Download Recruitment of lymphocytes to the human liver

Immunology and Cell Biology (2002) 80, 52–64
Special Feature
Recruitment of lymphocytes to the human liver
PATRICIA F LALOR, PHILIP SHIELDS, ALLISTER J GRANT and
DAVID H ADAMS
Liver Research Laboratories, University of Birmingham MRC Centre for Immune Regulation, Institute of Clinical
Research, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom
Summary This review discusses the function and localisation of lymphocytes resident within the human liver,
under both physiological and pathological conditions. Through description of the mechanisms that mediate
lymphocyte recruitment into tissues, this article explains how hepatic endothelial and epithelial cells regulate the
recruitment of specific lymphocyte subpopulations. We illustrate that the expression of adhesion molecules and
chemokines is crucial to the control of lymphocyte adhesion. Thus, in the normal liver, adhesion molecules such as
vascular adhesion protein-1 (VAP-1), intercellular adhesion molecule-1 (ICAM-1) and intercellular adhesion
molecule-2 (ICAM-2), and chemokines such as regulated on activation, normal T cell expressed and secreted
(RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α),
interferon γ inducible protein-10 (IP-10), MIG and interferon inducible T-cell alpha chemoattractant (ITAC) are
involved in lymphocyte binding to different endothelial compartments. However, in response to inflammation or
injury, additional expression of adhesion molecules such as VCAM-1, P-selectin and E-selectin, as well as higher
levels of chemokines, permits the attraction and retention of specific effector populations of lymphocytes. We also
discuss the expression and function of a newly defined adhesion protein, (VAP-1), and suggest that the unique
functions of this protein may provide therapeutic potential for the treatment of liver disease.
Key words: adhesion molecules, chemokines, endothelium, liver, lymphocyte homing.
Introduction
The normal liver contains a large number of lymphocytes that
include not only specialized NK and NKT cells, but also CD4
and CD8 T cells. Whereas some of these cells are terminally
differentiated effector cells that are destined to die by apoptosis, many of these cells are not and include immunocompetent cells that traffic through the liver to provide continuing
immune surveillance as well as epithelial-associated effector
T cells. In inflammatory liver disease the number of lymphocytes in the liver increases and the type and distribution of
these infiltrating cells will determine the nature of the inflammation, for instance, a predominance of parenchymal inflammation is a feature of lobular hepatitis associated with viral
hepatitis while a predominantly portal infiltrate centred on
bile ducts is a feature of the biliary diseases primary biliary
cirrhosis (PBC) and primary sclerosing cholangitis (PSC). In
this review we discuss the molecular mechanisms that regulate the entry of lymphocytes to the normal and inflamed liver
and thereby determine the patterns of inflammatory damage
in liver disease.
The liver vasculature
The liver has a unique dual blood supply with arterial blood
delivered via the hepatic arteries and venous blood from the
Correspondence: Dr PF Lalor, Liver Research Laboratories, University of Birmingham MRC Centre for Immune Regulation, Institute
of Clinical Research, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK. Email: [email protected]
Received 16 October 2001; accepted 31 October 2001.
gut via the portal veins. This complex supply is necessary to
allow nutrients from the gut to be transported to the liver,
which is thus constantly exposed to gut-derived antigens in
the portal blood. Because portal blood also presents a route
through which infectious organisms can enter the liver, specific mechanisms have evolved to allow rapid and selective
immune responses within this tissue. Thus, the liver contains
a large resident and migratory population of lymphocytes and
macrophages that provide immune surveillance against
foreign antigens (Fig. 1). This population can be rapidly
expanded in response to infection or injury by recruiting
leucocytes from the circulation, a process that is dependent on
the ability of lymphocytes to recognize, bind to and migrate
across the endothelial cells that line the hepatic vasculature.
The complex nature of the liver vasculature means that there
are several points at which lymphocytes can interact with
endothelium and be recruited into different anatomical compartments.
The hepatic microvascular system consists of portal
venules, hepatic arterioles, sinusoids, central venules and
lymphatics. Most blood entering the sinusoids comes from the
portal venules, the terminal branches of which enter into the
sinusoids at inlets that are protected by contractile sinusoidal
lining cells that act as a sphincter to regulate blood entry and,
hence, blood flow within the sinusoids. Arterial blood enters
the sinusoids through branches of the hepatic arterioles and
via occasional direct arterioportal anastomoses at the level of
the terminal portal venules.1 These structures are also contractile and, therefore, contribute to the control of blood within
the sinusoids. The sinusoids take the blood through the lobule
from the portal tract to the central (terminal vein). Close to the
Recruitment of lymphocytes to the human liver
53
Figure 1 The normal human liver contains functional T cells. (a) Staining of normal donor liver for CD8 reveals CD8 T cells both in the
parenchyma and portal tracts. (b) Freshly isolated T cells from normal or hepatitis C virus infected human liver were stimulated to secrete
cytokines detected by intracellular cytokine staining. A significant proportion of T cells from normal liver secreted either IFN- γ or IL-10,
suggesting that these cells are functionally active. (c) The phenotype of freshly isolated intrahepatic T cells reveals them to be activated as
demonstrated by the expression of CD69 and CD38, but with low numbers of proliferating/cycling cells as shown by CD25 and CD71
expression. (d) The majority of intrahepatic T cells are CD45RO +, suggesting that they are primed or memory T cells.
