in line with earlier studies with IfN-yR,-/- mice and

suggestss that the presence of an intact IfN-yR] provides an autoregulatory loop to regulate the

amountt of II-'N-y expressed both locally at the site of inflammation and at remote sites.25"27 These

dataa suggest that [FN-y is an important regulator of apoptosis and regeneration in the liver in

responsee to extrahepatic cholestasis, and that apoptosis protects the host against excessive liver

injuryy in this condition.

Mortalityy rates due to bile duct obstruction in wild type mice were similar as in previous

reports.. " while Ezure el al. reported mortality in cholestatic mice only alter more than 9 weeks

IFN-yR1+/++ IFN-TR.,-/-

Sham m

BDL L PV V

cvv • PVV A

4 4

Figuree Kl. Immunohistochemical staining ofPARP in liver sections from sham (A. B) ami bdl (C, D! animals {n = SS mice per groupi al 7 da_\s after surgery. The number of PARP-positive cells (arrows) «as decreased and the numberr of necroticc areas (arrowheads) «as increased in cholestatic IFN-yR -/- mice. PV = portal vein, CV = central vein.. Bar = 100 u,m.

afterr bdl." In this respect, the [00% survival alter bdl in wild type mice that we found is not

surprisingg although mice are less tolerant to bdl than rats.30"32

Thee extent ot TNF production during cholestasis is controversial. Some studies report elevated

serumm levels of TNF during cholestasis." 6 whereas others report no alterations due to

cholestasis."'"" ' In the present study, TNF concentrations increased after induction of cholestasis

andd were significantly higher m IfN-yR,-/- mice, likely due to the ongoing necrosis and

inflammation,, rather than directly caused bv the absence of a functional IfN-yR, considering that

IFN-yy enhances rather than inhibits cytokine production.8'39

Althoughh bdl IfN-yR,-/- mice showed significantly higher lL-6 plasma levels than their wild

II 29

Interferon--:protectsInterferon--:protects against cholestatic liver injury

typee counterparts, this cytokine could not sustain the compensatory increase in liver mass

commonlyy observed in chronic obstructive cholangiopathy. Ezure et al. demonstrated that

exogenouss IL-6 treatment can contribute to biliary tree integrity and maintenance of hepatocyte

masss during chronic liver injury by completely reversing loss of liver mass by stimulating

hepatocytee proliferation.4'1 The fact that we found higher II.-6 levels in bdl IFN-yRr/- mice than

inn bdl HN-yR|+/+ mice indicates that in the acute phase of cholestatic liver injury the initial

increasee in liver mass during the first week is not II.-6 but IFN-y dependent despite a brisk

upregulationn ofIL-6 within hours to days after bdl (Fig. 6).

Inn accordance with previous studies.424, PCNA expression at 7 days after induction o['

cholestasiss was higher in periportal and mid/.onal hepatocytes than in pericentral hepatocytes

indicatingg a wave of cell regeneration particularly in these /.ones. Akyol et al. did not find a

significantt relationship between cholestasis and PCNA positivity in samples of human liver in

variouss stages of disease.44 However, one should bear in mind that the high PCNA-LI found in

thee present study is a response to relative!} early cholestatic changes, most likely mediated not

onlyy by unconjugated bile salts but even more so by cytokines released by inflammatory cells.

Indeed,, proliferative activity is increased during progression of chronic hepatitis and decreases

duringg progression of cirrhosis.4'1 The significantly higher percentage of PCNA-expressing nuclei

inn livers from bdl IFN-yR|+/+ mice was also surprising, because TNF has been implicated to

playy a crucial role in liver regeneration,47"4'' and TNF levels were higher in bdl IFN-yR]-/- mice

thann in bdl IFN-yRi+/+ mice. It is conceivable, that spillover of toxic reagents after liver cell

necrosiss followed by a rapid and substantial release of pro-inflammatory mediators from Kupffer

cellss and circulating mononuclear cells resulted in a disturbed regenerative response.

