-
Gut, 1991,32, 1386-1391
Morphological and immunohistochemical analysis ofthe human liver
in chronic pancreatitis
R P Jalleh, J A Gilbertson, R C N Williamson, C S Foster
AbstractMorphological and immunohistologicalappearances of liver
biopsy specimens aredescribed in a personal series of 52
patientsundergoing operation for chronic pancreatitis.The findings
are compared with those in aseries of 10 histologically normal
liver biopsyspecimens from patients without pancreatitis.Alcohol
was the prime aetiological agent in 40ofthe 52 patients (77%). No
obvious damage tohepatic parenchymal cells or biliary structureswas
observed butminor morphological changesof alcohol associated liver
disease were seen in42% of specimens. The most consistentfinding,
present in 48 specimens (92%), was achronic inflammatory cell
infiltration of portaltracts. In all but one case, T
lymphocytespredominated, but a few B cells were present.In four
biopsy specimens, T cells spilled overinto adjacent hepatic
parenchyma, but therewas no evidence of T cell mediated
cytotoxicdamage to the parenchymal cells or biliaryepithelium. It
is suggested that these inflam-matory cells are in transit from the
pancreasthrough the liver via the portal circulation andmay reflect
the underlying pathogenesis ofchronic pancreatitis rather than
alcoholic liverdisease.
Departments of Surgeryand Histopathology,Royal
PostgraduateMedical School,Hammersmith Hospital,LondonR P JallehJ A
GilbertsonR C N WilliamsonC S FosterCorrespondence to:Dr C S
Foster, Department ofHistopathology, RoyalPostgraduate Medical
School,Hammersmith Hospital, DuCane Road, London W12ONN.
Accepted for publication21 January 1991
Chronic pancreatitis affects approximately threeper 100 000 of
the British population, althoughthe incidence may be increasing
along with thenational consumption of alcohol.'2 The con-tinuing
inflammatory process in the pancreas ischaracterised by
irreversible morphologicalchange, especially fibrosis and loss of
acini.Alcohol is the dominant aetiological factor inchronic
pancreatitis and contributes to severalforms ofliver disease,
notably alcoholic hepatitis,cirrhosis, and hepatocellular
carcinoma. Yetclinical evidence of a definite associationbetween
chronic pancreatitis and alcoholic liverdisease is lacking. 5
Indeed, in our ownexperience ofabout 100 alcoholic patients
under-going operation for severe chronic pancreatitis,the liver has
nearly always looked normal atlaparotomy.The observed
histopathological appearances
of the liver in chronic pancreatitis includeminimal non-specific
changes in architectureand are not characteristic of alcohol
related liverdisease.36 Some of these changes have beenattributed
to extrahepatic biliary obstruction,which is present in up to 27%
of patients withchronic pancreatitis.7- Therefore, we haveemployed
conventional histological techniques,immunohistochemistry, and
lymphocytemorphometry to characterise liver disease in aseries of
surgical patients with chronic pan-
creatitis of both alcoholic and non-alcoholicaetiology.
This study aimed to determine whether thelivers of patients with
chronic pancreatitisexhibited morphological changes of
ethanoltoxicity comparable with the severity of theirpancreatic
disease. If not, the objective was toidentify morphological
features, consistentlyexpressed, that might indicate alternative
patho-genetic mechanisms in chronic pancreatitis.
Methods
PATIENTSLiver biopsy specimens were taken from anunselected
personal series of 52 patients under-going operation for chronic
pancreatitis betweenDecember 1985 and June 1990 at Bristol
RoyalInfirmary or the Hammersmith Hospital,London. There were 40
men and 12 women witha mean age of41 2 years. Detailed social
historiesindicated that alcohol was the cause in 40patients, with
mean (SD) estimated daily ethanolconsumption of 216 (32) g over a
period of 12.8(1.4) years. Causes in the other 12 patientsincluded
previous acute pancreatitis in five andcholedochal cyst in one; no
cause was identifiedin the remaining six patients. Liver
functiontests were abnormal in only seven patients, andnone were
jaundiced at the time of operation.Operations were performed by a
single surgeon(RCNW) and comprised pancreatic resection(44),
pancreaticojejunostomy (5), and sphinc-teroplasty (3). Liver biopsy
specimens wereobtained intraoperatively using a Tru-cut
biopsyneedle (Travenol Laboratories, Illinois, USA),and more than
one specimen was obtained inmost cases.
