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Pamela
S. Tietz, Xian-Ming Chen, Ai-Yu Gong,Robert C. Huebert, Anatoliy
Masyuk, Tatyana Masyuk,Patrick L. Splinter, Nicholas F. LaRusso,
Center
for Basic Research in Digestive Diseases, Department of Internal
Medicine and Biochemistry and Molecular Biology, Mayo Medical
School, Clinic and Foundation, Rochester, MN 55905
Supported by grants DK24031 and DK57993 (N. F. LaRusso) from
the National Institutes of Health and by the Mayo Foundation
Correspondence to: Nicholas F. LaRusso，Center
for Basic Research in Digestive Diseases, Mayo Medical School,
Clinic and Foundation, 200 First Street, SW Rochester, MN 55905，United
States. larusso.nicholas@mayo.edu
Telephone:+1-507284-1006 Fax: +1-507284-0762
Received 2002-01-14 Accepted 2002-01-18
Abstract
Cholangiocytes-the epithelial cells which line the bile ducts-are
increasingly recognized as important transporting epithelia actively
involved in the absorption and secretion of water, ions, and
solutes. This recognition is due in part to the recent development
of new experimental models. New biologic concepts have emerged
including the identification and topography of receptors and flux
proteins on the apical and/or basolateral membrane which are
involved in the molecular mechanisms of ductal bile secretion.
Individually isolated and/or perfused bile duct units from livers of
rats and mice serve as new, physiologically relevant in vitro models
to study cholangiocyte transport. Biliary tree dimensions and novel
insights into anatomic remodeling of proliferating bile ducts have
emerged from three-dimensional reconstruction using CT scanning and
sophisticated software. Moreover, new pathologic concepts have
arisen regarding the interaction of cholangiocytes with pathogens
such as Cryptosporidium parvum . These concepts and
associated methodologies may provide the framework to develop new
therapies for the cholangiopathies, a group of important
hepatobiliary diseases in which cholangiocytes are the target cell.
Tietz
PS, Chen XM, Gong AY, Huebert RC, Masyuk A, Masyuk T, Splinter PL,
LaRusso NF. Experimental models to study cholangiocyte biology.
World J Gastroenterol 2002;8(1):1-4
INTRODUCTIONS
The
liver contains two types of epithelia:hepatocytes, accounting for ～65%
of the liver cell population; and intrahepatic bile duct cells, or
cholangiocytes, the epithelial cells that line the intrahepatic
biliary tree and account for ～5%
of the liver cell population.
In the past decade, interest in cholangiocyte
pathobiology has exploded due to: (i) the development of new
experimental techniques that allow hypotheses related to
cholangiocyte biology to be directly addressed (Figure 1); (ii) the
recognition that cholangiocytes are critically important to normal
liver function, especially solute and water transport, cell-cycle
phenomena, cell signaling, and interactions with other cells, matrix
components, foreign organisms and xenobiotics; and (iii) the
appreciation that cholangiocytes represent the major target of a
group of serious genetic and acquired diseases termed the
cholangiopathies. In a coordinated series of hypothesis-driven
studies, we have explored selective aspects of cellular processing
by cholangiocytes focusing on proteins (receptors, channels,
exchangers, transporters, junctional proteins, molecular motors,
pro/anti-apoptotic molecules) that we hypothesized were likely
critically important in cholangiocyte secretion, absorption,
intracellular transport, and structural modifications. Thus, we
briefly review here a variety of novel in vivo and in
vitro experimental models that have allowed us to expand our
understanding of how cholangiocytes function and what happens when
disease alters their normal physiology. Several more extensive
reviews on cholangiocyte pathobiology are referenced for readers
interested in more comprehensive reviews.
