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Geoffrey W.

McCaughan Molecular pathogenesis of liver


Mark D. Gorrell
G. Alex Bishop disease: an approach to hepatic
Catherine A. Abbott
Nicholas A. Shackel
inflammation, cirrhosis and liver
Peter H. McGuinness transplant tolerance
Miriam T. Levy
Alexandra F. Sharland
David G. Bowen
Denise Yu
Loubnah Slaitini
W. Bret Church
John Napoli
Authors addresses Summary: The hallmarks of chronic liver diseases are chronic inflamma-
Geoffrey W. McCaughan1, Mark D. Gorrell1, tion, cellular damage, regeneration and fibrosis. An appreciation of intra-
G. Alex Bishop1, Catherine A. Abbott1, Nicholas A. Shackel1, hepatic molecular expression patterns in normal and diseased liver pro-
Peter H. McGuinness1, Miriam T. Levy1, vides clues for understanding pathogenic pathways whilst studies of the
Alexandra F. Sharland1, David G. Bowen1, Denise Yu2, structure and function of molecules implicated in liver disease provide
Loubnah Slaitini3, W. Bret Church4, John Napoli5, insights into their potential as therapeutic targets. We have examined the
1A. W. Morrow Gastroenterology and Liver expression, function, molecular structure and structurefunction relation-
Centre, Royal Prince Alfred Hospital, Centenary ships of type IV dipeptidyl aminopeptidases. In particular, the roles of
Institute of Cancer Medicine and Cell Biology CD26/DPPIV in T-cell proliferation and chemotaxis and of fibroblast acti-
and The University of Sydney, Sydney, Australia. vation protein in human cirrhosis are discussed. We have investigated the
2Department of Cancer Genetics, Kolling pathogenesis of liver disease by characterising patterns of cytokine and
Institute of Medical Research, Royal North Shore growth factor expression in experimental and human cirrhosis. We have
Hospital, Sydney, Australia. quite recently expanded this approach to use differential gene expression
3Department of Pathology, The University of analyses to elucidate overall pathways of gene activation and suppression
Sydney, Australia. in human cirrhosis. In addition, our detailed molecular and cellular stud-
4Garvan Institute of Medical Research, Sydney, ies of the mechanisms of spontaneous liver transplant tolerance have gen-
Australia. erated novel insights into this process. This review touches on these
5Department of Gastroenterology, Royal North diverse aspects of liver function and disease.
Shore Hospital, Sydney, Australia.

Correspondence to: Introduction


G. W. McCaughan
Centenary Institute of Cancer Medicine and Cell Chronic liver diseases are characterised by chronic intrahepatic
Biology
inflammation, cellular damage, regeneration and tissue fibro-
Locked Bag No. 6
Newtown sis. There are few animal models that are able to study all these
NSW 2042 events in unison. A basic knowledge of patterns of intrahepatic
Australia
gene expression in normal and diseased liver tissue is a neces-
Fax: 61 2 9565 6101
e-mail: G.McCaughan@centenary.usyd.edu.au sary prerequisite to increasing our understanding of these pro-
cesses, particularly in human liver disease. Consequently, a pri-
Acknowledgements
mary goal of our laboratory has been to study the expression of
This research was supported by the National
Health and Medical Research Council of Australia various molecules in diseased and normal liver tissue. We have
and was carried out while all authors except taken three different but overlapping approaches toward this
W. B. Church worked at the A. W. Morrow
goal. Firstly, we have studied, at a fundamental level, the struc-
Gastroenterology and Liver Centre.
ture and function of molecules in the dipeptidyl peptidase
(DPP) IV family, initially concentrating on the prototypic mem-
Immunological Reviews 2000 ber of this family, DPPIV, which is expressed at high levels on
Vol. 174: 172191
Printed in Denmark. All rights reserved various cells within the normal liver. This work has led to an
understanding of the three-dimensional structure of this gene
Copyright Munksgaard 2000
family and the characterisation of dipeptidyl aminopeptidases
Immunological Reviews
ISSN 0105-2896 involved in liver remodelling. Secondly, we have directly exam-

