Journal of Gastroenterology and Hepatology (2000) 15, 244–253

REVIEW

Pathophysiology of the intrahepatic biliary epithelium

MARIO STRAZZABOSCO,* CARLO SPIRLÌ* AND LAJOS OKOLICSANYI †

*Department of Medical and Surgical Sciences,University of Padova and Azienda Ospedaliera di Padova,
Padova and †University of Parma, Parma, Italy

Abstract The intrahepatic bile duct epithelium modulates the fluidity and alkalinity of the primary
hepatocellular bile from which it reabsorbs fluids, amino acids, glucose and bile acids, while secreting
water, electrolytes and immunoglobulin A. The transport function of the intrahepatic biliary epithelium
is finely regulated by a number of gastrointestinal hormones, neuropeptides and neurotransmitters that
promote either secretion or absorption. The intrahepatic biliary epithelium appears to be a primary
target in a broad group of chronic cholestatic disorders that represent an important cause of morbid-
ity and mortality. The spectrum of cholangiopathies ranges from conditions in which a normal epithe-
lium is damaged by disordered autoimmunity, infectious agents, toxic compounds or ischaemia, to
genetically determined disorders arising from an abnormal bile duct biology, such as cystic fibrosis or
biliary atresia. Probably as a result of the known heterogeneity in cholangiocyte function, different por-
tions of the biliary tree appear to be preferentially affected in specific cholangiopathies. From a patho-
physiological point of view, cholangiopathies are characterized by the coexistence of cholangiocyte loss
(by apoptotic or lytic cell death) with cholangiocyte proliferation and various degrees of portal inflam-
mation, fibrosis and cholestasis. These basic disease mechanisms are discussed in detail. Better under-
standing of cholangiocyte pathophysiology, in particular the immune regulation of cholangiocyte
function, will help in designing newer genetic or pharmacological approaches to treat cholangiopathies.
© 2000 Blackwell Science Asia Pty Ltd

Key words: apoptosis, cholangiocytes, cholestasis, cystic fibrosis, cytokines.

PHYSIOLOGY OF THE BILIARY ments possessing different functional properties, with

EPITHELIUM
Bile formation requires the coordinated function of
hepatocytes and intrahepatic bile duct epithelial cells
(cholangiocytes). After its secretion by hepatocytes,
during its route towards the duodenum, the primary
bile is extensively modified by the bile duct epithelium
which can secrete fluid, bicarbonate, chloride and
immunoglobulin (Ig)A or reabsorb glucose, bile acids,
amino acids and electrolytes.1,2 The intrahepatic ducts
form an extensive network inside the liver and are
responsible for the production of approximately 30% of
bile volume, a percentage that can be rapidly increased
to meet changing physiological demands.3 Alkaliniza-
tion and fluidification of bile prevails in the intrahepatic
portion of the biliary tree, while bile concentration and
acidification mainly take place in the gall-bladder. The
intrahepatic biliary tree is further separated into seg-
hormone-regulated bile secretion being confined to the
interlobular and septal intrahepatic bile ducts.4 Diseases
of the bile ducts show a clear predilection for special-
ized sections of the biliary tree (Fig. 1); for example,
primary biliary cirrhosis (PBC) selectively involves the
interlobular bile ducts, the section where secretin-
stimulated secretion is supposed to be more active. In
comparison, drug-induced ductopenia is restricted to
the small cholangioles, suggesting that there may be
functional differences between major ducts and cholan-
gioles in terms of xenobiotic transport and metabolism.
The transport function of the intrahepatic bile duct
epithelium is finely regulated by a complex interplay of
gastrointestinal hormones, neuropeptides and neuro-
transmitters that promote either net secretion or ab-
sorption. In response to secretin, the intrahepatic bile
duct epithelium increases the hydration and alkalinity
of hepatocellular bile by secreting Cl– and HCO3–.

Correspondence: Dr M Strazzabosco, Department of Medical and Surgical Sciences, Clinica Medica I°, University of Padova,
Via Giustiniani, 2, 35100 Padova, Italy. Email: mstrazza@ux1.unipd.it
Accepted for publication 30 September 1999.