IN
VIVO MODELS
The rat biliary tree has been shown to undergo selective
proliferation in response to different experimental stimuli such as
bile duct ligation (BDL)[1], 70% hepatectomy[2],
carbon tetrachloride treatment[3], and feeding of α-naphthylisothiocyanate
(ANIT) [4,5]. As best we know, the proliferated
cholangiocytes retain a normal cholangiocyte phenotype. The bile
duct ligated rat model has been useful in initiating studies on the
effect of hormones on bile secretion. Infusion of secretin and
somatostatin results in a choleretic and cholestatic response,
respectively, in the bile duct ligated but not sham-operated rat,
due not only to an increased number of cells but an increased
expression of the receptors for secretin and somatostatin on
individual cholangiocytes[6-8]. Much less is known about
how the mouse biliary tree responds to hormones. To further explore
the regulatory mechanisms of ductal bile secretion, it was necessary
to develop additional novel in vitro experimental models.
IN
VITRO MODELS
Freshly Isolated Cells
The development of in vitro models of biliary epithelia
essentially began with the ability to isolate cholangiocytes of high
purity from normal rat, mouse and human liver[9, 10].
This experimental model allowed direct studies on the effects of
hormones on cholangiocytes which demonstrated among other things,
that secretin stimulated exocytosis via a cyclic AMP mechanism, and
that this effect was blocked by somatostatin[11].
The
technique of counter-flow elutriation has allowed refinement of cell
isolation techniques from normal or BDL rats[12, 13] by
allowing separation of subpopulations of cholangiocytes which differ
in size. Using this model, we have provided evidence that these
small, medium and large cholangiocytes originate from different
portions of the intrahepatic biliary ductal system. Using molecular
and physiological approaches, we have also demonstrated that these
subpopulations of cholangiocytes differ in their transport and
proliferative capabilities[12, 13].
Figure
1
Experimental in vitro models of biliary epithelia. (a)
A scanning electron micrograph of a group of isolated cholangiocytes
after separation using immunomagnetic beads. Note that the prominent
microvilli (arrowheads) are limited to one side of the cells. Mag =
4,400. (b) A
transmission electron micrograph cross-section of normal rat
cholangiocytes in culture demonstrates characteristics of polarized
cells with apical microvilli (arrowheads) and numerous basolateral
intercellular interdigitations near the collagen coated filter
denoted by *, (Bar = 2μm, Mag = 7,500). (c) (d) A transmission
electron micrograph of apical (c)
and basolateral (d)
plasma membrane domains revealed similar homogenous vesiculated
membranes of varied shapes and sizes without apparent contamination
of other organelles (Bar = 0.5 μm, Mag = 22,500). (e)
A three-dimensional reconstructed image of the intrahepatic biliary
tree isolated from normal rat liver. (f)
A light micrograph of an unstained isolated bile duct unit from rat
liver. After overnight culture, the two ends seal forming an
enclosed unit. A single layer of epithelial cells with a thin outer
layer of connective tissue surrounds the lumen. (g)
A microperfused intrahepatic bile duct unit isolated from rat liver,
manually dissected and cannulated with micropipettes. (h)
Transmission electron micrograph of C. parvum infection of cultured
human cholangiocytes. A parasitophorous vacuole contains a
developing parasite stage which is intracellular but
extracytoplasmic. A macrogamet is shown in the inset, demonstrating
development of sexual stages of the parasite. Bar = 1μm.
Cholangiocyte
cDNA Library
A unique cDNA library has been constructed from highly purified
cholangiocytes isolated from rats subjected to bile duct ligation
for 2 weeks[14]. Total cellular RNA was extracted from
cholangiocytes and poly(A)+ mRNA isolated. Subsequently,
the poly(A)+ mRNA was reverse transcribed and
directionally cloned. The cholangiocyte cDNA library allows
screening and sequencing of positive clones for numerous molecules
yet to be identified.Tietz PS, et al . Experimental models to
study cholangiocyte biology
Cultured Cells
Freshly isolated cells maintained in short-term culture provided the
opportunity to perform patch-clamp studies yielding novel insights
into the electrophysiology of cholangiocytes[15].
Established long-term cultured cholangiocytes can be grown on
semi-permeable cell culture filters by which they quickly reach
confluence, maintain morphologic polarity and develop adequate
transepithelial electrical resistance, making them a prototype for
use in transport studies. This model has been utilized to
characterize the uptake of glucose and bile acids and to study water
transport[16-19].