172
McCaughan et al Molecular pathogenesis of liver disease

ined the intrahepatic expression of various inflammatory


cytokines and growth factors during the evolution of experi-
CXCR3 Cleaved
mental and human cirrhosis. This work has led us to use differ- IP-10 CXCR3
CCR5 Th1 CCR5 IP-10
ential gene expression to probe pathogenic pathways in cirrho-
CCR1 CCR1
sis. Finally, we have had a long-term interest in the nature of X Cleaved
RANTES
DPP-IV RANTES
spontaneous liver transplant tolerance and we have used molec-
ular and cellular techniques to define novel mechanisms X
X Cleaved
involved in this process. This review covers these three areas of Eotaxin CCR1 CCR1 Eotaxin
work in our laboratory. MDC X
CCR3 Cleaved
Th2 CCR3
CCR4 CCR4 X MDC
SDF-1
Cleaved
CXCR4 CXCR4 X SDF-1
Role of type IV dipeptidyl aminopeptidases in inflammation
and liver fibrosis
Role of CD26/DPPIV in the inflammatory process Fig. 1. CD26/DPPIV and the Th1/Th2 balance. The proposition that
DPPIV alters the balance of chemokine activity towards stimulation and
CD26/DPPIV (EC 3.4.14.5) is expressed by T, B and natural attraction of Th1 cells is illustrated. CD26/DPPIV is preferentially
killer (NK) lymphocytes, macrophages, epithelial, endothelial expressed on Th1 cells (25) and cleaves RANTES, eotaxin, MDC, SDF-1a
and acinar cells and fibroblasts. High level expression, equiva- and SDF-1b. The cleavage products of these chemokines trigger Th1 but
not Th2-specific chemokine receptors (59, 11, 26).
lent to that on epithelial cells, is found on stimulated T cells.
CD26/DPPIV can bind to extracellular adenosine deaminase
(ADA) (EC 3.5.4.4) and can cleave N-terminal dipeptides from
substrates, generally targeting a penultimate proline, but the
contributions of these biological activities to T-cell functioning A role for CD26/DPPIV in leucocyte migration may also be
remain unclear (see reviews (14)). mediated by direct or indirect binding to collagen and
Substrates of the enzyme DPPIV that have relevance to im- fibronectin (1921) or the proposed endopeptidase activity of
mune function are primarily members of the chemokine fam- DPPIV on gelatin (22). However, we have been unable to detect
ily. DPPIV-mediated cleavage of RANTES (regulated on activa- collagen binding or gelatinase activities in recombinant human
tion normal T-cell expressed and secreted), eotaxin, monocyte- CD26/DPPIV (Y. Y. Peng, J. A. Werkmeister, J. A. M. Ramshaw,
derived chemokine (MDC) and stromal derived factor (SDF)- M. D. Gorrell, unpublished) (23). Observations using natural
1a and SDF-1b alters the receptor specificity of these chemo- DPPIV must be interpreted cautiously due to possible prolyl
kines (511). Such in vitro effects of CD26/DPPIV enzyme ac- endopeptidase contamination (24).
tivity on chemokine signalling and chemotaxis, which can be
ablated by mutation of the catalytic serine (5, 10), are an indi- Role of CD26/DPPIV in T-cell co-stimulation
cation that CD26/DPPIV potentially modulates T-cell and The ability of CD26/DPPIV to bind ADA has the potential to
monocyte extravasation and migration in vivo. The receptors for regulate cell surface and consequently intracellular levels of
these chemokines, CCR3, CCR4, CCR5 and CXCR4, are differ- adenosine (2, 3, 27). Adenosine levels are important in regu-
entially expressed by Th1 and Th2 cells (1217). Chemokine lating cellular proliferation, with high levels resulting in pro-
inflammatory protein (IP)-10 signalling via CXCR3, which is found inhibition and apoptosis. Indeed, ADA binding to
preferentially expressed by Th1 cells (13), is unaffected by CD26/DPPIV on the surface of T cells results in increased pro-
cleavage of IP-10 by DPPIV (5). A predicted outcome of DPPIV- liferation and interleukin (IL)-2 production (28, 29). On the
mediated cleavage of chemokines is therefore a differential re- other hand, the contribution of DPPIV enzyme activity to
duction in the stimulation of Th2 immune responses (Fig. 1) CD26/DPPIV mediated regulation of T-cell proliferation has
(11). Indeed, in leprosy DPPIV is highly expressed in the Th1- long been controversial (30, 31). Although DPPIV inhibitors
type lesions of tuberculoid leprosy but is undetectable in the modulate T-cell responses (3138), such inhibitors have the
Th2-type inflammatory infiltrate of lepromatous leprosy (18). same effects on cells that lack catalytically active CD26/DPPIV
The ability of DPPIV to alter or ablate the activities of substance (36, 38). Furthermore, rendering CD26 catalytically inert by
P, neuropeptide Y and endomorphin-2 (see review (1)) may mutation of the catalytic serine has no effect on its ability to
indicate a contribution by this enzyme to regulating interac- modulate T-cell proliferation in vitro (39) (M. D. Gorrell, L. Slai-
tions between the nervous and immune systems. tini, C. A. Abbott, I. De Meester, T. Khne, G. W. McCaughan

Immunological Reviews 174/2000 173


McCaughan et al Molecular pathogenesis of liver disease

unpublished). This question requires revisitation once inhibi-


tors that are quite specific for DPPIV are developed (see below).

CD26/DPPIV expression in liver diseases


As DPPIV is located on the bile canaliculus it not surprising that
serum levels are elevated in liver diseases (4042). However,
the sources of serum DPPIV are unknown (40, 43). In human
cirrhosis we have described intense expression of DPPIV on
proliferating bile ductules, neovascular structures and infiltrat-
ing leucocytes (44). In humans, hepatocyte expression is nor-
mally confined to zones 2 and 3 of the acinus but this restricted
expression is lost in regenerating nodules, where all hepato-
cytes are DPPIV positive. Furthermore, basolateral expression of
DPPIV is often seen in severe liver injury (Fig. 2B). In addition,
we have observed dramatically increased serum concentrations
of DPPIV during liver regeneration (40) but downregulation of
cell surface DPPIV in hepatoma cell lines (45). The contribu-
tion of CD26/DPPIV, with its ability to bind ADA at the bile
canalicular surface, to hepatocyte regulation of adenosine levels
has not been fully considered but clearly requires further exam-
ination (46).

Role of fibroblast activation protein in liver fibrosis


Fibroblast activation protein (FAP) is a post-proline cleaving
enzyme very closely related to DPPIV (23, 49, 55) (Table 1).
Unlike DPPIV, FAP expression is limited to activated fibroblasts Fig. 2. Expression of DPPIV and FAP in human liver. Cryostat sections of
and hepatic stellate cells at sites of tissue injury and remodelling normal (A) and cirrhotic (B, C) human livers were stained for DPPIV (A,
(Fig. 2C) (23, 48, 55). Interestingly, we have noticed a correla- B) or FAP (C). DPPIV is normally expressed on the bile canalicular domain
of the hepatocyte cell surface, capillary endothelium and activated
tion between levels of FAP expression and tissue injury in hep- mononuclear leucocytes (A). Altered expression by hepatocytes occurs in
atitis C fibrosis (87). Stellate cells have a central role in the cirrhotic liver; higher magnification (inset) shows the basolateral
pathogenesis of chronic liver injury and extracellular matrix expression of DPPIV seen in some patients (B). In contrast, FAP-specific
immunoreactivity occurs at the tissue-remodelling interface, i.e. the
(ECM)-digesting enzymes are primary mediators of this role. periseptal area of the regenerative nodule, and weaker immunoreactivity
FAP is unique in the stellate cell arsenal of ECM-digesting is seen in the fibrous septa (C). Magnifications: approximately 600 (A),
enzymes in that it is on the cell surface and dependent on nei- 100 (B), 200 (C). Immunoperoxidase stain, haematoxylin
counterstain.
ther preactivation nor metal ions. This contrasts FAP with the
matrix metalloproteinases, which are metal dependent, nearly
all secreted, and made as pro-enzymes requiring activation. and macrophage inhibitory peptide (MIP)-1b. The central un-
Both recombinant and natural human FAP exhibit gelati- answered question is whether the role of FAP in cirrhosis is
nase activity (23, 83), which is surprising because all other beneficial or detrimental. This question could be addressed us-
known substrates of prolyl oligopeptidase family members are ing FAP-deficient mice or a specific enzyme inhibitor.
small, containing less than 100 amino acids. Small substrate
size is consistent with our understanding of the tertiary struc- Structure of DPPIV-like peptidases
ture of this protein family (see below), so this paradox requires CD26/DPPIV is the prototypic type IV DPP. The DPPIV-like
investigation. The other natural substrates of FAP are unknown, gene family, distinguished by a pair of glutamates that are about
but as FAP is known to cleave N-terminal AlaPro-containing 430 residues N-terminal to the catalytic serine and are essential
substrates, some potential substrates can be predicted. These for enzyme activity (88), forms enzyme family S9b, related to
include erythropoietin, pancreatic polypeptide and the chemo- but distinct from prolyl endopeptidase (PEP) (family S9a) in
kines melanoma growth-stimulating activity (MGSA/GRO) b the prolyl oligopeptidase (S9) enzyme family (8991). CD26