CONTROL LIVER SPECIMENSTen histologically normal liver biopsy
specimenstaken from patients without pancreatitis wereused as
control tissues. Seven specimens werefrom patients with no known
focal or generaliseddisease who were undergoing surgery
aftertrauma. Two specimens were taken during distalpancreatectomy:
one for a solitary neuroendo-crine tumour and the other for removal
of abenign pseudocyst. One specimen was takenfrom the unaffected
lobe of liver during excisionof a benign simple hepatic cyst.
HISTOLOGICAL EXAMINATIONSpecimens were fixed in buffered neutral
formolsaline and embedded in paraffin wax. Tissuesections (2 lim)
were routinely stained withhaematoxylin and eosin (H&E) to
identify
1386
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Morphological and immunohistochemical analysis ofthe human liver
in chronic pancreatitis
TABLE I Histological and histochemicalfeatures of liverbiopsy
specimens
Controls Pancreatitis(n=10) (n=52)
Feature No (%) No (%)
Parenchymal hyperplasia 0 7 (13)Mallory's hyaline 0 - 0Steatosis
0 18 (35)Pericentral fibrosis 0 4 (8)Cholestasis 0 5 (10)PAS 0 0
-Iron 0 1 1 (21)Copper 0 2 (4)Copper-associated protein 0 0 -Portal
inflammation 3 (30) 48 (92)Marginal ductular proliferation 0 - 4
(8)Portal fibrosis 0 0Cirrhosis 0 0
PAS= periodic acid sehiff.
morphological features recognised to be associ-ated with ethanol
toxicity (Table I). Whereverfeasible, these features were assessed
usingnumerical grading systems which ranged from0-III according to
whether none, 75% respectively of hepatic paren-chyma was affected.
Parenchymal cell hyper-plasia was defined as hepatocyte plates two
ormore cells thick and present in more than 15% ofeach specimen.
Additional tissue sections werestained histochemically. Reticulin
was identifiedby a modified Gordon and Sweet's technique.Periodic
acid Schiff (PAS) staining before andafter diastase digestion
detected glycogen orintracellular glycoproteins including al
anti-trypsin. Perls' Prussian blue reaction was used toidentify
iron, which was quantified according tothe criteria of Braillon et
al. '° Copper and copperassociated protein were identified by
rhodanineand orcein respectively.
IMMUNOHISTOCHEMISTRYMonoclonal antibody UCHLI (anti-human Tcell)
was obtained as a gift from Dr PeterBeverley, University College
Hospital, London.Monoclonal antibody L26 (anti-human B cell)was
purchased from Dako Ltd (High Wycombe,UK). UCHL1 was diluted 1:10
and L26 1:100 in120 mmol/l sodium phosphate buffered saline(PBS, pH
7.4). Polyclonal rabbit antisera tolysozyme and to al antitrypsin
(to identify macro-phages) were purchased from Dako Ltd. Bothwere
diluted to 1:1000 in PBS. A conventionalindirect immunoperoxidase
staining techniquewas used to detect bound monoclonal
antibodies.Human tonsillar tissue was used as the positivecontrol.
Polyclonal antisera were detected by theperoxidase antiperoxidase
technique with humansplenic tissue as the positive control. These
lattertissue sections were digested with trypsin (0-1%w/v in
aqueous 0.1% CaC12 for seven minutes at37°C) after dewaxing.