Isolated
Organelles
In order to perform more rigorous transport studies and to generate
accurate kinetic transport parameters (i.e.,Km and Vmax),
we developed techniques for isolating vesicles enriched in either
the apical or the basolateral domain of cholangiocytes starting
either with whole rat liver or with cholangiocytes in culture[20,
21]. An initial application of these vesicles was to generate
kinetic values for sodium-dependent taurocholate uptake, a process
that we demonstrated occurs in apical but not basolateral vesicles[17].
This tool allows continued studies to localize and functionally
evaluate transport processes in cholangiocytes.
Intrahepatic
Bile Duct Units
Experiments using intact intrahepatic bile duct units isolated by
mechanical and enzymatic techniques from normal rats to study
biliary epithelial transport physiology have been previously
described[22]. An advantage of this model is that we can
not only control the lumenal contents (by manipulating components of
the perfusate) but we can independently and simultaneously modify
the basolateral milieu (by manipulating components of the bathing
buffer). With this approach, we were able to detect changes in
intralumenal pH and electrolyte concentrations in response to
agonists. We could also demonstrate water transport across this
epithelial barrier in response to osmotic gradients, the
characteristics of which (kinetics, temperature independence and
mercury sensitivity) all suggested the presence of water selective
channel proteins (i.e., aquaporins) (AQP) on the membranes of
cholangiocytes. Using quantitative computer-assisted image analysis
to measure expansion and reduction of lumenal area as a reflection
of water movement, we have demonstrated that water movement across
the bile duct units is transcellular and channel-mediated[23].
We have subsequently expanded this model using a
microperfusion technique and an epifluorescence detection system[24].
With this modification, we have demonstrated the movement of water
into (secretion) and out of (absorption) the lumen of the perfused
ducts in response to inward and outward osmotic gradients. The
calculation of both net water movement (Jv) and osmotic
water permeability (Pf) provide evidence that the
measured bi-directional fluxes reflect water movement through water
channels.
In
addition, we have demonstrated that isolated bile duct units
actively absorb solutes such as bile acids and glucose, and
transport ions such as bicarbonate. We have also shown that lumenal
perfusion of ATP and other nucleotides activates P2Y ATP receptors
on the apical cholangiocyte plasma membrane and induces increases in
[Ca2+] i and net ductular alkalization, suggesting that
ductal bile secretion is regulated by these signaling molecules[25].
This
technique has now been adapted to the mouse in which we can
reproducibly isolate and microperfuse intact bile duct units from
normal mouse liver with the anticipated application to transgenic or
knockout mouse models[26]. Transgenic mice continue to be
developed in which there is a selective knockout of one or more
aquaporin water channels[27]. The isolated and perfused
bile duct unit model would allow us to test the hypothesis that
knockout mice lacking AQP1 and AQP4 water channels or perhaps other
naturally expressed aquaporins may have substantially reduced
choleretic and cholestatic responses to hormones since the flux of
water molecules through these water channels should be absent.
The isolation of intact, polarized intrahepatic bile duct
units from both rat and mouse allows the direct study of secretory
and absorptive activities of the bile ducts in a way which most
closely approximates the normal biliary ductal system.
Three-Dimensional
Modeling
Although cholangiocyte functional and morphological heterogeneity
likely contributes to the selective involvement of different
portions of the biliary tree in the cholangiopathies, our
understanding of the nature and mechanisms for normal and abnormal
anatomical remodeling of the biliary tree is limited. To better
define the heterogeneous nature of the biliary tree, we utilized a
computer-aided three-dimensional imaging technique, first described
in a study of the normal human biliary tree[28], to
perform quantitative anatomical studies of the rat intrahepatic
biliary system[29].
Computer generated three-dimensional reconstruction of the
intrahepatic biliary tree using microscopic-computed tomography
scanning and sophisticated software allowed us to generate key
biliary tract dimensions (length, surface area, duct diameter,
volume) and branching patterns (distance from the junction of intra-
and extrahepatic ducts, number of bile duct branches and branching
angles).