portal tract the sinusoids form an interconnecting network
that becomes organized into parallel vessels connected via
short intersinusoidal sinusoids. Before leaving the sinusoids
and entering the central vein, blood flows through outlet
sphincters that control the rate of blood flow entering the
central veins. Matsumoto renamed hepatic lobules as hepatic
microvascular subunits because each lobule is supplied by
one inlet venule and its associated hepatic arteriole. 2 Thus, not
only is the vasculature within the liver lobule complex, but
there are also several points where blood flow can be regulated by the myofibroblast sinusoidal lining cells. This means
that blood flow through the sinusoids can vary greatly both
between different compartments and with time or injury.
Intravital studies suggest that transient plugging of some
sinusoids, particularly those in the periportal areas, by
flowing leucocytes is a normal feature of liver blood flow. 3,4
Heterogeneity of endothelium within the liver
The endothelial cells that line the different vascular beds
within the liver show marked heterogeneity. The most
obvious difference is between the vascular endothelium that
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lines the portal vessels and terminal veins and the specialized
fenestrated endothelium that lines the sinusoids. However,
there is also evidence for differences between portal and
hepatic venous endothelium and differences within the sinusoids.5–9 The sinusoidal endothelial cells (SEC) are morphologically unique and are characterized by an absence of tight
junctions between cells, a lack of basal lamina and by the
presence of open fenestrations that are arranged into characteristic sieve plates.4,10 These ultrastructural features have
evolved to facilitate solute transport between the blood and
the underlying hepatocytes, which are separated from the
endothelial cells by the space of Disse. Sinusoidal endothelial
cells express the scavenger receptor for oxidized low-density
lipoprotein (LDL), CD36, which is absent from vascular
endothelium in the portal tracts and they lack factor VIII
related antigen and Wiebel–Palade (WP) bodies, two characteristic features of vascular endothelium. In contrast, vascular
endothelial cells lining the hepatic arterial branches, portal
and central veins are continuous with an underlying basement
membrane, express factor VIII related antigen and contain
WP bodies.5–7,11,12
There are also important differences in the expression of
adhesion molecules and the secretion of chemokines between
vascular and sinusoidal endothelium. Consistent with the lack
of WP bodies, SEC fail to express P-selectin in vivo and
in vitro and in addition show markedly reduced or absent
expression of E-selectin.7 This absence of selectins on sinusoidal endothelium extends to inflammatory conditions such
as PBC, in which E- and P-selectin are strongly induced on
portal vascular endothelium.13 This may reflect a lack of requirement for selectin-mediated tethering in the low-flow environment of the sinusoid, where leucocyte recruitment in vivo has
been shown to occur in the absence of selectins. 14 Sinusoidal
endothelial cells express low levels of an antigen (CD31) that
is strongly expressed at cell–cell junctions in vascular
endothelium and the lack of such junctions in sinusoidal
endothelium might explain this difference. In contrast to
CD31, intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed at high levels on sinusoidal endothelium
compared with the low basal expression on non-inflamed
vascular endothelium.6
Chemokine expression also varies between the vascular
beds in the liver. In the non-inflamed human liver, several
chemokines, including regulated on activation, normal T cell
expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory
protein-1α (MIP-1α) and IL-8, can be detected on vascular
endothelium in portal tracts and this expression increases
markedly with inflammation.15,16 However, little chemokine
is detected on non-inflamed sinusoidal endothelium and the
pattern seen with inflammation differs from that seen on
portal vascular endothelium.17 The interferon-inducible
chemokines, interferon γ inducible protein-10 (IP-10), interferon inducible T-cell alpha chemoattractant (ITAC) and
human monokine induced by interferon γ (HuMig) are
detected on sinusoids in association with necroinflammatory
activity, whereas there is relatively little induction of
RANTES, MIP-1α or IL-8 on sinusoidal endothelium. The
reasons for these differences are unclear. One possibility is
that they reflect fundamental differences in the response of
sinusoidal versus vascular endothelium to inflammation.