174 Immunological Reviews 174/2000


McCaughan et al Molecular pathogenesis of liver disease

Table 1. Comparison of DPPIV with FAP

Functional characteristic DPPIV References FAP References


Hydrolysis of H-Gly-Pro-x 47 23
Hydrolysis of H-Ala-Pro-x 23, 48, 49
Cleavage of chemokines 59 ?
Gelatinase activity 23 23, 50
Binding to adenosine deaminase 5153 23
Anti-DPPIV MAb bind 54 54
Anti-FAP MAb bind 23, 48, 55 23, 48, 55
Expression in normal adult tissues ubiquitous 56, 57 nil 58, 59
Expression on activated fibroblasts in
healing wounds and tumours 60, 61 48, 58, 62
Expression by foetal mesenchymal cells 63 48, 62
Expression on activated hepatic stellate cells ? 23
Expression on activated T cells, B cells,
NK cells, macrophages 6470 58, 59
Physical characteristic
Number of amino acids 766 7175 760 55, 76
Number of extracellular amino acids 738 74 733
Nucleotide identity 63%
Amino acid identity 54%
Amino acid similarity 70%
Number of exons 26 77, 78 26 49
Gene size 90 kb 79 90 kb 49
Gene location 2q 24.3 77 2q 23 80
a/b hydrolase domain 81 55
Catalytic triad serine, aspartate, histidine 82 83
Intron near catalytic serine 77 49
Potential N-linked glycosylation sites 9 71, 72 6 55
Cysteines 12 71, 72 8 55
Catalytically active as dimer, not monomer 84 23, 50
Monomer mobility on SDS-PAGE 110 kDa 95 kDa 62
Dimer mobility on SDS-PAGE 150 kDa 21, 67, 84, 85 180 kDa 23, 48, 50
Diethyl pyrocarbonate produces monomer ? 50
Heating produces monomer 67, 84, 85 48, 50
Predicted 7-blade b propeller domain 54, 86 86

and FAP have very similar structures (Table 1). They have lack of covalent bonding between blades 1 and 7 of the propel-
polypeptide chains of about 80 kDa that produce two extracel- ler is predicted to allow some structural flexibility that may be
lular domains: an a/b hydrolase domain similar to that of PEP involved in enlarging the central pore for substrate entry (92).
and a non-catalytic domain which we have predicted to be a Consideration of this structure provides some insights into
seven-blade b propeller (Fig. 3) (54, 86). Sequence homologies the nature of epitopes on CD26. CD26 has at least five epitopes
indicate that all members of the S9a and S9b families have the (93, 94) and most anti-CD26 mAbs stimulate T-cell prolifera-
same overall topology. Such a structure places the substrate tion (1). The most interesting epitope is that of the six mAbs
entrance and antibody and ligand-binding sites on the lower that inhibit ADA binding (54, 9397). MAb epitopes on b pro-
face of the b propeller, the enzyme surface that is furthest from pellers depend on tertiary structure (98). Concordantly, we
the cell surface (Fig. 4). The position of the catalytic site, inside have shown that the ADA-blocking epitope of CD26/DPPIV is
this hollow enzyme and about 20 from the entrance, presum- discontinuous and involves propeller blades 4 and 6 (54, 86).
ably restricts substrates to those capable of gaining access. The All other mAbs except 2F9 bind extracellular residues N-termi-

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McCaughan et al Molecular pathogenesis of liver disease

Enzyme activity C
ADA binding
7 N
Upper face Antibody binding
ADA & Ab binding
C 6
1
N 1 2 3
4

K441 E206
E205
T440
2
5

L294 L294
E205 L340
E206 V341 V341
K441 L340
R343 T440
Central 4 R343 3
Lower face
pore

Fig. 3. Structurefunction relationships in the ADA-binding domain of numbered from the centre. Unlike WD repeat proteins (181), prolyl
CD26/DPPIV. Ribbon diagram of the predicted b propeller domain with oligopeptidase blades 1 and 7 are not bound covalently. The hydrolase
spheres showing positions of point mutations of interest. Mutations domain is anticipated to be above the propeller in the side view (left) and
ablating enzyme activity are blue, a mutation ablating ADA binding is behind the propeller in the view towards the lowerface (right). Substrate
yellow, mutations that diminish binding by mAbs that inhibit ADA entry into the central cavity is probably via the central pore of the propeller
binding are red and the mutation that affects both mAb and ADA binding (54, 88, 92). The figure was prepared using MOLSCRIPT (100) and
is orange (54, 88). Each propeller blade consists of four b-strands, Canvas v. 5.03.

nal to the ADA binding site (54, 94, 99). MAb 2F9 binds near the prolyl bond, these enzymes have a mixture of characteris-
the C-terminus (94). The other epitopes have been localised, tics (105). T cells express DPP8, QPP and attractin. Like FAP
using chimaeric and truncated molecules, to long sections of and CD26/DPPIV, DPPIVb and NAALADase II are cell surface
sequence (54, 94, 99), suggesting that these epitopes are glycoproteins. In contrast, DPP8 and QPP are cytoplasmic.
dependent on tertiary structure. Our model of CD26/DPPIV Separate cDNAs encode surface and soluble forms of attractin.
(Fig. 3) predicts that these epitopes involve propeller blades 1 Whereas DPP8, with 27% identity to DPPIV, and QPP, with
and/or 2, but this requires confirmation. 42% identity to prolyl carboxypeptidase, contain the a/b
hydrolase fold characteristic of the SC enzyme clan, NAALAD-
Other post-proline dipeptidyl aminopeptidases ase is structurally unrelated to DPPIV and attractin is unrelated
The target of DPPIV enzyme inhibitors is not always DPPIV (36, to any known enzyme. Indeed, it is not clear that attractin is a
38). Searches for other enzymes that cleave GlyPro or AlaPro protease (105), and it is possible that the reported low-level
amino-terminal dipeptides have discovered DPP8 (C. A. Abbott, hydrolysis of GlyPro substrates is indirect, perhaps via bind-
D. Yu, G. W. McCaughan, M. D. Gorrell, unpublished); quies- ing to a DPP. There is no structural data on DPPIVb. The dis-
cent peptidyl peptidase (QPP) (101); attractin (102); a coveries of carboxypeptidases and DPPIV-like enzymes that
glutamate carboxypeptidase, N-acetylated a-linked acidic cleave DPPIV substrates indicate that DPPIV inhibitors require
dipeptidase (NAALADase) II (103); and DPPIVb (104). Curi- re-evaluation.
ously, despite their common catalytic activity of hydrolysing