Pathophysiology of the biliary epithelium
Figure 1 The biliary epithelium is composed of multiple
segments of increasing diameter that possess different func-
tional properties. Colangiopathies show a clear predilection
for specialized sections of the biliary tree. Primary bilary
cirrhosis selectively involves the interlobular bile duct, drug-
induced ductopenia is restricted to small cholangioles, while
primary biliary cholangitis may affect any section of the biliary
epithelium, from the small cholangiocytes to the extrahepatic
tree.
Bicarbonate secretion is a major function of the biliary
epithelium, being important, not only to meet digestive
needs, but also to regulate biliary pH and thus the
absorption of weakly lipophilic organic acids.
In the last decade our understanding of the molecular
mechanisms underlying secretory/absorptive functions
of cholangiocytes has increased tremendously (Fig. 2).
Here we will briefly summarize current knowledge on
the molecular physiology of cholangiocyte secretion; the
reader is referred to a number of recent reviews for a
more detailed description.1,5,6 A number of different ion
carriers and channels have been identified The basolat-
eral Na+/H+ exchanger isoform 1 (NHE1), Na+:HCO3–
symporter7 (or a Na+-dependent Cl–/HCO3– exchanger
in humans)8 intrude HCO3– into the cell; Na+-K+-
ATPase maintains the Na+ gradient and, together with
K+ channels, determines the membrane potential differ-
ence. A Na+/K+/2Cl– cotransporter9 actively transports
Cl– ions into the cell and appears to be a major deter-
minant of fluid secretion. On the apical pole of the cell,
a Na+-independent Cl–/HCO3– exchanger (AE2),7
extrudes bicarbonate into the bile, according to the intra-
cellular pH and the in-to-out Cl– gradient imposed
by a cAMP-stimulated low conductance Cl– channel
(CFTR).10 Application of patch–clamp techniques has
led to the identification of at least two more types of Cl–
channels in cholangiocytes: a Ca2+-dependent Cl– con-
ductance and a Ca2+- and cAMP-independent high
conductance Cl– channel inhibited by pertussis toxin
which binds guanosine triphosphate (GTP)-binding
proteins.11 More recent work has demonstrated the
expression of apical isoforms of the Na+/H+ exchanger
(NHE2),12 consistent with a role for intrahepatic cholan-
giocytes in Na+ reabsorption. Other carriers, such as the
Na+-dependent glucose transporter (SGLT1),13 the glu-
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245
Figure 2 Mechanisms of fluid and electrolyte transport in
cholangiocytes. At the basolateral membrane, acid extrusion
is carried out by (1) the Na+/H+ exchanger and (2) Na+:HCO3–
symport or (3) Na+-dependent Cl–/HCO3– in humans. The
main acid loader, a Cl–/HCO3– exchanger, is located at both
the basolateral (4) and apical (5) membrane, where its activ-
ity is stimulated by cAMP/protein kinase A (PKA). (6) A
Na+/H+ exchanger (NHE-2) is located at the apical membrane
and is involved in Na+ reabsorption. (7) The cystic fibrosis
transmembrane conductance regulator (CFTR) is localized at
the apical membrane and is stimulated by secretin and cAMP.
(8) A water-selective channel, aquaporin 1, located at both the
apical and the lateral membrane, mediates osmotic water
movement and may be regulated by secretin. In addition to
CFTR, a Ca2+-activated Cl– channel (9) is present on the
apical pole of the cell and may be activated by luminal puriner-
gic nucleotides. (10) A basolateral K+ channel and Na+/K+-
ATPase establish the membrane potential and Na+ gradient.
Chloride uptake is mediated by Na+/K+/2Cl– cotransport (11).
The intrahepatic biliary epithelium also plays a role in Na+-
dependent reabsorption of taurocholate (TCA) via an apical
carrier similar to the ileal bile acid transporter (12). iBAT, ileal
bile acid transporter.
tamate transporter14 and the ileal bile acid transporter
(iBAT)15 are expressed on the apical membrane of
cholangiocytes and participate in the reabsorption of
glucose, glutathione breakdown products and conju-
gated bile acids. Finally, a water-selective channel, aqua-
porin (AQP-1)16 is expressed both at the apical and
basolateral cell membrane and mediates water move-
ment according to the osmotic gradients.
After binding to a G-protein-coupled receptor and
stimulation of the cAMP/protein kinase A (PKA)-
dependent signalling pathway,17 secretin, the principal
stimulatory hormone, promotes Cl– efflux, HCO3–
secretion and membrane vesicular transport, result-
ing in increased ductular choleresis.18 In addition to
secretin, a number of gastrointestinal hormones
and neuropeptides stimulate (e.g. vasoactive intestinal
peptide (VIP)19 and bombesin20) or inhibit (somato-
statin and gastrin) ductal secretion.21 A rich peptider-
gic plexus is present in the liver parenchyma; in
particular, bombesin immunoreactivity is localized in
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246 M Strazzabosco et al.