Some evidence supports this because sinusoidal endothelial
cells secrete IP-10 in response to IFN-γ in vitro.17 However,
it is also possible that the differences are explained by an
effect of the local microenvironment. Other cell types
influence gene transcription in sinusoidal cells as part of a
complex process of autocrine cross-talk. For example, hepatocytes support SEC differentiation by secreting vascular
endothelial growth factor (VEGF).18 Thus, cytokines from
stellate cells, hepatocytes or other local cell types may determine the chemokines secreted by sinusoidal endothelium.
Alternatively, the chemokines detected on sinusoidal endothelium might be secreted by other cell types, such as fibroblasts
or inflammatory cells, in the immediate local environment,
which are subsequently transported across the SEC to be
presented by binding to endothelial surface proteoglycans. 19
This model in which chemokines secreted elsewhere are
‘posted’ on the endothelium has been demonstrated in other
systems and is regulated by active processes of chemokine
uptake, transcytosis and presentation by endothelial cells. 20–23
Thus, the unique phenotype of sinusoidal endothelium is probably due to the local microenvironment in the sinusoids as well
as differences in differentiation of the endothelium.
Lymphocyte adhesion to endothelial cells
The nature of the lymphocytes recruited to the liver will be
determined by the ability of specific leucocyte subsets to
recognize and bind to hepatic endothelial cells under different
conditions. The molecular basis of lymphocyte endothelial
interactions has been studied in many systems, resulting in the
acceptance of a ‘generic’ model that is broadly applicable to
most tissues and situations in which leucocytes bind endothelium. However, it is emerging that the fine details of the
molecules involved differ between tissues depending on the
local requirements.
In the generally accepted model,24–26 rolling receptors
expressed on endothelial cells capture fast moving lymphocytes from the bloodstream. These receptors may be members
of either the selectin family of adhesion proteins27 or the immunoglobulin superfamily.28–30 Once captured, the adherent cell
detects activating messages presented in the form of chemokines presented on proteoglycans in the endothelial glycocalyx.31 These chemokines activate leucocytes via specific
cell surface G-protein-coupled receptors, resulting in a
cascade of intracellular signals involving the small GTPases
Rho and Rac and PI3Kinase.32,33 These signals trigger the
conformational activation of leucocyte integrins, cytoskeletal
reorganization and cellular motility,34 bringing the leucocyte
to a halt and allowing it to respond to migratory signals 31,35
that direct it across the endothelium and into tissue in which it
follows a hierarchy of chemokines to the focus of inflammation (see Fig. 2).
Thus, it is the combination of molecules on the endothelium
and receptors on lymphocytes that act together to determine
whether a lymphocyte is recruited. This provides several
points at which diversity can be introduced to permit tissuespecific recruitment of different leucocyte populations.
Subsets of lymphocytes with tropism for specific tissues can
be defined by their expression of particular adhesion molecules and chemokine receptors, which allow them to respond
to a combination of molecules on the endothelium that confer
Recruitment of lymphocytes to the human liver
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Figure 2 Lymphocyte adhesion to endothelial cells occurs in a series of stages. Free flowing lymphocyte subpopulations in the
bloodstream (A + B) express different profiles of adhesion and chemokine receptors. Cells expressing appropriate counter receptors are
captured by tethering or rolling receptors expressed on endothelial cells (1). Chemokine proteins localized on the glycocalyx of the
endothelial cell are detected by specific, G-protein-linked receptors expressed on the leucocyte (2). This results in a conformational change
in lymphocyte integrins, which permits firm adhesion of the lymphocyte to endothelial-expressed immunoglobulin adhesion molecules (3).
Chemokine recognition also results in cytoskeletal reorganization within the adherent lymphocyte, which facilitates migration across the
endothelial monolayer and into tissue (4). Once within the tissue the leucocyte follows a chemotactic gradient of chemokine signal towards
the site of inflammation. Other cells in the microenvironment can affect the molecules expressed by the endothelium either by altering the
differentiation in a tissue-specific manner (e.g. hepatocyte-derived growth factors drive the differentiation of sinusoidal endothelium), by
secreting activationg factors, such as cytokines, that activate endothelium or by secreting chemokines that are then transported through the
endothelium and presented within the glycocalyx.
a molecular address for that particular tissue. 36,37 This paradigm has been used to explain why naive T cells are selectively recruited to lymph node and why mucosal lymphocytes
are selectively recruited to the gut (see Fig. 3).
T cells circulate continuously between the blood and the
tissues and can be divided into naive or virgin cells that have
not been exposed to antigen and primed cells that have been
activated by specific antigen presented in lymphoid tissue.
