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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). .
1001 1
> >
If) If)
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
ËË 600 ^ i i
~~ 400 z z Li. . ~~ 200
|i-actin n
~JJ P< 0.05
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,
• »» IFN-yR,
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 /-
Sham m
BDL L —>.. . '- >
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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
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