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Fig. 4. A model of CD26/DPPIV action at the T-cell surface. The central nucleus via the TCR/CD3 pathway (66, 108, 109), which includes
pore of the b-propeller domain, the likely site of substrate entry (see phosphorylation of p56lck, Fyn59, ZAP70 and c-Cbl (30, 37). The
Fig. 3), points away from the cell surface. The substrates of DPPIV include cytoplasmic domain of CD26 is too short to transduce a signal, so it is
the chemokines RANTES, MDC and eotaxin (see Fig. 1). The ADA-binding presumed that the transduction mechanism is indirect. CD45 is a candidate
activity of CD26 may contribute to the control of purine levels at the T-cell intermediary molecule (110) and has two cytoplasmic phosphatase
surface, in addition to CD38, CD39, CD73 (5' nucleotidase) and adenosine domains. Our model of residues 133 to 766 of CD26/DPPIV is depicted
receptors (3, 106, 107). CD26-triggered signals are transmitted to the here (M. D. Gorrell, W. B. Church, unpublished).

The studies of type IV DPPs, which began as a fundamental regeneration using semi-quantitative polymerase chain reac-
characterisation of an enzyme family expressed in normal liver tion (PCR). Our early studies in this area examined the tissue
tissue, have led to a unique understanding of molecules that expression of a large range of cytokines in human cirrhosis and
play a significant role in inflammatory processes and fibrosis. human liver allograft rejection (111, 112). Although the
These studies now complement our more direct examination results in allograft rejection were limited, probably due to
of the intrahepatic expression of cytokines and growth factors immunosuppressive therapy, the studies of human cirrhosis
in liver disease. revealed marked upregulation of mRNA of various cytokines,
particularly IL-2, IL-6 and IL-8 (111). Autoimmune hepatitis
(AIH) was characterised by increases in these inflammatory
Intrahepatic gene expression of inflammatory cytokines and cytokines, whilst primary biliary cirrhosis (PBC) had marked
growth factors in human and experimental liver disease upregulation only of IL-2, IL-8 and tumour necrosis factor-a
Intrahepatic gene expression of inflammatory cytokines in but not interferon-g (IFN-g).
human chronic liver disease These studies did not however examine earlier stage dis-
Virtually all liver diseases are characterised by tissue inflamma- ease or the Th1/Th2 paradigm of tissue inflammation. In order
tion, cellular injury, cellular regeneration and tissue fibrosis. In to do this we used hepatitis C virus (HCV) infection as a proto-
our laboratory we have concentrated on examining the gene type disease where both of these parameters could be studied.
expression of potential mediators of chronic inflammation and Our work clearly showed that HCV liver injury was associated

Immunological Reviews 174/2000 177


McCaughan et al Molecular pathogenesis of liver disease

Fig. 5. Inflammation and fibrosis correlate with T1


cytokine content in HCV liver. Linear regression
A analyses show IFN-g mRNA expression (number of
IFN-g molecules per 107 aldolase B molecules) versus
severity of histological grading of HCV liver biopsy
IFN-

specimens. A. Total Scheuer score (SS) versus IFN-.


B. Fibrosis component of SS versus IFN-. C. Portal
inflammation component of SS versus IFN-.
[From (113)]

Scheuer score (total)

B
IFN-

Scheuer score (fibrosis)

C
IFN-

Scheuer score (portal)

with an increase in the hepatic expression of the Th1 cytokines chronic HCV may be attenuated (120). We have recently dis-
IL-2 and IFN-g (Fig. 5) (113). There was little IL-4 or IL-10 cussed the apparent paradox of impaired anti-HCV immunity
detected. Furthermore, IL-2 and IFN-g expression levels corre- in the setting of an aggressive intrahepatic cell-mediated Th1
lated with each other, and both correlated with the degree of response (117). Perhaps the paradox can be resolved by under-
tissue inflammation and fibrosis (Fig. 5). We have subsequently standing that much of the hepatic inflammation in chronic
shown marked upregulation of the IFN-g-inducing cytokine IL- HCV disease may not be antigen specific. This is supported by
18 and documented an increase in an activated macrophage the apparent recognition of non-HCV-infected cells by anti-
population during HCV liver disease (Fig. 6) (114). HCV-specific CTLs in vitro (121). If this is the case in vivo the
We documented that progressive HCV disease was not enigma of what drives the non-specific inflammatory response
associated with an accumulation of intrahepatic viral load remains to be addressed.
(115). These studies have been complementary to data indicat-
ing significant and persisting intrahepatic immune responses Intrahepatic gene expression of growth factors
including anti-HCV cytotoxic T-lymphocyte (CTL) responses in in chronic liver injury
chronic HCV disease (Table 2) (116118). This has allowed us Although the pathogenesis of liver fibrosis and aspects of
to put forward a model of HCV immune pathogenesis particu- hepatic inflammation are well understood, the molecular trig-
larly related to liver injury (117). The recent recognition of gers of chronic liver regeneration during the cirrhotic process
CD81 as a potential HCV cell surface receptor and the known are unclear. Our studies of human cirrhosis showed significant
autoimmune features of HCV infection have also been incorpo- upregulation only of epidermal growth factor (EGF) (122).
rated into the model (Fig. 7) (119). However, a sequential analysis of experimental cirrhosis indi-
The model proposed above needs to be examined in light cated early but unsustained upregulation of the intrahepatic
of the data suggesting that anti-HCV CD4 T-cell responses in expression of hepatocyte growth factor which was followed by

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McCaughan et al Molecular pathogenesis of liver disease

Fig. 6. Macrophages in HCV liver. Immunostaining of


HCV-positive human liver tissues with CD68 and MAC387
monoclonal antibodies. A. HCV-negative normal tissue
stained with CD68. B. HCV-negative normal tissue
stained with MAC387. C. Chronic hepatitis tissue stained
with CD68. D. Chronic hepatitis tissue stained with
MAC387. E. Chronic hepatitis tissue stained with CD68.
F. Chronic hepatitis tissue stained with MAC387.
G. Cirrhotic tissue stained with CD6. H. Cirrhotic tissue
stained with MAC387. Immunoperoxidase method with
haematoxylin counterstain (approximately 200
magnification).