Primed cells consist of ‘effector’ cells, which home to inflammatory sites to mediate immune responses, and memory cells,
which are long-lived cells that have reverted to a lessactivated phenotype and provide both immunological
memory and the ability to respond rapidly to subsequent
encounters with an antigen.38 Naive T cells migrate exclusively to lymph nodes, which they enter using combinations
of the adhesion molecule L-selectin and the chemokine
receptor CCR7. This allows the T cells to interact with the
endothelial receptor peripheral node addressin and the chemokine SLC, which are largely restricted to lymph node high
endothelial venules.39,40 After activation by antigen, primed
and memory lymphocytes acquire a different complement of
adhesion receptors that promote their recruitment into tissue.
Some of these receptors, including the chemokine receptors
CXCR3 and CCR5, appear to play a role in recruitment into
inflammatory sites, whereas others promote tissue-specific
recruitment. For example, CCR9 and the integrin α4β1 are
selectively expressed on mucosal lymphocytes and allow
them to bind to vessels in the gut that express the chemokine
TECK and the α4β1 ligand MAdCAM-1.37,41 The absence of
these endothelial ligands from most other sites means that
these lymphocytes are selectively recruited to the gut,
whereas lymphocytes that express CCR4 and CLA bind to
dermal vessels and are recruited to the skin. 42–44 These processes insure that primed lymphocytes recirculate through the
organ from which its stimulatory antigen originated, thereby
increasing the likelihood that it will re-encounter its cognate
antigen.
Lymphocyte recruitment to the liver
The normal liver contains resident large granular lymphocytes
(pit cells) in the sinusoids. These cells probably enter the liver
from the circulation via the sinusoidal endothelium and are
retained in the sinusoids where they provide protection
against viral infections and tumour cells. 45–48 In addition,
the normal human liver contains significant numbers of T
lymphocytes in the portal tracts and scattered through the
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Figure 3 Recirculation of lymphocyte subpopulations is regulated by differential expression of adhesion molecules and chemokine
receptors. After leaving the thymus naive lymphocytes, which have not yet encountered antigen, recirculate continuously between the
lymphoid system and the bloodstream in the absence of antigen. This is facilitated by their expression of L-Selectin, CCR7 and low levels
of LFA-1 integrin, which can bind to PNAd, the chemokine SLC and ICAM-1, respectively, expressed on lymphoid high endothelial
venules (HEV). When naive cells encounter antigen within the lymphoid environment they differentiate into memory/effector cells. These
cells lose the molecules that permit entry into tissue draining lymph nodes and acquire new molecules that promote recruitment to tissue
and specifically in the case of the gut and skin to the organ from which their stimulatory antigen was derived. This process is facilitated by
the expression of specific homing receptors on subsets of lymphocytes and tissue-specific adhesion molecules and chemokines on
endothelium in target tissue. Thus, skin homing lymphocytes express CLA and CCR4, which bind to E-selectin and TARC expressed on
skin vessels, whereas gut homing lymphocytes express α4β7 integrin and CCR9, which recognize MAdCAM-1 and the chemokine TECK,
which are largely restricted to mucosal vessels. To date no specific liver homing lymphocyte populations have been identified, but it is
possible that such cells would express high levels of the receptor for vascular adhesion protein-1 (VAP-1), and may express CXCR3 for
binding IP-10, Mig and ITAC.
parenchyma (Fig. 1). Animal models suggest that terminally
differentiated effector T cells are removed by the liver, where
they die by apoptosis,49–51 and there is evidence from studies
using radiolabelled lymphocytes that a similar phenomenon
occurs in man.52–54 However, in normal human liver relatively
few liver-infiltrating lymphocytes express apoptotic markers
and, in addition to terminally differentiated T cells, other
subsets including CD45RA+ cells can be detected, suggesting
that many of the T cells in the liver are involved in providing
local immune surveillance.38
Infiltration of the liver by lymphocytes occurs in response
to many insults, including not only the hepatitis viruses, but
Recruitment of lymphocytes to the human liver
57
Figure 4 This figure shows molecules that may be involved in lymphocyte recruitment to different compartments of the human liver. The
establishment of inflammation requires both recruitment via the endothelium and retention in tissue. Some of the molecules that have been
reported to be involved under normal and inflamed conditions in different liver compartments are shown. These include molecules involved
in recruitment via endothelium and those involved in attracting lymphocytes to and retaining them at the epithelial surface of the bile ducts.