Endogenous peroxidasewas blocked with 0 3% H202 in distilled
waterfor 30 minutes at room temperature. In all cases,sections were
incubated with primary antibodiesovernight at 4°C. The resultant
immuneperoxidase complexes were developed in
0.05%3,3'-diaminobenizidine hydrochloride (DAB,Aldrich, Gillingham,
Dorset, UK) in PBS con-taining 0.03% (w/v) H202. Sections
werecounterstained with haematoxylin and mountedin Pertex
(Histolab, Hemel Hempstead, UK).
TABLE II Immunohistochemical staining ofmononuclearcells
Controls Pancreatitis(n= 10) (n=52)
Antibody No (%) No (%) p valueUCHL1(T lymphocytes) 3 30 47 90
p0.5
al Antitrypsin(macrophages) 7 70 34 65 p>0. 5
MORPHOMETRYB and T lymphocytes in portal areas werecounted using
a lOx 10 mm graticule within themicroscope ocular and a x400
magnificationobjective. For each specimen, the number oflymphocytes
in three representative portal areaswere counted and a mean value
was calculated.
STATISTICSX2 and unpaired Student's t test were used
forstatistical analysis of data.
Results
MORPHOLOGY AND HISTOCHEMISTRYThe morphological and histochemical
appear-ances of 52 liver biopsy specimens from patientsundergoing
operation for chronic pancreatitisand from the 10 control liver
biopsy specimensare shown in Table I. Immunohistochemicalstaining
patterns of T cells, B cells, and macro-phages are presented in
Table II.
In all liver specimens examined, the hepaticarchitecture was
intact and normal vascularrelations were preserved. Parenchymal
cellhyperplasia was not identified in any of thecontrol specimens
but was present in seven ofthespecimens (13%) from chronic
pancreatitispatients. Mallory hyaline bodies were notobserved in
any specimen.
In all biopsy specimens, the three portal tractelements were
intact. No inflammation of bileducts was seen and no vasculitis was
identified.There was no appreciable increase in fibrousconnective
tissue, and cirrhosis was not presentin any of the specimens. The
most strikingfeature was the relative absence of inflammationfrom
portal tracts of the control specimenswhereas 47 of the 52 (90%)
specimens frompatients with chronic pancreatitis containedportal
tracts that had been infiltrated by increasednumbers of mononuclear
cells. The nature ofthese cellular infiltrates is detailed
below.
STEATOSISMacrovesicular steatosis of the type associatedwith
alcohol induced liver disease was notobserved in any ofthe controls
but was present in18 (35%) of biopsy specimens from patients
withchronic pancreatitis. In 11 of these specimens,steatotic
hepatocytes occupied less than 50% ofthe lobules (grade I). In the
remaining sevenspecimens, fatty change occupied between 50and 75%
ofthe hepatic lobules (grade II). In 15 of
1387
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18alleh, Gilbertson, Williamson, Foster
the 18 (83%) specimens showing fatty change,hepatic parenchyma
and portal tracts containedless than 10 polymorph neutrophils per
highpower field.
PERICENTRAL FIBROSISA mild increase in pericentral venous
fibrosiswas present around some, but not all, centralveins of the
contained lobules in four (8%) liverbiopsy specimens from patients
with chronicpancreatitis. No consistent association wasfound
between perivenous fibrosis and any otherof the commonly accepted
characteristics ofalcoholic liver disease.
CHOLESTASISCholestasis was present in only five of the 52
liverbiopsy specimens. In all ofthese, the distributionof bile was
periportal and intracanalicular. Nopolymorph neutrophils were
present, theappearances being those of non-inflammatorycholestasis.
In only one of these five specimenswas marginal ductular
proliferation seen. Noother morphological features of chronic
biliaryobstruction were identified in any of the speci-mens. Of the
five patients in whom morpho-logical features of cholestasis were
identified,none were jaundiced and only two had twofoldincreases in
serum bilirubin and alkalinephosphatase. However, all five had
radiologicalevidence (endoscopic retrograde
cholangio-pancreatograph (4) hepatic iminodiacetic acid(HIDA) scan
(1)) of extrahepatic bile ductobstruction.