the portal triads in close contact with the bile duct

epithelium.22 Nerve fibres are also present in the wall of
bile ducts; vagal (cholinergic) stimulation increases
HCO3– output after acetylcholine binding to M3
muscarinic receptors on the basolateral membrane
of cholangiocytes and potentiates the rise in cAMP
induced by secretin.23 The effects of secretin and acetyl-
choline, including their net effect on secretion, are
additive, an observation that may bear physiological rel-
evance, as the former stimulates bile duct cells during
periods of parasympathetic activity.23
Regulation of ductal secretion also takes place at the
apical membrane of cholangiocytes through paracrine
factors present in the bile. Among them, ATP, a potent
secretagogue in a number of epithelia, is present in
micromolar concentrations in bile24 where it is released
by hepatocytes.25 Luminal ATP binds to P2Y2 puriner-
gic receptors present at the apical pole of cholangiocytes
and activates luminal Cl– efflux through Ca2+-activated
Cl– channels26 and basolateral HCO3– influx via NHE-
1.27 The effects of ATP and cAMP appear to be
additive in terms of stimulation of HCO3– efflux.
Furthermore, cholangiocytes regulate the concentration
of osmotically active compounds in bile. One such sub-
stance is glutathione, a tripeptide that represents both
the major determinant of bile salt-independent bile flow
and a major hepatic detoxifying mechanism.3 The apical
membrane of cholangiocytes is enriched with g-
glutamyltranspeptidase, an ectoenzyme able to catabo-
lize biliary glutathione to glutamate and cysteinyl-
glycine; which are then taken up by a glutamate carrier
and by a dipeptide transporter located at the apical pole
of cholangiocytes, respectively (H Daniel, pers. comm. Figure 3 Theoretical scheme for paracrine communications
1999), thus realizing an important cycle for the con- between cholangiocytes and non-epithelial cells in the liver.
servation of glutathione. Other important biliary con- Cholangiocytes synthesize and secrete a number of peptides
stituents that are recycled by cholangiocytes are the and mediators that are likely to enable the biliary epithelium
conjugated bile acids which are taken up by an apical to communicate extensively with other liver cells, such as
carrier similar to the ileal bile acid transporter.15,28 The hepatic stellate cells, portal fibroblasts and inflammatory cells,
physiological role of this carrier is still unclear, but it by stimulating the fibrogenic response. However, peptides and
may act as a regulator of the final concentration of mediators, either released in the portal spaces by immune
monomeric bile acids in bile. Apical uptake of glucose cells, macrophages (Mf) and mesenchymal cells or produced
from the bile via SGLT1 may also regulate biliary by the epithelium itself, may have profound effects on epithe-
osmolarity;13 in fact, as SGLT-1 transports approxi- lial cell function, inducing expression of molecules of major
mately 210 water molecules for each Na+ and glucose histocompatibility complex (MHC) class II antigen expression
molecule taken up, this carrier may function as a mo- or affecting transport properties. MCP-1, monocyte chemo-
lecular water pump.29 tactic protein-1; NO, nitric oxide; ET-1, endothelin-1; FGF,
Finally, cholangiocytes synthesize and secrete a fibroblast growth factor;TGF-b1, transforming growth factor-
number of peptides and mediators, such as interleukin b1; PDGF, platelet-derived growth factor; HGF, hepatocyte
(IL)-6,30 transforming growth factor-b2 (TGF-b2,31 growth factor; IFN-g, Interferon-g; CIV, collagen IV; CXVII:
endothelin-1,32 monocyte chemotactic protein-1 collagen XVII; IL, interleukin; TNF-a, tumour necrosis
(MCP-1),33,34 platelet-derived growth factor (PDGF)- factor-a; MMP-1 matrix metalloproteinase-1; PMN, poly-
B chain,31 tumour necrosis factor-a (TNF-a35 and morphonuclear cell. Modified and adapted from Shuppan and
nitric oxide (NO),36 that likely enable the bile duct Hahn.85
epithelium to communicate extensively with other liver
cells, including hepatic stellate cells (HSC), portal
fibroblasts and inflammatory cells (Fig. 3). Interest- BASIC DISEASE MECHANISMS IN
ingly, the biliary epithelium does not produce these CHOLANGIOPATHIES
paracrine factors under normal conditions, but actively
synthesizes them in many forms of acute and chronic A derangement in the physiological processes outlined
liver injury. Thus, activated cholangiocytes are likely to constitutes the pathophysiological basis for cholan-
play an important role, not only in reparative processes, giopathies. This is a group of chronic, progressive, liver
but also in the progression of chronic liver damage in disorders, characterized by the clinical syndrome of
chronic cholestatic syndromes. chronic cholestasis, which affects the biliary tree and is