Ikl ll IFN-yR|+/+ mice exhibited higher rates of mitoses that were paired with higher rates of

apoplosiss as compared with bdl IFN-yR r / - mice, so that the number of hepatocytes remained

constantt and tissue homeostasis was maintained, which is in agreement with previous findings/

Duringg chronic and subacute liver diseases, cell death and compensatory regeneration occur

simultaneously,, and the present study proves that IFN-y plays an important role in maintaining

liverr homeostasis during acute liver injury.

Decreasedd apoptosis and regeneration of hepatocytes accompanied the increased necrosis in bdl

IFN-yR,-/-- mice. Apoptosis occurs in normal liver, albeit in low levels, and in various forms oi

liverr disease The cytotoxic effect oi' IFN-y on hepatocytes is known to involve apoptosis.' '""̂

thuss the lower apoptotic rate in bdl IFN-yRr/- mice can be explained by the lack of effects of

IFN-y.. Previous studies have shown that involvement of IFN-y leads to more programmed cell

deathh and less necros is /04 further supporting this interpretation of our data.

Inn conclusion, our results provide evidence that bdl in mice leads to increased levels of

endogenouss Il ;N-y. which is able to induce the apoptotic pathway to prevent necrosis In this

wav.. liver damage is limited in cholestatic mice with a functional IFN-y receptor, in contrast with

overtt necrosis in IFN-yRr/- mice Further insight into the mechanisms ol' IFN-y-mduced

apoptosiss and subsequent proliferation of hepauvytes may help providing additional strategies

forr the treatment of cholestatic liver diseases.

130 0

ChapterChapter 7

REFERENCES S

1.. Armstrong CP. Dixon JM, Taylor TV', Da\ ies GC. Surgical experience ol deeply jaundiced patients with bile ductt obstruction. Br J Surg 1984: 7 1:234-238.

2.. Reynolds J V. Murchan P, Redmond HP. Wat son R\V. Leonard N. Hill A et al. Failure of macrophage activation inn experimental obstructive jaundice: association with bacterial translocation. Br J Surg 1995: 82:534-538.

3.. Clements WD. McCaigue M. Erwin P. Halliday I. Rowlands BJ. Biliarv decompression promotes Kuptler cell recoveryy in obstructi\e jaundice. Gut 199ft: 38:925-93 1.

4.. Grove J\V. Maessen JG. Tiebosch T. Buurman WA. Gouma DJ. Prevention of postoperative complications in jaundicedd rats. Internal biliary drainage versus oral lactulose. Ann Surg 1990: 212:221-227.

5.. Hunt DR. Allison MH, Prentice CR. Blumgarl LH. Endoloxemia. disturbance of coagulation, and obstructive jaundice.. Am J Surg 1982: 144:325-329̂

6.. Karsten TM. Allema JH. Reinders M. van Gulik TM, de Wit ET, Verbeek PC et al. Preoperative biliary drainage,, colonisation ofbile and postoperative complications in patients with tumours of the pancreatic head: aa retrospective analvsis of 241 consecutive patients. Eur J Surg 1996: 162:88 1 -888.

7.. Coulter KR. Doseff A. Sweeney P. Wang Y. Marsh CB. Wewcrs MD et al. Opposing effect by cytokines on fas-mediatedd apoptosis in A549 lung epithelial cells. Am J Respir Cell Mol Biol 2002: 26:58-66.

8.. Boehm I . Klamp T. Groot M. Howard iC. Cellular responses to interferon-gamma. Annu Rev Immunol 1997: 15:749-795. .

9.. Bach HA. Agtiet M. Schreiher RD. The IFN gamma receptor: a paradigm for cytokine receptor signaling. Annu Revv Immunol 1997: 15:563-591.

10.. Peslka S. Kotenko SV. Mulhukumaran G. l/.otova I.S. Cook JR. Garotta G. The interferon gamma (IFN-gamma)) receptor: a paradigm lor the multichain cytokine receptor. Cytokine Growth Factor Rev 1997: 8:189-206. .