Under inflammatory conditions broader profiles of adhesion molecules and chemokines permit recruitment of a wider range of lymphocyte
subsets.
also autoimmune and toxic damage such as that mediated by
alcohol. The distribution of hepatic infiltration by lymphocytes varies depending on the inflammatory stimulus. In
acute allograft rejection and biliary diseases such as PBC,
lymphocytic infiltration is localized predominantly to the
portal tracts, whereas lobular inflammation and infiltration of
the parenchyma characterize acute viral hepatitis and autoimmune hepatitis. The factors that determine the distribution
of hepatic infiltration in any given situation are poorly understood, but are likely to be determined by the site of entry of
lymphocytes into the liver and factors that retain lymphocytes
at a particular location.55 The recognition that the expression
of adhesion molecules and chemokines is not uniform
throughout the liver vasculature begins to provide a molecular
explanation for the distinct patterns of lymphocytic infiltration in liver disease (Fig. 4).
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Lymphocytic infiltration of the portal tracts
Lymphocytes are seen in portal tracts in the normal liver, and
infiltration increases in many inflammatory diseases. In
normal liver both CD4 and CD8 T cells are found in portal
tracts albeit at low numbers and a population of cells with the
characteristics of intraepithelial lymphocytes are found in
association with biliary epithelium.56 Furthermore, cells with
a dendritic morphology can be detected in portal tracts and
animal studies suggest that in response to infection dendritic
cells (DC) migrate via the sinusoids to the portal tracts, where
they stimulate the development of portal-associated lymphoid
tissue.57,58 We have recently reported similar findings in
human inflammatory disease. These observations suggest that
the portal tracts serve at least two immunological functions:
(i) as a site of mucosal protection for antigens entering via the
biliary epithelium; and (ii) as a form of lymphoid tissue to
coordinate and orchestrate antigen-driven responses in the
liver. This may explain why even in diseases such as viral
hepatitis, in which the antigen is predominantly localized in
hepatocytes, a portal infiltrate is a universal finding. Thus,
lymphocyte recruitment into portal tracts will be important
for normal immune surveillance as well as a factor in pathological inflammation. The exact route of entry for lymphocytes into portal tracts is unclear. The portal vessels include
hepatic arteries and their portal tract capillaries and portal
veins, which do not have classic postcapillary venules, but
instead empty into the sinusoids. During chronic inflammation
new microvessels develop in portal tracts that have morphological and phenotypic similarities with the secondary high
endothelial venules and their development during inflammation is likely to facilitate the recruitment of lymphocytes. 59
Under non-inflamed conditions portal vessels express vascular adhesion protein-1 (VAP-1) and ICAM-2 and low levels
of several chemokines, including RANTES, MIP-1α and
MIP-1β.55 The ability of VAP-1 to support lymphocyte
capture under some conditions suggests that appropriate
signals for lymphocyte recruitment are present even in normal
liver, although the range of lymphocyte subsets recruited may
be restricted.60 In inflammatory conditions, the portal vessels
express P-selectin, E-selectin, and vascular cell adhesion
molecule-1 (VCAM-1), none of which is detected on noninflamed portal endothelium, and high levels of ICAM-1. 13
In addition, strong staining with the chemokines MIP-1α,
MIP-1β, RANTES and MCP-1 can be detected. 16 Thus, the
selectins induced on inflamed portal vessels could promote
primary adhesion and tethering of lymphocytes with subsequent secondary adhesion occurring via integrins binding
to endothelial VCAM-1 and ICAM-1. In support of this,
increased expression of both very late antigen-4 (VLA-4) and
leukocyte function related antigen-1 (LFA-1) have been
reported on lymphocytes infiltrating inflamed liver. 61 Few
lymphocytes in inflamed liver express either L-selectin or
E-selectin ligands,62 suggesting that these pathways are less
important, but they do express the P-selectin ligand, PSGL-1,
and P-selectin is strongly induced on portal endothelium.
P-selectin binding is a characteristic feature of Th1 cells,
suggesting that it might be involved in recruiting specific
functional subsets to the portal tracts. 63 In addition to the
selectins, VAP-1 expression is maintained on inflamed portal
endothelium where it can also support lymphocyte binding.