Pathophysiology of the biliary epithelium
responsible for a significant morbidity and mortality
among paediatric and adult populations.37,38 The spec-
trum of cholangiopathies ranges from conditions in
which the biliary epithelium is damaged by (i) dis-
ordered immunity (PBC, graft vs host disease,
primary sclerosing cholangitis); (ii) infectious agents
(cytomegalovirus or Cryptosporidium infections); (iii)
ischaemia (post-transplant hepatic artery stenosis,
chronic transplant rejection); (iv) toxic compounds
(many common drugs); or (v) genetically transmitted
or developmental diseases arising from an abnormal
bile duct biology (e.g. cystic fibrosis, Alagille syndrome
and biliary atresia).
From a pathophysiological point of view, common to
all cholangiopathies is the coexistence of cholangiocyte
death (lytic or apoptotic), cholangiolar proliferation,
various degrees of portal inflammation and fibrosis and
qualitative/quantitative changes in bile production
(cholestasis) (Fig. 4). These basic disease mechanisms
are present, albeit to a different extent, in all kinds of
cholangiopathies. Cytokines and other proinflamma-
tory mediators released in close proximity to the biliary
epithelium contribute to these processes through (i)
stimulation of cholangiocyte apoptotic and proliferative
responses;39 (ii) activation of fibrogenetic processes;40
(iii) induction of damage to the peribiliary circulation;
(iv) induction of histocompatibility antigen expres-
sion;41 and (v) alteration of biliary epithelium transport
function.
Cholangiocyte death (lytic or apoptotic)
A vanishing bile duct syndrome (i.e. a decrease in the
number of bile ducts per portal tract) is the end result
of most cholangiopathies. Ductopenia can be consid-
ered as the result of bile duct loss prevailing over bile
duct proliferation. The mechanisms by which cholan-
giocytes die are not well known, but considerable inter-
est is being placed on apoptotic phenomena, the role of
which in bile duct morphogenesis is well established. In
Figure 4 Pathobiology of the
biliary tree.
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247
addition to being involved in ductal plate structure
regression,42 apoptotic phenomena are also involved in
tissue regression after induced hyperplasia (e.g. the
disappearance of proliferated ducts after the relief of
biliary obstruction).43 The role of apoptosis in cholan-
giopathies is, however, unclear and because of impor-
tant methodological problems (apoptosis is a transient
phenomenon and apoptotic bodies may be eliminated
by shedding into the bile duct lumen), the question is
difficult to address. Available morphometric studies
indicate that in PBC, apoptotic and lytic cell deaths
coexist. Apoptosis of cholangiocytes can be induced in
vitro by Cryptosporidium infection.44,45 The effects of
cytokines are also unclear: TNF-a, a major pro-
apoptotic cytokine, is not toxic to cholangiocytes,46
while IL-6, a proinflammatory cytokine and a known
autocrine growth factor,30 protects cultured cholangio-
cytes from ceramide-induced apoptosis by changing the
balance of apoptosis regulatory genes towards an anti-
apoptotic equilibrium (reduction of Bax, increase of
bcl-2).47
Hydrophobic bile acids can induce apoptosis in hepa-
tocytes via a Fas ligand-independent CD95 (Fas)
dimerization, induction of the mitochondrial transition
pore and activation of cathepsin B,48 but whether bile
acids can trigger apoptosis in cholangiocytes is pre-
sently unknown. The bile duct epithelium is a potential
target for bile acid toxicity, as shown by the presence of
extensive ductular damage and proliferation with portal
inflammation and fibrosis in mdr-2-deficient mice, an
animal model for progressive familial cholestasis type
III (MDR3 deficiency).49,50 In these conditions, the
absence of phospholipids in the bile exposes the biliary
epithelium to the unrestrained effects of high mono-
meric concentrations of bile acids. Under normal
conditions, the biliary epithelium is capable of absorb-
ing (passive, non-ionic diffusion, or active transport
through iBAT), metabolizing and recirculating bile
acids through the peribiliary circulation. Bile acids may
damage the epithelium, promote bile duct cellular pro-
liferation, or act as secretory signals. According to
Alpini et al., expression of iBAT is restricted to major