11 1. Hanger J, Garotta G. Pestka S. Interferon receptors. Biotherapy 1996; 8:163-174. 12.. Rashidbaigi A. Hanger J A. Jung V. Jones C, Morse HG. Tischfield J A et al. The gene lor the human immune

interferonn receptor is located on chromosome 6. Proc Natl Acad Sci USA 1986: 83:384-388. 13.. Jung V, Rashidbaigi A. Jones C. Tischfield JA. Shows TB. Peslka S. Human chromosomes 6 and 21 are

requiredd for sensitivity to human interferon gamma. Proc Natl Acad Sci L'SA 1987: 84:4151-4155. 144 Huang S. Hendriks W. Althage A. Hemmi S. Bluethmann H, Kamijo R et al. Immune response in mice that

lackk the interferon-gamma receptor. Science 1993: 259:1742-1745. 15.. Sewnath MH. van der Poll T. ten Kate JWF. Van Noorden CJ. Gouma DJ. Interleukin-1 receptor typo I gene-

deficientt bile duct-ligated mice are partially protected against endotoxin HEPATOUOGY 2002: 35:149-158. 16.. Sewnath MH. Hevels JHM. Oude Hlferink RP. Van Noorden CJF. ten Kate JWF. van Deventer SJH et al.

Hndotoxin-inducedd mortality in bile duct-ligated rats after administration of reconstituted High-Densitv lipoprotein.. Hepatology 2000: 32:1 289-1 299.

17.. Kerr JH. Gobe GC, Winterford CM. Harmon BV. Anatomical methods in cell death. Methods Cell Biol 1995: 46:1-27. .

18.. Heist M. Gantner H. Bohlinger I. Gennann PG. Tiegs G. Wendel A. Murine hepatocyte apoptosis induced in vitroo and in vivo by TNH-alpha requires transcriptional arrest. J Immunol 1994: 153:1778-1788.

19.. Itoh S. Matsu/aki Y. Kimura T. Ikegami T. Shoda J, Fujiwara M et al. Cytokine profile of liver-infiltrating CD4++ T cells separated from murine primary biliary cirrhosis-like hepatic lesions induced by graft-versus-host reaction.. J Gastroenterol Hepatol 2000: 15:443-451.

20.. Hechner AJ. Velasquez A. Knudsen KR. Johanns CA. Tracy TFJr. Matuschak GM. Cholestatic liver injury increasess circulating TNF-alpha and IH-6 and mortality alter Escherichia coli endotoxemia. Am J Respir Crit Caree Med 1998; 157:1550-1558.

21.. Sekiya H. Komatsu T. Isono H. Eurukawa M, Matsushima S. Yamaguchi N et al. Decrease in the prevalence off IH-4-producing CD4+ T cells in patients with advanced stage of primary biliar\ cirrhosis. Am J Gastroenterol 1999:94:3589-3594. .

22.. Tilg H. Wiltner A. Vogel W. Herold M, Nolchen B. Judmaier G et al. Serum levels of cytokines in chronic liver diseases.. Gastroenterology 1992: 103:264-274.

23.. Munder M, Mallo M. Hichmann K. Modolell M. Murine macrophages secrete interferon gamma upon combined stimulationn with interleukin (IE)-12 and IE-18: A novel pathway ol'autocrine macrophage activation. J Exp Medd 1998: 187:2103-2108.

24.. Camoglio H. te Velde AA. de Boer A. ten Kate HJ. Kopt M. van Deventer SJH. Hapten-induced colitis associatedd wiih maintained Thl and inflammatory responses in IHN-gamma receptor-deficient mice. Eur J

131 1

Interferon--;Interferon--; protects against cholestatic liver injury

Immunoll 200»: 30: 1486-1495.