PAS STAININGHistochemical staining with PAS did not
showintracellular PAS positive, diastase resistantglobules within
hepatocytes of any of the liverspecimens examined. Nevertheless, in
onebiopsy specimen, prominent staining for et,antitrypsin involving
the hepatocytes indicated aprobable heterozygous al antitrypsin
deficiency(an incidence of2% in this series).
SIDEROSISHepatocellular siderosis was noted in 11 patients(21%)
and all were grade I. In three of thesepatients, Kupffer cells also
contained stainableiron. Only two cases of siderosis were
associatedwith steatosis, and in both the steatosis was mild.
Figure 1: Portal region ofliver biopsy specimen stainedusing
monoclonal antibodyUCHLI . The portal tract isdensely infiltrated
withlymphocytes ofT celllineage. There is spillage ofT lymphocytes
into theadjacent parenchymawithout evidence ofcytolyticactivity to
portal structures orto adjacent hepatocytes(original
magnificationx480).
COPPER AND COPPER ASSOCIATED PROTEINIntracellular copper was not
found in any of thecontrol biopsy specimens and was identified
inonly two specimens from pancreatitis patientsusing a rhodanine
technique. In both specimens,staining was scanty (grade I). The
appearanceswere not those of a congenital copper storagedisease.
Distribution was confined to a fewperiportal hepatocytes. No
correlation wasapparent between this staining and the presenceof
cholestasis, steatosis, or iron deposition.Orcein staining did not
show stainable copperassociated protein in any of the control
orpancreatitis specimens.
PORTAL INFLAMMATIONIn none of the 10 control liver biopsy
specimenswere acute inflammatory cells or eosinophilsidentified. In
three of these, chronic inflam-matory cells were seen. The
phenotypes of thesecells were determined by immunohistochemis-try.
However, 48 ofthe 52 specimens (92%) frompatients with chronic
pancreatitis containedincreased numbers of inflammatory
cells.Neutrophils were present in only 15 specimens.Several
eosinophils were identified within theportal tracts ofone specimen
in which cholestasiswas also present. Neither neutrophils
noreosinophils made more than a scanty contributionto the
inflammatory infiltrate within theirrespective specimens.
MARGINAL DUCTULAR PROLIFERATIONMarginal ductular proliferation
was not presentin any of the control biopsy specimens but
wasidentified in four specimens from the patientswith pancreatitis.
In one of these specimens,marginal ductular proliferation was
associatedwith cholestasis, while in the remaining three
nocholestasis was evident.
PORTAL FIBROSISNo increased portal fibrosis or cirrhosis
wereidentified in any specimens from controls orpancreatitis
patients.
IMMUNOHISTOCHEMISTRYImmunohistochemistry was performed
toidentify and phenotype the mononuclearinflammatory cells in the
liver biopsy specimens.Using the monoclonal antibody UCHL1,
Tlymphocytes were identified in only three of 10control specimens
(30%) with a mean (SEM)density of 24 (4) cells per portal tract. T
lympho-cytes, however, were identified in 47 of 52 (90%)specimens
from pancreatitis patients. These cellswere almost entirely located
in the portal areas(Fig 1). The mean (SEM) number ofT lympho-cytes
per portal tract was 45 (5) cells. In fourspecimens, isolated T
lymphocytes were seen insinusoidal and canalicular regions (Fig 2).
Ineach of these specimens, there was an especiallydense portal
infiltration of T cells: mean (SEM)130 (17) cells per portal tract.
The relativedensity of T cell infiltration is shown in
TableIII.
1388
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Morphological and immunohistochemical analysis ofthe human liver
in chronic pancreatitis
Figure 2: Focal collectionsofT lymphocytes withinhepatic
parenchyma andwithout evidence ofcytolyticactivity to
adjacenthepatocytes. No furtherorganisation ofthese smallcellular
aggregates wasobserved and no granulomaswere identified
(originalmagnification x480).