248
ducts;28 if so, cholangioles would be more prone to the
toxic effects of hydrophobic bile acids. Hydrophilic bile
acids (e.g ursodeoxycholic acid, UDCA), however, are
of benefit in cholangiopathies such as PBC and CF.
Recent data indicate that UDCA counteracts the apop-
totic effects of hydrophobic bile acids on hepatocytes.
It is unknown whether UDCA possess anti-apoptotic
effects on cholangiocytes. Interestingly, Koga et al.
reported a much lower rate of apoptosis in PBC patients
treated with UDCA.51
The mechanisms leading to cell damage in immune-
mediated cholangiopathies are poorly known. Whether
antimitochondrial auto-antibodies have a pathogenic
role in PBC is also unknown. The finding that AMA
antibodies react against the apical membrane of cho-
langiocytes in PBC patients52,53 suggests that their mol-
ecular target is either a mistargeted/truncated form
of pyruvate-dehydrogenase complex-E2 (PDC-E2),
immune complexes or a cross-reactive epitope derived
from an extrinsic (bacterial?) molecule. If this were the
case, its presentation by major histocompatibility
complex (MHC) class II molecules would allow activa-
tion of CD4+ T cells and, consequently, of the invading
effector CD8+ cells. However, as B7 costimulatory mol-
ecules seem not to be expressed during the early stages
of the disease, it is presently unclear to what extent
cholangiocytes may act as professional antigen-present-
ing cells. The presence of IgA antimitochondrial anti-
bodies (AMA) in bile of PBC patients and the
observation that, while PDC-E2 is an ubiquitous
antigen, damage is restricted to biliary cells (i.e. to cells
able to transport IgA transcellularly) have prompted the
hypothesis that IgA AMA may penetrate the cell and
induce liver damage by preventing the correct targeting
of PDC-E2 and, thus, interfere with mitochondrial
function, an effect that may also result in apoptotic cell
death.54 However, this sequence of events remains
highly speculative.
Cholangiocyte proliferation
Cholangiocytes possess latent mitotic capabilities and
proliferate following a number of liver injuries (ductu-
lar reaction). This is an important step in the develop-
ment of chronic liver damage, as ductular reaction is
considered as the pace-maker of portal fibrosis.55 In
PBC, as in several other cholangiopathies, destruction
of interlobular bile ducts is associated with a vigorous
proliferation of bile ductules; atypical ductules at the
border of the cirrhotic nodules show neuroendocrine
features56 and transiently express phenotypic features
that are present during fetal bile duct development,
such as expression of the anti-apoptotic protein bcl-2
and of the neural adhesion molecule (NCAM; L Fabris
et al. submitted). These cells are negative for the prolif-
erative marker Ki67, which is consistent with the origin
of atypical ductules from metaplasic hepatocytes. Fol-
lowing cholestatic injury, it seems that hepatocytes from
the limiting plate undergo ductular metaplasia, in a
manner similar to the ontogenetic response of hepato-
blasts that differentiate into primordial cholangiocytes
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M Strazzabosco et al.
when in contact with the undifferentiated mesenchyma
surrounding the portal vein terminations. The mecha-
nisms responsible for bile duct cell proliferation and/or
induction of metaplasia of portal hepatocytes into
cholangiocytes, are not well known and their elucida-
tion is an area of major effort. A number of growth
factors, such as epidermal growth factor (EGF), hepa-
tocyte growth factor (HGF) and IL-6 in humans and
insulin-like growth factor-1, EGF and HGF in rats
have been shown to stimulate cholangiocyte growth
in vitro.30 A role for bile acids has been postulated4,57
and awaits experimental demonstration. The role of
inflammatory mediators, locally released cytokines and
changes in extracellular matrix should also be inves-
tigated. These studies will be facilitated by the ability
to isolate NCAM-positive cells from diseased livers
(L Fabris et al. sumbitted).
Portal inflammation and fibrosis
Most cholangiopathies are associated with signifi-
cant amounts of inflammatory infiltrate in the portal
spaces; few studies, however, have addressed the pat-
tern of cytokines produced in the liver in the course
of immune-mediated cholangiopathies.58 Results are
equivocal and the interpretation is hampered by dif-
ferences in the experimental protocols. Studies in PBC
have shown increased production of both T helper type
1 (Th1) cells (IL-2), interferon-g (IFN-g)) and T helper
type 2 (Th2) cells (IL-4, IL-5 and IL-6) cytokines; the
Th1 pattern being predominant in later stages of the
disease.59 Tumour necrosis factor-a was also shown to
increase in PBC.60 An emerging concept is that bile duct
epithelial cells are active participants in inflammatory
diseases and, in pathological conditions, secrete proin-
flammatory and chemotactic cytokines, such as IL-6,
TNF-a,35 IL-8 and MCP-161 together with growth
factors able to activate mesenchymal cells and matrix
production (endothelin-1, PDGF, TGF-b2), or to
decrease matrix production and leucocyte adhesion
(NO; Fig. 3). These peptides and mediators, either
released in the portal spaces by immune cells,
macrophages and mesenchymal cells or produced by
the epithelium itself,41 may have profound effects on
epithelial cell function; for example, IFN-g promotes
MHC class II antigen expression by biliary cells,41
affects the transport properties of the epithelium and
stimulates induction of NO production by cholangio-
cytes.36 In contrast, IL-6 acts as a potent growth factor
for biliary cells. Interestingly, in septic patients, cho-
lestasis is associated with the pathological picture of
cholangitis lenta in which proliferation of marginal duc-
tules coexists with evidence of inspissated bile in bile
ductular structures at the edges of the portal tracts.62
Compared with hepatocellular cholestasis, which is
not associated with progressive fibrosis, in chronic
cholangiopathies there is an extensive fibrotic response
in the portal tracts. Fibrogenesis is a dynamic process
that depends on the extent and duration of parenchy-
mal damage: the persistence of the original noxa causes
a prolonged activation of tissue repair mechanisms that