In some chronic inflammatory liver diseases, including
PBC, chronic hepatitis C infection and PSC, portal tract
infiltrates organize into lymphoid follicles containing B and T
lymphocytes, DC and new CD34+ vessels with the morphology of high endothelial venules.64,65 The development of new
lymphoid tissue in inflammatory sites at times when normal
lymph node development is complete is termed lymphoid
neogenesis,66 and provides a microenvironment for the
recruitment and retention of lymphocytes at sites of chronic
inflammation.67,68 Recent evidence suggests that these follicles express chemokines such as SLC, which are usually
restricted to lymph nodes, and may therefore provide a
specialized microenvironment for the continuing recruitment
and differentiation of lymphocytes within the chronically
inflamed liver. In addition to lymphoid follicles, the fibrous
septa, which are a characteristic feature of cirrhosis, also
contain newly formed blood vessels and have been referred to
as ‘fibrovascular membranes’.69 These vessels express high
levels of adhesion molecules and chemokines and provide an
additional pathway for lymphocyte recruitment. Because they
are located at the periphery of portal tracts at the interface
with the parenchyma they may be critical for the development
of the lesion of interface hepatitis and the spreading of inflammatory damage from the portal tract to the parenchyma. 70
Lymphocyte recruitment via the sinusoids
Intravital microscopy has revealed that leucocyte recruitment
to the hepatic parenchyma can occur through the sinusoids in
a process that involves direct adhesive interactions with
sinusoidal endothelium.14,51,71,72 Moreover, the heterogeneity
within the sinusoidal bed suggests that recruitment through
the perivenular and periportal sinusoidal endothelium might
be differentially regulated.73 Several features of the sinusoidal
bed are profoundly different from other vascular beds and
will affect how lymphocytes interact with sinusoidal endothelium. The sinusoid is a low blood flow environment in which
leucocytes come into contact with the sinusoidal lining cells
as they make their way through the liver microvasculature.
These conditions are likely to reduce the need for molecules,
such as selectins, that mediate capture of fast-flowing lymphocytes in other vascular beds and might explain why
selectins are largely absent from the sinusoids. 14,62 Several
other adhesion molecules are constitutively expressed on
hepatic sinusoidal endothelium including ICAM-1 and VAP-1,
both of which support lymphocyte adhesion. 60,61 VAP-1 can
mediate lymphocyte capture under some conditions, although
recent evidence suggests that it is also required for transendothelial migration.74 This latter function may be particularly
important in the sinusoids that lack a basal lamina, tight
junctions and junctional adhesion molecules such as CD31
that have been shown to regulate transmigration across other
vascular endothelial cells.75–77
Increased expression of adhesion molecules and chemokines is detected on sinusoidal endothelium during inflammation when ICAM-1 is increased, and VCAM-1 and CD31
induced. In addition, the development of chronic inflammation can stimulate the development of neovessels at areas of
interface hepatitis in which the sinusoidal endothelium undergoes neocapillarization with the development of a rudimentary basement membrane and expression of microvascular
Recruitment of lymphocytes to the human liver
markers, such as CD34.78,79 In inflammatory conditions, the
expression of VAP-1 remains constant, even in diseases in
which portal vessels express few selectins or no E-selectin or
P-selectin expression is detected on the sinusoids. In these
inflammatory conditions, primary adhesion to sinusoidal
endothelium could also be mediated by VAP-1 or VCAM-1,
which are induced and can support the capture of lymphocytes via α4-integrins. Subsequent firm adhesion appears
to be mediated predominantly by β2 integrins and ICAM-1.
Role of chemokines in regulating lymphocyte recruitment to
liver compartments
The detection of chemokines on sinusoidal endothelium also
changes with inflammation and there is evidence that specific
chemokine/chemokine receptor interactions regulate the
recruitment of activated T cells to different compartments of
the inflamed liver.17 Furthermore, the pattern of chemokine
secretion in the liver may determine the distribution and
severity of the T-cell inflammatory infiltrate. CC chemokines
with activity for T cells such as MIP-1α and MIP-1β are
found predominantly on vascular endothelium within portal
tracts, whereas the CXC chemokines, Mig and IP-10, which
bind to CXCR3, a receptor that is upregulated on activated
lymphocytes, are preferentially expressed by sinusoidal
endothelium. Furthermore, the expression of IP-10 is associated with localized lymphocyte lobular infiltrates in areas of
necroinflammatory activity in hepatitis C and parenchymal
damage in chronic allograft rejection. 19 Hepatic sinusoidal
endothelial cells secrete IP-10 and Mig in vitro in response to
IFN-γ and increased IP-10 mRNA has been detected in
inflamed livers in association with local inflammation. In
addition, hepatocytes stimulated with cytokines 17,80,81 and
activated Kupffer cells19 also secrete CXCR3 ligands, suggesting that chemokines sequestered in the sinusoidal glycocalyx are likely to reflect the production by several cells types
within the local microenvironment (Fig. 2). Further evidence
for the importance of IP-10 and Mig is provided by the high
levels of their receptor CXCR3 on liver-infiltrating T cells
compared with autologous peripheral blood T cells. 17,19,82,83
The requirement for IFN-γ to induce CXCR3 ligands, and the
finding that high levels of CXCR3 are often associated with
Th1 responses,84,85 all suggest that this pathway is tightly
regulated and a critical factor in the progression of Th1
responses in the hepatic parenchyma.