a marked increase in the expression of EGF and fibroblast Recent advances in differential gene expression analysis
growth factor (FGF) (123). There was little increase in trans- techniques applied to human cirrhosis
forming growth factor-a. At the protein level there was marked Differential gene expression: an overview
expression of FGF in areas surrounding proliferating bile Differential gene expression is a common approach to facilitate
ductules. Although the molecular pathways of acute liver regen- understanding of gene function. This approach correlates gene
eration are being increasingly well understood, the important expression differences with cell or tissue phenotypes. Differen-
players in the chronic regenerative process, which is a hallmark tial gene expression can be examined using sequence intensive
of the cirrhotic process, have received insufficient attention. methods such as serial analysis of gene expression (SAGE)
Although the above approaches have given us insights into (124), electronic subtraction (125) and DNA sequencing
disease pathogenesis, they have been limited by an examination chips, but these methods are limited to well-resourced institu-
of a restricted set of molecules and known or potential disease tions (126). The more common and accessible differential
pathways. New molecular techniques, such as suppression sub- gene expression methods compare mRNA pools using cDNA
tractive hybridisation and gene array analysis, have the poten- arrays or PCR-based methods, such as representational differ-
tial to find new and interesting molecules and novel pathogenic ences analysis (RDA) (127) and differential display (128).
processes that may be implicated in liver tissue injury. We have cDNA array analysis is a powerful technique for examining
recently used both of these techniques to explore human cir- expression profiles of hundreds to thousands of genes simulta-
rhosis. neously. Three types of arrays are commonly used: 1) mem-
brane-based arrays that are typically hybridised with radiola-
belled probes; 2) high-density microarrays using dual fluoro-
chrome-labelled probes; and (3) gene-chip technology, which
uses high-density arrays of oligonucleotides. Representational

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McCaughan et al Molecular pathogenesis of liver disease

Fig. 7. Compartmentalisation model of chronic


HCV infection. Continued liver injury is associated
T1 T2 with a persistent T1 response within the liver.
HCV Immune recognition events within the spleen, lymph
DC nodes and peripheral blood leads to T2 cytokine
production and associated persistent anti-HCV
Liver antibody production. Autoantibodies and associated
immune complex disease are possibly induced by
T1
direct CD81 B-cell stimulation (117).
T1/T2
Lymph
nodes Spleen
Blood

Table 2. Evidence for intrahepatic Th1


HCV liver damage ? response in chronic HCVa
HCV
? T2 response
CTL/macrophage
activation Increased intrahepatic IL-2, IFN-g, IL-2R expression
Direct
Increased intrahepatic activated macrophages
HCV antibody CD81
T1 response B-cell Detectable intrahepatic anti-HCV CTL response
stimulation
Control of viral load Increased Th1 related adhesion molecule expression
?
Autoantibodies Increased Th1 related chemokines and receptors
Immune pressure
No increase in viral load with disease severity
Viral mutation
Immune complex disease
a From (114116)

differences analysis and differential display compare mRNA involved in the pathogenesis of cirrhosis, specifically genes
pools using PCR-based methods with subsequent differential involved in immune-mediated liver injury. Comparisons were
expression screening or by resolution of differentially made between hepatic liver injury secondary to HCV, AIH, PBC
expressed products on a gel (129). These techniques are more and primary sclerosing cholangitis (PSC) and non-diseased
labour intensive than array analysis but enable identification of liver (donor liver biopsies and normal liver). The diseased tis-
novel genes. sue was taken at the time of liver transplantation from patients
In liver disease most differential expression experiments with end-stage cirrhosis. Initially, mRNA pools were compared
identify and examine individual candidate genes, as described using suppression subtractive hybridisation, which enriches
above. Approaches aimed at profiling multiple genes or identi- for differentially expressed genes by using adapter ligation and
fying novel genes in human liver are limited (130), with a sig- subsequent PCR amplification of differentially expressed spe-
nificant portion of genes identified being tumour related (131, cies (CLONTECH PCR Select, Palo Alto, CA, USA). Table 3 sum-
132). Liver cDNA array analyses have appeared as abstracts, but marises the results of the subtraction analysis. Having identified
few are published. Additionally, most differential expression candidate genes, further characterisation involved confirma-
experiments using these techniques compare cell lines rather tion of differential expression with real-time reverse trans-
than tissue samples. The liver, with its genetic diversity, pleth- criptase (RT)-PCR using an ABI Prism 7700 sequence detector
ora of functions and well-characterised disease phenotypes, (Perkin Elmer, Roche, New Jersey, USA) and Sybr Green 1. Sybr
represents an ideal tissue to examine differential gene expres- Green 1 is a double-stranded DNA-specific dye which enables
sion. PCR product to be quantified as it is produced (134). One
sequence identified as upregulated using subtraction hybridis-
Differential gene expression in human cirrhosis ation was the tumour antigen CO-029 (135, 136), which has
Intrahepatic differential gene expression in human cirrhosis not been examined in non-tumour tissue. The real time quan-
was examined using cDNA array analysis and a modification of titative RT-PCR supported the subtraction result, confirming
the classical RDA technique, suppression subtractive hybridisa- significant upregulation, greater than 1.8-fold, of the CO-029
tion (133). Our aim was to identify new genes or pathways mRNA in HCV compared to normal liver. Subsequent CO-029

180 Immunological Reviews 174/2000


McCaughan et al Molecular pathogenesis of liver disease

Table 3. Suppression subtractive hybridisation

Tester vs driver Clones screened Differentially expressed clones Novel clones Examples of interesting clones
AIH vs PBC 81 >4 9 Human NK-cell enhancing factor
Endothelial monocyte-activating polypeptide II
PBC vs AIH 81 >10 2 NK-cell-activating factor
HCV vs AIH 182 >8 1 Oligoadenylate synthetase
AIH vs HCV 182 >25 2 Interleukin-8
Matrin-3 homologue
HCV vs normal 84 > 49 1 Interferon-induced 15 kDa/17 kDa protein
CO-029 tetraspanin
Normal vs HCV 84 > 33 1 a-1-antichymotrypsin mRNA
Protein disulphide isomerase

Suppression subtractive hybridisation utilises paired forward and reverse subtrac- cDNA following reverse transcription or by labelling the subtracted sample with
tions enriching for differentially expressed mRNA in a tester sample compared to a 32P-dCTP. Differential expression was considered to be a 10-fold or greater differ-

driver sample. Differential expression screening was performed using dot-blot ence in signal intensity. The 145 clones successfully sequenced represented 89
hybridisation with 32P-2'-deoxycytidine 5'-triphosphate (dCTP)-labelled first strand unique clones.