Pathophysiology of the biliary epithelium
leads to tissue fibrosis. Hepatic stellate cells are the
main connective tissue producing cells and, during
tissue repair, undergo a process of activation from the
quiescent to the highly proliferative myofibroblast
phenotype.63 This activation, which is also charac-
terized by an increased synthesis of collagen types I
and III, follows a complex interplay between extracellu-
lar matrix components, polypeptide growth factors,
cytokines and other soluble mediators,63 (Fig. 3). As
outlined, most cholangiopathies are associated with the
proliferation of marginal ducts.55 Several lines of evi-
dence suggest that cholangiocytes play an active role in
stimulating the fibrogenic response, possibly activating
the usually quiescent portal fibroblasts and HSC. In
chronic liver diseases, in situ hybridization and immuno-
histochemical studies show that cholangiocytes produce
paracrine factors such as TGF-a,30 endothelin-1,32
MCP-1,33,34 PDGF-B and TGF-b2,31 which are able to
stimulate HSC activation and matrix production.
Furthermore, bile duct cells synthesize basement
membrane proteins and collagen type IV.64,65 The close
association between bile duct proliferation and
mesenchymal activation is also present in cholangio-
carcinomas, neoplasias that frequently show a strong
desmoplastic reaction. It is still unclear whether the
changing epithelial phenotype directly induces an
alteration of the mesenchymal cells or if the changes
in the extracellular matrix induce the phenotypic shift
in the biliary epithelium. Modulation of the fibrogenic
response may be helpful in avoiding obliteration of
the bile ducts and in slowing the evolution to biliary
cirrhosis.
Cholestasis
The role of the biliary epithelium in bile produc-
tion is such that, while damage to the biliary epithelium
will result in the reduced flow of bile, impaired
fluid secretion by cholangiocytes will induce qualita-
tive changes in the biliary fluid that may predispose
the biliary epithelium itself to damage from other
concurrent events. Cholestasis may originate from the
fibro-inflammatory obliteration of bile ducts, from
mutations in transport proteins, or from altered secre-
tory responses induced by inflammatory mediators.
Complete biliary obstruction is rarely seen in clinical
practice; this condition is associated with vigorous pro-
liferation of well-differentiated cholangiocytes, a phe-
nomenon that may have a compensatory function. In
fact, available information suggests that, in contrast to
hepatocytes where the basolateral sodium-taurocholate
transporter polypeptide (ntcp) is decreased,66 chronic
extrahepatic cholestasis does not impair ion transport
in cholangiocytes.67 Rather, the CFTR Cl– channel is
more highly expressed10,67 and the choleretic response
to secretin administration is much higher, partly as a
result of an increased expression of the secretin recep-
tor gene.68 These findings might explain the well-known
occurrence of colourless bile enriched with water and
electrolytes after bile duct ligation.67 Increased secretin
receptor gene expression and Cl–/HCO3– exchanger
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249
activity and increased choleretic responses have also