B lymphocytes were not identified in any ofthe control biopsy
specimens. In contrast to theprevalence of T cells in the liver
biopsy speci-mens from patients with chronic pancreatitis,however,
B lymphocytes were identified in onlyeight specimens (15%). The
pattern ofinfiltrationwas similar to that of T cells in that
theselymphocytes were aggregated in portal regions.The density of
this lymphoid infiltration wassubstantially less than that for T
cells, with amean (SEM) of 10 (1) cells per portal area(p100 3
-
Figure 3: Liver biopsy specimen stained using
polyclonalantiserum to lysozyme. The identified macrophages
andhistiocytes were predominantly lining sinusoids ofparenchymal
zone I. A few morphologically identicalsinusoidal lining cells
remained unstained (originalmagnification x480).
pancreatitis and those with other causes arepresented in Table
IV. Statistical analysis, wherepossible, has shown no differences
between thetwo groups except that T lymphocytes weremore frequent
in alcoholic patients (p
-
9Jalleh, Gilbertson, Williamson, Foster
TABLE IV Histologicalfeatures ofalcoholic and nonalcoholic
patients with chronic pancreatitis
Alcoholic Non-alcoholic(n =40) (n= 12)No (%) No (%) p value
Portal fibrosis 0 (0) 0 (0) -Steatosis 12 (30) 6 (50)
05>p>01Cholestasis 3 (8) 2 (17) 05>p>0-1Iron 10 (25) 1
(8) 05>p>0-1Copper 2 (5) 0 (0) -UCHL1 (T cell) 38 (95) 9 (75)
005>p>0O02L26 (B cell) 8 (20) 0 (0)Lysozyme 34 (85) 9 (75)
05>p>01ctl Antitrypsin 25 (63) 9 (75) 0-5>p>0-1
increase in fibrous connective tissue withinportal tracts nor
was there evidence of cirrhosis.The remarkable absence of severe
liver damage,especially in the group of heavy drinkers, standsin
sharp contrast to the severe changes of chronicpancreatitis
observed in nearly every case.The commonest hepatic abnormality
was
steatosis, which was present in 35% of patientsbut severe in
only 13%. In only one patient wasthis associated with abnormal
liver function.Hepatocellular fat is labile and may
thereforereflect the patient's alcohol intake shortly
beforeoperation rather than any significant liver celldysfunction.
11The 10% incidence of cholestasis is in keeping
with the recent Glasgow experience.6 Cholestasisprobably
reflects extrahepatic biliary obstruc-tion, which was
radiologically confirmed in allfive of our patients.7 Cholestasis
was not seen infive other patients with biochemical, or
radio-logical, or both, evidence of bile duct stenosis.Marginal
ductular proliferation, anotherindicator ofrecent bile duct
stenosis, was presentin only one of the five specimens
showingcholestasis but was identified in an additionalthree
patients. Intrahepatocyte copper, a featuresuggesting a chronic
cholestatic disorder in theabsence of cirrhosis,'5 was not observed
in any ofour cases with cholestasis.
In this present series, we have defined thecharacteristic
features of inflammatory infil-tration of the liver in chronic
pancreatitis.Lymphoid infiltration, seen predominantly inportal
areas, comprised T lymphocytes ratherthan B lymphocytes. Only four
patients had adetectable parenchymal distribution of Tlymphocytes.
Since all four specimens containeda very dense portal infiltration,
these paren-chymal lymphocytes probably reflect a 'spillover'
mechanism. In the absence of featuresassociated with hepatocyte
toxicity, our datasuggest that the presence of T lymphocyteswithin
the parenchyma and portal tractsprobably reflects an upward
migration of inflam-matory cells from the pancreas rather than
adirect hepatotoxic process.The presence and density of
macrophages
within liver biopsy specimens from patients withsuspected
alcohol associated chronic pancreatitisis also ofnote. Several
studies have shown cells ofthe monocyte macrophage system.to be
pheno-typically heterogeneous. 16 l' Human hepaticmacrophages are
known to originate fromcirculating monocytes of bone marrow
origin.'8They form a self replicating population ofresident
macrophages which remains stable
unless a major inflammatory stimulus affects theliver.'9
Immunohistological examination hasindicated the numbers of lysozyme
positivehepatic sinusoidal macrophages to be decreasedin alcohol
associated liver disease.20 During thatstudy, an undefined portion
of sinusoidal liningcells was found to be negative for lysozyme.