mAb immunostaining localised immunopositivity to hepato- in non-cirrhotic HCV liver injury compared to other forms of
cytes and biliary epithelium without significant stromal stain- chronic hepatitis (139).
ing in both normal and diseased tissue. CO-029 belongs to the The differential expression of two genes was identified by
tetraspanin family of molecules, which includes CD81, a puta- both suppression subtractive hybridisation and cDNA array
tive receptor for HCV. Initial identification of genes such as CO- analysis. The upregulation of IL-8 and endothelial monocyte-
029 using these techniques is now more rapid than subsequent activating polypeptide II in cirrhotic liver was detected by sup-
characterisation. pression subtractive hybridisation. Subsequent cDNA array
Array analysis used two cDNA arrays (Human Gene Array analysis found greater than 3.5-fold upregulation of these
1.0 and Cytokine/Receptor Array, CLONTECH) representing a inflammation-associated molecules in both HCV and AIH liver.
total of 664 unique genes. Probes were made from pooled These preliminary results indicate that differential gene
mRNA (each pool was derived from four patients) from nor- expression using the techniques of cDNA array analysis and
mal, donor, AIH, PBC, HCV and PSC liver tissue. Genes on each subtractive hybridisation has the potential to rapidly increase
array were quantified using a phosphoimage analyser and our knowledge of pathogenic pathways in experimental and
adjusted for ubiquitin housekeeper gene expression. Table 4 lists human liver disease. We are currently completing the analysis
some preliminary data on genes identified in the inflammatory of our gene array data and pursuing new genes identified by
and fibrotic pathways in chronic HCV cirrhosis. Fig. 8 gives an suppression subtractive hybridisation. We believe these results
example of a cDNA array hybridisation. A proportion of the will reveal novel insights into HCV infection, PBC, AIH, PSC
array results was consistent with previous semi-quantitative and the cirrhotic process in general.
PCR results generated by other researchers and by us. For exam- The third area of interest in our laboratory has been to
ple, inflammatory mediators such as IL-8, IFN-g receptor b sub- investigate the mechanisms of spontaneous liver transplant tol-
unit and IL-2 receptor were found to be upregulated in HCV erance. These investigations arose from our initial studies of
liver on array analysis, concordant with studies that used other human liver allograft rejection, which identified a role for CD8-
methods (111, 137). In addition, a 1.7 to 5.1-fold upregula- positive lymphocytes in the destruction of the biliary epithe-
tion of gene expression in hepatitis C liver demonstrated by lium and the microvasculature in chronic rejection (140, 141).
cDNA array analysis was confirmed by quantitative real time
RT-PCR with Sybr Green 1. Other interesting genes involved in
liver regeneration, signal transduction and apoptosis were An analysis of liver transplant tolerance
found to be upregulated in HCV compared to normal liver for Liver passenger leucocytes are important
the first time (data not shown). Molecules such as MHC Class I in liver transplant tolerance
C-4 subunit were demonstrated to be upregulated in HCV cir- The spontaneous acceptance, without evidence of rejection, of
rhosis compared to normal, an increase previously demon- liver allografts has been well documented in many animal spe-
strated in cholestasis (138), which is an effect of cirrhosis, and cies. A number of mechanisms have been proposed for the

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McCaughan et al Molecular pathogenesis of liver disease

Fig. 8. A cDNA array analysis of HCV liver. An ATLAS cytokine/receptor


array probed with 32P-labelled cDNA made from HCV cirrhotic liver
mRNA (four patients). This array contains cDNAs for 265 genes spotted
in duplicate. The boxed portion of the cDNA array is magnified on the
right: this segment of the array hybridised with probes derived from
normal (top) and HCV (bottom) liver samples are presented to show two
examples of altered expression. Upregulation of genes 2 and 3 was seen in
HCV compared to normal using a housekeeper gene (gene 1) signal as a
reference.

Table 4. cDNA array analysis grouping genes with pathogenic processes

Gene HCV:normal ratio Pathogenic process


Interleukin-8 4.7 Inflammation
Monocyte chemotactic protein-1 precursor 2.6 Inflammation
MHC Class I C-4 antigen a subunit 2.8 Inflammation
Lymphotoxin > 2.2 Inflammation
Fibronectin receptor >-subunit 5.7 Fibrosis
Fibroblast growth factor receptor-1 precursor 2.2 Fibrosis
Transforming growth factor->-3 4.8 Fibrosis
Epithelial discoidin domain receptor-1 2.0 Fibrosis
Helix-loop-helix protein 2.2 Signalling
Tristetraproline 2.4 Signalling
Ephrin A3 precursor 2.6 Signalling
Glutathione-S-transferase M1 3.2 Stress response
Proto-oncogene c-jun 6.2 Oncogene

Hybridisation signals from the cDNA arrays were quantified by phosphoimage anal-
ysis. The ratios compare signals from individual arrays adjusted for background and
housekeeping gene signals. Normal liver was non-diseased tissue from transplant
donor biopsies or non-tumour tissue from patients having hepatic metastases
resected.