been reported in other conditions associated with
proliferation of differentiated cholangiocytes, such as
alpha-naphthly-isothiocyanate intoxication69 and partial
hepatectomy.67
The role of altered ion transport systems in the
pathogenesis of cholangiopathies is demonstrated in
cystic fibrosis, a frequently occurring genetic disease in
which mutations in CFTR lead to impaired cAMP-
dependent Cl– transport and to pulmonary, pancreatic
and liver disease. In contrast, in hepatocytes, in which
a number of genetically determined defects in mem-
brane transport systems (such as Dubin–Johnson syn-
drome, progressive familial intrahepatic cholestasis,
benign recurrent intrahepatic cholestasis, etc.)70 have
been identified, CF-associated liver disease is the only
known liver disorder affecting cholangiocyte transport
proteins. Clinical liver decompensation is infrequent,
but bile ductular cell damage can also be demonstrated
in asymptomatic cases, well before hepatocellular
damage appears.71 Cystic fibrosis results from a number
of mutations in the gene encoding for CFTR, a low con-
ductance Cl– channel which in the liver is restricted to
cholangiocytes and mediates the choleretic effects of
secretin.71 In addition to its role as a cAMP/PKA acti-
vated Cl– channel, CFTR also participates in the regu-
lation of other membrane transport proteins, such as
Na+ channels (EnaC),72 K+ channels,73 outward rectify-
ing Cl– channels,74 and Cl–/HCO3– exchangers.75 The
CFTR also appear to regulate cellular secretion of
ATP,76 intracellular vesicle acidification77 and process-
ing and trafficking of certain proteins.78 In other epithe-
lia CFTR may be permeable to HCO3– and allow a
substantial bicarbonate conductance. For example,
studies in the duodenal mucosa of CFTR (–/–) mice79
suggest a dual pathway for HCO3– secretion: one
component is electrogenic via CFTR-mediated HCO3–
conductance, a second component is electroneutral
via CFTR-dependent Cl–/HCO3– exchange and is
closely associated with carbonic anhydrase activity, an
enzyme that is highly expressed in duct cells. Indeed,
cholangiocytes isolated from CF patients undergoing
liver transplantation show reduced cAMP-stimulated
Cl– efflux and impaired bicarbonate secretion via
Cl–/HCO3– exchange (A Zsembery et al., submitted).
Although the molecular basis of the CF is reasonably
well understood, the pathogenesis of liver damage in
CF is not entirely clear; we speculate that in the pres-
ence of a mutated CFTR, reduced bile hydration and
alkalinity precipitates a series of events that damages
the biliary epithelium, such as retention of cytotoxic
bile acids and xenobiotics and a reduction in na-
tural defences against microbiological pathogens.80 In
response to epithelial damage, release of inflammatory
mediators and cytokines leads to chronic portal inflam-
mation, that, depending on the immunogenetic back-
ground of the individual and other concurrent factors,
may lead to pathognomonic focal biliary cirrhosis
(Fig. 5).71 Furthermore, the lower incidence of CF liver
disease, with respect to pancreatic damage, suggests
that in the liver, other secretory mechanisms and signals
may play a vicarious role in conditions in which the
cAMP-mediated secretory pathway is impaired. As