In various forms of liver disease, including the
cholangiopathies, proliferation of cholangiocytes is a common
pathological response[1, 30, 31]. The anatomical basis
and remodeling process which occurs in response to various stimuli
remain unclear. We have since applied the three-dimensional
reconstruction of the biliary tree to rats in whom selective
cholangiocyte proliferation was induced by ANIT feeding[4,5].
The anatomical remodeling and quantitative observations after
selective cholangiocyte proliferation suggest that the proliferation
process involves sprouting of new side branches. Recently
three-dimensional modeling has been applied to generate data for the
hepatic artery and portal vein within the same liver (Masyuk,
LaRusso unpublished). This descriptive study allowed key findings on
the length of vascular segments, diameter of blood vessel segments
and volume of the hepatic artery and portal vein, associated with
experimentally-induced cholangiocyte proliferation.
Three-dimensional reconstruction will provide complementary
data that, at a minimum, will yield structural information on
biliary tract architecture and biliary mapping (i.e.,which branches
of the biliary tree are involved in absorption or secretion, the
mechanisms by which ducts proliferate in response to injury, and the
impact of these modifications on the peribiliary vasculature.) The
significance of these studies relates not only to the intrinsic
value of understanding cholangiocyte physiology but also to
providing a biologic rationale for why only certain segments of the
biliary tree are involved in the individual cholangiopathies.
Primary biliary cirrhosis, for example, leads to destruction of
interlobular bile ducts, while intrahepatic cholestasis induced by
drugs affects principally the cholangiocytes that line small bile
ducts[30, 32, 33].
In
Vitro Infection Model of Biliary Cryptosporidiosis
Using
monolayers of human cholangiocytes derived from normal liver and
immortalized by SV40 transformation, we developed an in vitro infection
model of biliary cryptosporidiosis[34]. Cryptosporidiosis
is an infectious disease caused by Cryptosporidium parvum ( C.
parvum ), an emerging parasite which causes self-limited
diarrhea in immunocompetent subjects and potentially
life-threatening syndromes in immunocompromised individuals,
primarily those with acquired immunodeficiency syndrome (AIDS).
Despite the magnitude and severity of cryptosporidial infection, the
pathogenesis is poorly understood, and there is currently no
effective therapy. Using this novel infection model, we found that C.
parvum sporozoites (derived from oocysts excysted in vitro )
attach to the apical surface of biliary epithelia, invade the cells,
reside in a parasitophorous vacuole, and undergo both sexual and
asexual development.
C.
parvum attachment to cholangiocytes involves interactions
between specific glycoproteins on cholangiocytes and C. parvum
sporozoite lectins while the invasion into cholangiocytes works
through actin-dependent membrane spreading mediated by cortactin and
RhoGTP-binding proteins[34-36]. While C. parvum is
cytopathic to uninfected cells adjacent to infected cholangiocytes
via Fas/FasL-dependent apoptosis, the organism prevents cell death
of infected cells by inhibiting apoptosis via activation of NF-κB[37,
38]. This model provides a useful system for the development
of novel therapeutic strategies for C. parvum induced
enterology and AIDS-cholangiopathy.
CONCLUSION
Advances
in medical knowledge most often are preceded by advancements in
technology and experimental models. Results from experiments using
these new models and methods have clarified which flux proteins are
expressed in cholangiocytes, their intracellular topography and
segmental distribution, what molecules control their cellular
compartmentalization, and their physiologic relevance to ductal bile
formation. In addition, the information has provided a theoretical
framework for development of novel therapeutic strategies for the
cholangiopathies, a group of cholestatic genetic/acquired
hepatobiliary diseases in which the cholangiocyte is the principal
target of diverse destructive processes.
Abbreviations
ANIT
a-naphthylisothiocyanate
AQP
aquaporin
BDL
bile duct ligation
C. parvum
Cryptosporidium parvum
Acknowledgement
We greatly appreciate the secretarial assistance of Deb Hintz in
preparation of this manuscript.
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