182 Immunological Reviews 174/2000


McCaughan et al Molecular pathogenesis of liver disease

resistance of the liver to rejection. These include production of generation of oral tolerance (151153), we have sought to
protective factors by the liver such as soluble MHC class I mol- characterise liver leucocytes in the rat. The liver has a large pop-
ecules that can neutralise cytotoxic T cells directed against the ulation of passenger leucocytes such as B cells, T cells and mye-
liver donor MHC (142) or a soluble form of Fas that blocks loid cells such as monocytes and MHC class II-expressing cells
apoptotic death of hepatocytes mediated by Fas-ligand-express- with dendritic morphology (147). It also contains populations
ing effector T cells (143). There is also a soluble factor, appar- of atypical lymphocytes such as NK T cells (154). Liver leuco-
ently unrelated to either of these proteins, which appears in the cyte phenotypes, however, still remain poorly characterised in
serum of tolerant liver-transplanted rats and which can specif- the rat, despite the prominence of this species in studies of liver
ically suppress in vitro alloreactivity (144). However, efforts to transplant tolerance. Interestingly, we have been unable to iden-
induce transplant tolerance with soluble factors have met with tify NK T cells in the rat by the standard phenotypic markers
little success (145). used in mice. In mouse strains expressing the NK R-P1 subfam-
In contrast to the relative ineffectiveness of soluble factors ily of NK markers, NK T cells can be identified as bearing both
in prolonging allograft survival, there is considerable evidence this marker and the ab T-cell receptor (TCR). NK T cells in the
that mobile cellular elements of the transplanted liver are cen- mouse can also be identified by intermediate ab TCR expres-
tral to the liver tolerance process. One line of evidence that sion, or the phenotype TCRpos CD4/CD8 double negative or
donor cells played a role came from studies in which donor liv- TCRpos CD4low (155). None of these characteristic phenotypes
ers were parked in intermediate hosts to replace passenger leu- could be demonstrated in the rat (D. G. Bowen, G. W. McCaughan,
cocytes of donor strain with those of the intermediate host G. A. Bishop, unpublished data). It is feasible that the pheno-
(146). Livers of PVG rat strains were parked in DA rat strain type of the NK T cell differs markedly in the rat from that
intermediate hosts. Although the DA strain is unrelated, the seen in mice or humans; however, the possibility exists that this
liver is not rejected, and liver leucocytes are exchanged with leucocyte subset is truly absent in the rat, and its putative im-
the recipient. The livers thus contained PVG parenchyma and munomodulatory functions (156, 157) are subsumed by other
DA passenger leucocytes, while DA livers parked in PVG inter- cell subtypes.
mediate hosts contained DA parenchyma and PVG passenger The role of the liver in immune function remains unclear.
leucocytes. Transplantation of these livers to PVG strain recipi- It has been postulated that the liver functions as a graveyard
ents showed that tolerance was markedly reduced in those liv- for activated T cells, with activated T cells congregating in the
ers in which the donor leucocytes matched the recipient. liver to undergo apoptosis (158). However, we have studied
Direct evidence that donor passenger leucocytes were recirculation of liver leucocytes in the rat and have demon-
important in liver transplant tolerance came from experiments strated that the liver-derived T cells remain capable of recircu-
in our laboratory in which these cells were reduced by irradia- lation, tending to home to the lymph nodes and spleen, and are
tion of the liver donor prior to transplantation of the liver still readily identifiable 1 week after transfer. Whilst liver-
(147). Recipients of passenger leucocyte-depleted livers rap- derived T cells do not recirculate selectively to the liver, we
idly rejected the liver in strain combinations in which non-irra- found that liver-derived NK cells (pit cells) home specifically
diated livers are normally accepted (147). Reconstitution of to the liver. This probably relates to differences in expression of
donor passenger leucocytes by parking of the leucocyte- adhesion molecules such as CD11a and CD 18 (159), as well as
depleted liver in a normal donor for 48 h (147) or by injection CD 11b/c (D. G. Bowen, G. W. McCaughan, G. A. Bishop,
of donor leucocytes (148) restored acceptance of the liver. unpublished). The specific homing of pit cells to the liver is
These experiments show the importance of passenger leuco- unsurprising, given the differences in morphology and cyto-
cytes of donor origin in promoting liver acceptance and fulfil toxicity that exist between pit cells and NK cells found in other
Kochs postulates for demonstrating the requirement for these tissues (160). Further phenotypic examination of this leuco-
cells for acceptance. The role of liver leucocytes in liver accep- cyte subset may lead to hypotheses on function. A broader
tance has been confirmed by different groups using other strain range of phenotypic markers would facilitate studies of the rat.
combinations (149, 150). Despite the differences in phenotype between liver leuco-
cytes and leucocytes found in other organs, we have shown that
Characterisation of rat liver leucocytes it is unlikely that leucocytes unique to the liver have a unique
Given this potential role of passenger liver leucocytes in the ability to induce tolerance as spleen cells are as efficient as liver
generation of spontaneous tolerance to liver transplants, as well leucocytes in their ability to reconstitute acceptance of leuco-
as data suggesting that liver leucocytes may contribute to the cyte-depleted livers (148, 149).

Immunological Reviews 174/2000 183


McCaughan et al Molecular pathogenesis of liver disease

Paradoxical immune activation in lymphoid tissues emerged that the involvement of IL-2 and IFN-g in transplant
in liver transplant tolerance tolerance is not confined to liver transplantation alone as toler-
We have observed that when the lymphoid tissues of allograft ance induced by co-stimulatory blockade using anti-CD40
recipients are compared there is a surprising increase of IL-2 ligand antibody and CTLA4-Ig cannot be induced in IL-2 or
and IFN-g mRNA in the spleen and draining lymph nodes of IFN-g knockout recipients (170, 171). This is clear evidence
liver-tolerant animals which is reduced or absent in the corre- that at least some forms of transplantation tolerance are depen-
sponding lymphoid tissues of recipients of liver, kidney or skin dent on immune activation and the expression of IL-2 and
allografts in the process of rejection (161164). This finding is IFN-g.
unusual because IL-2 and IFN-g are associated with rejection
and their level of expression in the rejecting organ can increase Apoptotic cell death of activated T cells in liver transplant
over 1,000-fold at the mRNA level (162, 163). There is, how- tolerance
ever, a major difference between tolerance and rejection in the Apoptosis of antigen-reactive T cells is one means by which
site and timing of IL-2 and IFN-g expression. We have shown immune responses are limited, and mice that are deficient in
that they reach peak expression 1 day after transplantation in molecules such as Fas or Fas-ligand, which are involved in apop-
the lymphoid tissues of tolerant animals, while they appear tosis, die of autoimmune disease (reviewed in (172)). A con-
later and their expression is confined principally to the graft in sistent liver transplantation tolerance finding (from our labora-
animals rejecting the transplanted organ (162, 163) (reviewed tory and others) is that in the early stages after transplantation
in (165)). there are more apoptotic cells in transplanted organs in the pro-
Even more surprising is the finding that exogenous IL-2 cess of tolerance than in those undergoing irreversible rejection
prevents liver transplant tolerance when given at the time of (173175). This paradoxical situation of greater cell death in
liver transplantation (150, 166). The paradox thus arises that tolerant grafts than in those that are rejected is partly explained
early expression of IL-2 and IFN-g mRNA in lymphoid tissues by the observation that these dying cells are concentrated in the
is associated with liver graft acceptance but at the same time areas of leucocyte infiltrate, indicating that they may be recipi-
administration of exogenous IL-2 prevents acceptance. This is a ent leucocytes which have infiltrated the graft and then died
central issue in understanding the process involved in the out- (173, 174). Thus there are two processes that lead to the
come of the immune response to the liver, and one that has appearance of apoptotic cells within the graft. One involves
ramifications outside of liver transplantation. The immune death of graft parenchymal cells during rejection (176) and the
response must apparently proceed through steps of activation other involves death of alloreactive graft-infiltrating leucocytes
before it subsequently matures to either reject or accept the during development of tolerance.
liver. This concept is novel and challenges our established view Proof that many of the graft-infiltrating cells in tolerant liv-
of the ways in which tolerance to transplanted organs is ers are apoptotic leucocytes came from studies in which sec-
achieved. Almost all clinical and experimental methods devel- tions were double stained for co-expression of surface CD8 and
oped to obtain long-term survival of transplanted organs in markers of apoptosis (177) or by purification of graft-infiltrat-
humans or in animal models are based on the assumption that ing cells from the transplanted organ and analysis by multico-
the immune response must be inhibited by all means possible. lour flow cytometry (162). These experiments showed that
All immunosuppressive drugs in current clinical use have been there were many apoptotic leucocytes in tolerant livers in the
selected for their ability to block immune activation in T cells first 2 weeks after transplantation. Examination of the subsets
or to remove components of the T-cell repertoire. of apoptotic leucocytes showed that both CD8 (162, 177) and
Immune activation leading to tolerance is not unique to CD4 T cells (162) were apoptotic, and that greatest apoptosis
liver transplantation and the process is well documented in was in the activated T-cell population (162).
many models of immune tolerance. In a landmark paper The close link between apoptosis of alloreactive leucocytes
describing tolerance to minor lymphocyte-stimulating (Mls) and subsequent allograft tolerance has been confirmed by a
superantigen, Webb & Sprent described immune activation of number of studies (162, 173, 174). These show that high lev-
Mls-specific transgenic T cells preceding their clonal deletion els of leucocyte apoptosis were observed in liver tolerance but
(167). Subsequently, many studies have shown that under cer- not rejection of kidney allografts, and that induction of liver
tain circumstances T-cell activation is followed by death of the rejection by administration of exogenous IL-2, by irradiation of
responding T cells, and that IL-2 is involved in this deletional the liver donor or by peri-transplant corticosteroid therapy
tolerance mechanism (168, 169). Recently, evidence has markedly reduced the level of apoptosis. This close association