25.. Cock field S M . Ramassar V. Nouja im .1. \an der Meide PH. Halloran I5!-. Regulation ot I FN-gamma expression

inn vivo. IFN-gamma up-regulates expression of its m K N A in normal and lipopolysacchaiide-stimulated mice.

JJ Immunol 1 W : 150:717-725.

26.. Halloran PF. Autenned P. Ramassar V. I 'rmson .1. Cock Held S. I .ocaI T cell responses induce widespread M H C

expression,, Fvidence that IFN-gamma induces its own expression in remote sites. J Immunol 1992: 14S:3S37-

3846. .

27.. D i Vlar / io P. Puddu P. Conti L Belardelli P. Gcssam S. Interferon gamma upregulaics its own gene expression

inn mouse peritoneal macrophages. .1 hxp Med 1994; 1 79:1731-1 73d.

28.. Bemelmans M H . Ciouma l )J . Greve JW, Buurman W A . Cytokines tumor necrosis factor and intcr lcukin-6 in

experimentall b i l iary obstruction in mice. H F P A T O F O G Y 1992: 15:1 132-1 136.

29.. F./tire T, Sakamoto T . Tsuj i H. F u n / JCi. Murase N. Fung .1.1 et al. The de\elopment and compensation of

bi l iaryy cirrhosis in mterleukin-6-deficient mice. Am .1 Pathol 2000: 156:1627-1639.

30.. Kimmings A N . van Deventer S.1, Obertop H, Clou ma IXI. Treatment wi th recombinant bacteiïcidal/pcrmeabilily-

increasingg protein to prevent cndotoxin-induccd mortal i ty in bile duct-l igated rats. .1 Am Col l Stug 1999:

189:374-379. .

3 1 .. Diamond T. Rowlands B.I. Fndotoxaemia in obstructive jaundice. HPB Surg 1991:4:81-94.

32.. K immings A N . van Deventer S.I. Obertop H. Rauvvs FA. Gouma. D.I. Inflammatory and immunologic effects

off obstructive jaundice: pathogenesis and treatment.. I A m Col l Surg 1995: ISF567 -5S1 .

33.. I.echner A.I. Velasquez A. Knudsen KR. Johanns C A . Tracy 'I'FJr. Matuscliak CiM. Cholestatic l iver in jury

increasess circulat ing TNF-alpha and IF 6 and mortality after Fschcnchia coli endotoxemia. A m .1 Respir C'rit

Caree Med 199S: 157:1550-1558.

34.. O'Nci l S. Hunt J. Fi lk ins .1. Gamel l i R. Obstructive jaundice in rats results in exaggerated hepatic production

off tumor necrosis factor-alpha and systemic and tissue tumor necrosis factor-alpha levels after endotoxin.

Surgeryy 1997; 122:281-286.

35.. Swain M G . Appleyard CB. Wallace .IF. Marie M. TNF-alpha facilitates inflammation-induced glucocorticoid

secretionn in rats wi th bi l iary obstruction. ,1 Hepatol 1997: 26:361-368.

36.. Bemelmans M H , Gouma DJ. Buurman WA. Tissue distribution and clearance of soluble murine TNF' receptors

inn mice. Cytokine 1994: 6:608-615.

37.. Beierle HA. Vaulhey JN. Moldawer I T . Cope I and F M 3 . Hepatic tumor necrosis factor-alpha production and

distantt organ dysfunction in a murine model of obstructive jaundice. A m J Surg 1996: 17 1:2()2-206.

38.. Leon LR. White A A . Kluger M.I. Role of IF-6 and T N F in thermoregulation and survival dining sepsis in mice.

Amm J Physiol 1998;275:R269-R277,

39.. Chung IY . Benveniste F N . Tumor necrosis faclor-alplia production by astrocytes. Induction by

lipopolyy saccharide. IFN-gammu. and II -1 beta. .1 Immunol 1990; 144:2999-3007.