250
Figure 5 Current working hypothesis for the pathogenesis of
liver diseases in cystic fibrosis patients. Reduced bile hydra-
tion and alkalinity precipitates a series of events able to
damage the biliary epithelium: reduced biliary clearance
increases the exposure to pathogens and toxic compounds.
Furthermore, as hypothesized in the respiratory epithelium,80
the altered volume and quality of bile may impair the
epithelium’s innate immune system. This is the first line of
defence against pathogenic insult and includes the integrity
of the epithelium, the continuous flow of bile and the secre-
tion of yet to be identified multiple substances with anti-
inflammatory and antimicrobial properties. *In the airways
epithelium this includes lysozymes, phospholipase A2, defens,
cathelicidin, complement and immunoglobulin A.
already discussed, a candidate mediator is biliary ATP,
which binds to luminal P2Y2 receptors, activates apical
Ca2+-dependent Cl– channels other than CFTR, and
increases the activity of NHE-1, possibly resulting in
stimulation of HCO3 efflux.27 In fact, our own data
show that, in cholangiocytes isolated from CF livers, an
increase in intracellular Ca2+ concentration, induced by
administration of ionomycin leads to parallel activation
of Cl– channels and HCO3– extrusion. These results
indicate that, in CFTR-deficient cells, secretion of
bicarbonate can be restored by alternative activation of
intracellular Ca2+-dependent Cl– channels, an observa-
tion that may have therapeutical relevance.81
The molecular pathogenesis of cholestasis in
immune-mediated cholangiopathies remains unclear.
Cholestasis may be due to obliteration of bile ducts, but
it is usually present well before the onset of ductopenia
and it can be seen even in the absence of canalicular
dilatation and bile plugs. It has been suggested that
alterations in biliary electrolyte transport may con-
tribute to the pathogenesis of cholestasis in primary dis-
orders of bile ducts. For example, defective expression
of the gene coding for the AE-2 anion exchanger
and reduced immunoreactivity for the apical anion
exchanger have been reported in patients with PBC.82,83
Changes in cholangiocyte transport function may also
be secondary to the inflammatory condition. In contrast
to experimental cholestasis, where inflammation is
minimal, most human cholangiopathies are associated
with portal inflammation and to the release of a number
of proinflammatory cytokines that may have profound
effects on epithelial cell function, and even alter its
transport properties.84 For example, endotoxin admin-
istration decreases bile flow, an effect that is inhibited
by antibodies against TNF-a. Our own data show that
mixtures of different proinflammatory cytokines (IL-6,
14401746, 2000, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1440-1746.2000.02091.x by UFPE - Universidade Federal de Pernambuco, Wiley Online Library on [11/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
M Strazzabosco et al.
IFN-g and TNF-a) inhibit cAMP-dependent fluid
secretion and Cl–/HCO3– activity in polarized rat
cholangiocytes.86 These data are of interest because they
demonstrate that cholangiocyte secretory function is
deeply affected by the inflammatory microenvironment
in the absence of duct obliteration and advocate the use
of anti-inflammatory agents in cholangiopathies.
CONCLUSIONS
The role of the biliary epithelium in the pathogenesis
of biliary tract disease is just beginning to be unveiled.
The emerging idea is that cholangiocytes are active
participants, rather than innocent bystanders, in the
development of cholangiopathies. Whether genetically
determined, induced by extrahepatic causes or by
inflammatory mediators, the epithelium will respond
to the different noxae via apoptotic or lytic cell death
programmes, by activating mitosis and producing a vast
array of mediators that enable paracrine communica-
tion with non-parenchymal and inflammatory cells and
by changing its transport capability. Reduced fluid
and electrolyte transport by cholangiocytes (ductular
cholestasis) is a central step because it will predispose
the biliary epithelium to further damage. The continu-
ing inflammatory/fibrotic/cholestatic response will have
a key role in the progression of the disease. The field of
immune regulation of cholangiocyte function will prove
to be very rewarding in the future, particularly in view
of the potential to pharmacologically block the action
of specific cytokines. Furthermore, better understand-
ing of cholangiocyte physiology will help in the design
of newer genetic or pharmacological agents that are able
to target specific cholangiocyte functions and are likely
to provide effective and safe therapeutic strategies in the
near future.
ACKNOWLEDGEMENTS
The financial support of Telethon (grant E-873) and
of Ministero Dell’ Università e della Ricerca Scientifica
(Cofinanziamento 1998, Grant 9806210866) is grate-
fully acknowledged.
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