184 Immunological Reviews 174/2000


McCaughan et al Molecular pathogenesis of liver disease

Fig. 9. Possible mechanisms of liver


transplant tolerance induction by donor
leucocytes. Donor leucocytes transplanted
with the liver play a major role in liver
transplant tolerance. These cells migrate
Donor leucocytes
rapidly to recipient lymphoid tissues such
as spleen and lymph nodes draining the
AICD of recipient T cells in graft. There they are associated with
spleen and lymph nodes marked immune activation followed by
apoptosis of T cells in the graft and
Transplanted liver recipient lymphoid tissues. Possible
Possible immunomodulatory immune mechanisms for this are: A)
interactions in lymphoid tissues Immunomodulatory donor leucocytes
T form a modified stimulatory complex with
DC
donor antigen presenting cells and
IM recipient alloreactive T cells. B) Activation
of recipient T cells by donor antigen
A. Aberrant stimulation
presenting cells occurs normally but the
T
activated T cells then encounter donor
T
DC
IM
leucocytes, leading to aberrant stimulation.
T C) There is competition between donor
B. Sequential stimulation antigen-presenting cells, which deliver a
T-cell signal for activation and survival of
T recipient T cells, and donor leucocytes,
IM death
which deliver a signal for activation and
IM T
IM
death.
T
DC
T
T

C. Competition

IM Donor immunomodulatory cell T


T Recipient T cell

DC Donor dendritic cell T Recipient activated T cell


T

between liver transplant tolerance and death of infiltrating migrate to the graft and increase its cytokine expression there,
T cells, especially activated T cells, is further evidence that liver induces immediate activation, cytokine expression in recipient
transplant tolerance is akin to activation-induced cell death lymphoid tissues and consequent apoptotic death.
(AICD). Another possible mechanism by which recipient alloreac-
tive T cells are neutralised is that the donor dendritic cells
Mechanisms of AICD in recipient lymphoid tissues deliver an activating signal to recipient T cells as normal. These
Possible immune pathways by which donor leucocytes might activated T cells subsequently encounter donor immunomodu-
induce recipient T-cell activation then apoptotic death in recip- latory cells in recipient lymphoid tissues, causing them to
ient lymphoid tissues are shown in Fig. 9. Recipient T cells are increase their expression of IL-2 and IFN-g mRNA and subse-
normally activated in recipient lymphoid tissues by donor den- quently die (Fig. 9B). These events could be similar to the
dritic cells (178, 179) and then migrate to the graft and sequential stimulation involved in AICD, where stimulation via
increase their expression of IL-2 and IFN-g at that site. One pos- the TCR leads to activation of the T cell and progression into
sible mechanism by which liver passenger leucocytes induce cell cycle. Subsequent re-engagement of the TCR of these acti-
tolerance is that immunomodulatory cells of the donor form a vated T cells results in apoptotic cell death (180).
complex with the donor dendritic cell and alloreactive T cells A third possibility is that dendritic leucocytes and immu-
of the recipient in the recipient lymphoid tissues (Fig. 9A). The nomodulatory cells compete to stimulate alloreactive T cells of
modified stimulatory complex is postulated to deliver an aber- the recipient. Immunomodulatory donor leucocytes might
rant stimulatory signal to the responding recipient T cell. This operate by delivering signal 1 through their engagement of the
aberrant signal, instead of stimulating the responder T cell to recipient TCR but not signal 2, which is delivered by co-stim-

Immunological Reviews 174/2000 185


McCaughan et al Molecular pathogenesis of liver disease

ulatory molecules, such as B7, which are present on dendritic the presence of donor leucocytes in lymphoid tissues and, if so,
cells but are lacking on many leucocyte populations. In this what population of donor leucocytes can most efficiently
case, the pool of alloreactive precursor T cells can either be induce AICD. Finally, there are many potential applications of
stimulated by dendritic cells, after which they effect graft these findings to clinical transplantation. It may be possible to
destruction, or by co-stimulatory molecule-deficient donor induce tolerance to liver or other organ grafts by AICD-like
leucocytes, after which they increase expression of IL-2 and mechanisms using donor leucocytes purified from the spleen
IFN-g and then die. In this case, if the numbers of donor immu- or from the liver perfusate of the organ donor. An unresolved
nomodulatory leucocytes are sufficiently large, then the pro- question is to what degree current immunosupression inhibits
portion of alloreactive T cells that receive an apoptotic signal AICD mechanisms that might otherwise be occurring during
will greatly outnumber those that receive an activating signal clinical liver transplantation.
from dendritic cells and the alloreactive T-cell pool will be
depleted (Fig. 9C).
Conclusions
In summary, liver transplant tolerance, one of the most
powerful models of peripheral tolerance to transplanted Our laboratory has examined three overlapping themes in the
organs, is associated with a process that superficially resembles study of chronic liver injury using molecular and cellular tech-
AICD. Although the basic processes involved in AICD have been niques. These approaches have provided unique insights into
demonstrated during liver transplant tolerance, there is still the role of the prolyl oligopeptidases and inflammatory cyto-
much evidence required to firmly establish AICD as the princi- kines in the pathogenesis of liver injury. Current work has
pal tolerising mechanism. It is of primary importance to estab- delineated new members of the prolyl oligopeptidase gene
lish which cells in recipient lymphoid tissues are stimulated to family, described novel pathways in the pathogenesis of liver
increase expression of IL-2 and IFN-g following liver transplan- disease and defined new approaches to liver allograft tolerance.
tation. It is also of great interest to know whether this is due to

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