40.. F/ure T . Sakamoto T . Tsuj i H. F u n / JCi. Murase N. Fung .1.1 et al. The development and compensation of

bil iarvv cirrhosis in intei lcukin-6-dcficient mice. A m .! Pathol 2000: 156:1627-1639.

4 1 .. Fiu / , Sakamoto ! ' . Yokomuro S. F/ure T. Suhbotin Y. Murase N el al . Acute obstructive cholangiopaihy in

interleukin-66 delicient mice: compensation by leukemia inhibitory factor (I.IF') suggests importance ol gp-l 30

signalingg in the duclular reaction. Liver 2000: 20:114-1 24.

42.. Gaudio F. Onor i P. Pannarale L. Alvaro D. Hepatic microcirculat ion and peribi l iary plexus in experimental

biliarvv cirrhosis: a morphological study, Gastroenterology I 996 : 111:1118-1124

43.. Barone M. Francavil la A. Polnncno L. lerardi I;. Ronianell i I ) . Berloeo Pel al . Modulat ion of rat liepatocyte

prol i ferationn by bile salts: in vitro and in v ivo studies. Hepatology 1996: 23:1 I 59-1 I 66.

44.. A k v o t G . Dursun A. Povra/ A. Fluoglu O. Ataoglu O. Fdaly N et al. P53 and proliferating cell nuclear antigen

(PCNA)) expression in non-tmnoral liver diseases. Pathol Int 1999 ;49 :214 -221 .

45.. Harrison RF. Reynolds G M . Rowlands DC. Immunohistoehemieal evidence for the expression of proliferating

celll nuclear antigen (PCNA) by non-proliferating hepatocyles adjacent to metastatic tumours and in

inf lammatoryy conditions. J Pathol 1993: 171:1 15-122.

46.. Nakajima T . Kagavva K. l e d a K. Ohkavvara T . Kimura H. Kakusii i M et al. Fvaluation of hepatic prolil'eralive

activityy in chronic l iver diseases and hepatocellular carcinomas by proli ferating cell nuclear antigen (PCNA)

immunohisiochcmicall staining of methanol-fixed tissues. J Gastroenterol 1994. 29:450-454.

47.. Satoh M , Yama/ak i M . Tumor necrosis factor stimulates D N A synthesis ol ' mouse hepatocytes in primary

culturee anil is suppressed bv transforming growth factor beta and interleukin 6. J Cell Physiol 1992: I 50:1 34-

139. .

48.. Fauslo N. Growth factors in liver development, regeneration and carcinogenesis. Prog Growth Factor Res 1991:

3:219-234. .

132 2

ChapterChapter 7

49.. kiusto N. Mead ill. Regulation of liver growth: protooncouenes and transforming urowth (actors I ah Invest 1989:60:4-13. .

.Ml.. Hall PA. Coates PJ. Ansari B. Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract:

thee importance oi'apoptosis. J Cell Sci 1994: 107:3569-3577.

.11.. Vlorita M. W atanahe Y. Akaike T. Protective effect of hepatocyte growth factor on interferon-gamma-induced cytotoxicityy in mouse hepatocUes. Hepatologv 1995; 21:15X5-1593.

52.. Okamura U. Tsutsi H. Komalsu T. Yutsudo VI. Hakura A. Tanimoto T el al. Cloning of a new cvtokine (hat inducess II ;\-gaiiima production by T cells. Nature 1995: 37<S:KX-9].

53.. Miyoshi H. Rust C, Rubens PJ. Burgart 1 J. Gores GJ. Hepatocyte apoptosis alter bile duct ligation in the mouse imubess las. Gastroenterology 1999: 117:669-677.

54.. Muschen M. Warskulal I . Peters-Regehi T. Bode JG. Kubit/ R. Haussinger D. Involvement of CD95 (Apo-1/Pas)) Iigand expressed by rat Kupffer cells in hepatic immiinore»ukition. Chistroenlerolosiv 1999' 1 16 666-677. .

133 3