The different patterns of chemokine expression between
portal vessels and sinusoids suggest that IP-10 and Mig are predominantly involved in recruitment via the sinusoids, whereas
interactions between the CC chemokines MIP-1α and MIP1β and CCR5 may be involved in the recruitment of T cells to
portal areas. Because these CCR5 ligands can be detected on
portal vessels in normal human liver (albeit at lower levels
than in inflammatory disease) they provide a mechanism for
the recruitment of CCR5high memory T cells into portal areas
in normal liver as well as for recruitment in inflammatory
liver diseases.15 Chronic hepatitis confined to portal areas
(‘chronic persistent hepatitis’) is generally associated with a
favourable outcome, whereas extension of the inflammatory
process into the adjacent liver parenchyma in the form of
interface hepatitis (‘piecemeal necrosis’) is associated with
destruction of hepatocytes and progressive periportal fibrosis
59
(‘chronic active hepatitis’).86–88 The induction of the interferondependent chemokines IP-10, Mig and ITAC on sinusoidal
endothelium may be an important mechanism for the direct
recruitment of CXCR3high T cells to the liver parenchyma and
the development of progressive liver damage.
Role of non-endothelial cell types in lymphocyte
recruitment to the liver
Endothelial chemokines regulate the recruitment of lymphocytes into tissue, but the development of chronic inflammation requires that cells are retained at sites of inflammation. 89
Chemokines on stromal or epithelial cells not only attract
infiltrating cells, but also retain them in tissue by activating
integrin-mediated adhesion, thereby promoting the development of chronic inflammation.89 Many chemokines are
detected on inflamed bile ducts in vivo and secreted by
cytokine-stimulated biliary epithelium in vitro, including IL-8,
MCP-1 and RANTES. These chemokines may be important
in localizing infiltrating lymphocytes to the portal tracts and
retaining them in close proximity to bile ducts 90,91 in biliary
diseases, such as PBC, or allograft rejection. 19 Non-inflamed
biliary epithelium expresses high constitutive levels of SDF, 92
a chemokine that has been shown to be a potent inducer of
sustained integrin activity in lymphocytes. 67 This chemokine
has been implicated in B lymphopoesis in the liver during
fetal development,92 but it may also be important in the
normal adult liver. Liver-infiltrating lymphocytes express the
SDF receptor CXCR4 and cells entering the non-inflamed
liver may be attracted and retained at the biliary epithelium,
where they can provide immune surveillance against pathogens entering via the biliary tract.19
Activated stellate cells that drive the development of
fibrosis and cirrhosis in the inflamed liver 93 are a potent
source of several chemokines including IP-10 and MCP1.94–96 The ability of fibroblasts to recruit, retain and promote
the survival of infiltrating lymphocytes has been proposed to
be an important step in the evolution from acute to resolving
inflammation in the joints,89 and stellate cells may play a
similar role in the liver.89,93 In addition to stellate cells, other
inflammatory cells, including DC, infiltrating leucocytes and
Kupffer cells, will all contribute to the chemokine milieu
within the liver. One particularly important chemokine in this
context is MIP-3α, which recruits activated CD4+ T cells by
binding to the receptor CCR6. CCR6 is expressed on CD4 T
cells infiltrating the human liver and MIP-3α is detected in
periportal areas associated with DC and macrophages.
Furthermore, MIP-3α secretion by DC can be triggered by
culturing them with apoptotic cells, suggesting that local
tissue damage will lead to DC secretion of MIP-3α and
recruitment and retention of activated CD4 T cells at sites of
injury. The fact that CCR6 is coexpressed with CCR5 on
activated CD4 T cells provides them with a mechanism for
recruitment via endothelium as well as localization within
inflamed tissue.97
Role of vascular adhesion protein-1 in lymphocyte
recruitment to the human liver
One of the most obvious differences between hepatic sinusoidal endothelium7 and endothelium in other tissues is the lack
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of a role for selectins in the liver vasculature. 14,62 In the
absence of selectin interactions, other tethering molecules are
required to capture lymphocytes on hepatic endothelium and
we have proposed that VAP-1 is a candidate for this role.