Asubsequent study confirmed these findings andshowed portal tract
macrophages to be increasedin liver biopsy specimens exhibiting
only steatosiswhereas lysozyme positive hepatic
sinusoidalmacrophages were decreased.2' Heterogeneity ofliver
macrophage phenotypes was confirmed bythe disparity of numbers of
macrophagesidentified using three different immunohisto-chemical
markers.
In this study, the numbers of macrophages incontrol liver biopsy
specimens and in those frompatients with chronic pancreatitis were
notsignificantly different. These observations aredistinct from the
findings in alcoholic liverdisease20 21 and hence provide
additional evidencethat alcoholic liver disease and alcohol
associatedchronic pancreatitis do not involve exactly
similarpathogenetic mechansims. Differences innumbers of
macrophages identified using anti-sera to lysozyme and to a1
antitrypsin furthersubstantiate the phenotypic heterogeneity
ofhepatic macrophages in both control and pan-creatitis biopsy
specimens. Differences in aeti-ology and subsequent pathogenesis of
chronicpancreatitis, thus differentially modulating thephenotype of
hepatic macrophages, may accountfor the absence of lysozymal
staining in nine ofthe specimens from patients with
pancreatitis.
It is not understood why patients with alcoholicdamage to the
pancreas so seldom have seriousliver disease. Although nutritional
factors havebeen implicated,22 23 recent reports do notsupport this
hypothesis.2"26 The original sugges-tion that a diet rich in fat
and protein mightpredispose to alcoholic liver injury of
thepancreas was contradicted by the subsequentdietary studies.
Also, alcohol consumption isnotoriously difficult to quantify,
particularly inrelation to nutritional status. If chronic
pan-creatitis occurs earlier than alcoholic liverdisease, affected
patients might not have time todevelop hepatic cirrhosis.3
Similarly, ethanolintake might change with the onset of
pancreaticpain or the threat of operation. Yet evidence ofresidual
hepatic disease secondary to alcoholwould be expected, but this was
not found. Sincecertain human leukocyte antigen (HLA) haplo-types
are associated with either alcoholic liverdisease or chronic
pancreatitis, genetic factorscould help to determine which disease
processdevelops; however, current data are inconclusiveand require
further elaboration.27"29 Lastly, it isknown that in chronic
pancreatitis liver andpancreatic damage proceed
independently.30
Current evidence indicates that chronicpancreatitis is not a
single defined entity, but adisease process comprising a group of
differentaetiologies and pathogenetic mechanisms whichshare a few
common morphological end points.If a single agent, such as alcohol,
is the mostimportant aetiological factor in the genesis of
thisdisease, then the exocrine pancreatic tissues ofaffected
patients, when compared with hepatic
1390
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Morphological and immunohistochemical analysis ofthe human liver
in chronic pancreatitis 1391
parenchymal tissue, seem to be unduly sensitiveto its toxic
effects. Such sensitivity is eithercongenital or acquired in
origin. The finding ofraised numbers of T lymphocytes
infiltratinghepatic portal tracts, particularly in the absenceof
acute inflammatory cells, might indicate thata cell mediated immune
component is involvedin human chronic pancreatitis. We suggest
thatethanol is not acting as a simple toxin in a doserelated manner
but rather as an adjunct or'trigger' that renders exocrine
pancreatic tissuesensitive to cell mediated cytotoxicity.
We are grateful for the assistance of Professor J W B
Bradfield,Department ofHistopathology, University of Bristol in
permittingunreserved access to the archived specimens at the
RoyalInfirmary.
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