VAP-1 is a homodimeric, class II transmembrane protein 98,99
that supports lymphocyte adhesion to high endothelial venules
in lymph nodes.100 Outside the lymph node, in the absence of
inflammation, endothelial expression of VAP-1 is largely
confined to hepatic vessels, where it supports lymphocyte
adhesion.60,61 This adhesion is sialic acid dependent and
operates under shear stress in vitro,61 suggesting that VAP-1
could act as a capture receptor to support the tethering
interaction between lymphocytes and hepatic sinusoidal
endothelium in the absence of selectins. Further evidence for
VAP-1 as a capture receptor comes from structural modelling
of the D4 domain of human VAP-1, which reveals six sites
for glycosylation, on the top of the molecule, from which
carbohydrate chains would extend from the surface of the
molecule into the vascular lumen.101 VAP-1 is not as effective
as the selectins in promoting leucocyte rolling, but the architecture of the liver and the low blood flow within the liver
microcirculation may circumvent the requirement for rolling
receptors operating in the classical sense (i.e. permitting rapid
capture of a fast moving cell). Thus, a lymphocyte moving
slowly through the narrow, irregular sinusoids might be more
readily captured via VAP-1. Our recent data suggest that
VAP-1 plays a more complicated role in lymphocyte adhesion to hepatic endothelium. Once captured by, and firmly
adherent to hepatic endothelium, a lymphocyte must breach
the endothelial barrier and migrate into the tissue. In most
circumstances this involves passage through interendothelial
tight junctions,102 a process that can be facilitated by molecules, such as CD31, which are concentrated at these sites. 77,103
However, as outlined above hepatic sinusoidal endothelial
cells form a discontinuous barrier without tight junctions and
they express low levels of CD31 compared with vascular
endothelium, suggesting that other factors are involved.
Using a flow-based adhesion system and hepatic endothelial
cells we have shown that antibodies to VAP-1 inhibit not only
adhesion, but also transendothelial migration. The regulation
of VAP-1 expression is poorly understood and like
MAdCAM-1 it is not readily inducible by cytokines in vitro,
although it is expressed in vivo in inflamed skin, joints and
gut. Expression on the cell surface appears to involve mobilization of protein from the cytoplasm as well as de novo
expression.104 VAP-1 is shed from the endothelial cell surface
and can be detected in serum in a circulating form. In vivo the
great majority of sVAP-1 is derived from the hepatic vascular
bed and the highest levels of circulating VAP-1 are found in
chronic inflammatory liver diseases, further supporting its
unique role in the liver.105,106 Neither the mechanisms of VAP-1
cleavage nor the function of soluble VAP-1 are known.
The recent molecular characterization and cloning of
VAP-1 revealed that it has close sequence homology to the
copper-dependent semicarbizide-sensitive amine oxidases
(SSAO).107,108 It has been known for over 20 years that
chronic liver disease is associated with increased monoamine
oxidase (MAO) activity in serum109–111 and we have now
shown that this enzymatic activity is in fact SSAO activity
derived from soluble VAP-1 protein because SSAO activity
in human serum is abolished by immunodepletion of
sVAP-1.106 Thus, soluble VAP-1 is responsible for the vast
majority of SSAO activity in human serum both in health and
in inflammatory liver disease. These studies provoke several
questions. Why does an adhesion protein have enzymatic
activity? Are these functions related or evolutionary distinct?
Other molecules have been reported to share adhesive and
enzymatic properties including CD26, CD73 and the Adamalysins, but VAP-1 is the only adhesion molecule with amine
oxidase activity. Salmi and colleagues have recently proposed
that the active site of SSAO in the VAP-1 molecule is directly
involved in the adhesion.112,113 They suggest that VAP-1 binds
lymphocytes via a sialic acid dependent epitope and subsequently utilizes the catalytic reaction between the SSAO and
an amine on the lymphocyte surface to link the endothelial
cell and the lymphocyte by a transient covalent bond. In
addition, protein-bound aldehydes that are formed on the
lymphocyte as a result of the enzymatic reaction could
modulate further interactions with endothelial cells. Whatever
the precise mechanism the ability to block VAP-1 function
with small molecule enzyme inhibitors has enormous therapeutic potential.
Conclusion
Lymphocyte recruitment to the human liver is mediated by
distinct combinations of molecules depending on whether
recruitment occurs via the portal vascular endothelium or the
hepatic sinusoids. Under non-inflamed conditions the molecules involved are relatively restricted and result in selective
recruitment of lymphocytes, whereas with inflammation the
repertoire of molecules available for recruiting lymphocytes
increases. The hepatic sinusoids are a distinct vascular bed
with unique morphological, rheological and phenotypic characteristics and in this environment VAP-1 appears to play an
important role both in lymphocyte capture in the absence of
selectins and in transmigration. The interferon-inducible
chemokines that bind the lymphocyte receptor CXCR3 are
strongly expressed on inflamed sinusoids, where they are
associated with necroinflammatory activity. These chemokines may be critical for the development of progressive liver
injury in diseases such as chronic hepatitis C. Understanding
the precise function of these molecules may permit the
development of specific anti-inflammatory strategies aimed at
preventing effector cell recruitment to particular liver compartments while leaving general lymphocyte recirculation intact.
Acknowledgements
We are grateful to the Wellcome Trust, the European Commission QLG7-CT-1999–00295, the MRC and the Sir Jules
Thorn Trust for research funding.
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