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
and hepatic stellate cells at sites of tissue injury and remodelling
(Fig. 2C) (23, 48, 55). Interestingly, we have noticed a correla-
tion between levels of FAP expression and tissue injury in hep-
atitis C fibrosis (87). Stellate cells have a central role in the
pathogenesis of chronic liver injury and extracellular matrix
(ECM)-digesting enzymes are primary mediators of this role.
FAP is unique in the stellate cell arsenal of ECM-digesting
enzymes in that it is on the cell surface and dependent on nei-
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.
Both recombinant and natural human FAP exhibit gelati-
nase activity (23, 83), which is surprising because all other
known substrates of prolyl oligopeptidase family members are
small, containing less than 100 amino acids. Small substrate
size is consistent with our understanding of the tertiary struc-
ture of this protein family (see below), so this paradox requires
investigation. The other natural substrates of FAP are unknown,
but as FAP is known to cleave N-terminal AlaPro-containing
substrates, some potential substrates can be predicted. These
include erythropoietin, pancreatic polypeptide and the chemo-
kines melanoma growth-stimulating activity (MGSA/GRO) b
Fig. 2. Expression of DPPIV and FAP in human liver. Cryostat sections of
normal (A) and cirrhotic (B, C) human livers were stained for DPPIV (A,
B) or FAP (C). DPPIV is normally expressed on the bile canalicular domain
of the hepatocyte cell surface, capillary endothelium and activated
mononuclear leucocytes (A). Altered expression by hepatocytes occurs in
cirrhotic liver; higher magnification (inset) shows the basolateral
expression of DPPIV seen in some patients (B). In contrast, FAP-specific
immunoreactivity occurs at the tissue-remodelling interface, i.e. the
periseptal area of the regenerative nodule, and weaker immunoreactivity
is seen in the fibrous septa (C). Magnifications: approximately 600 (A),
100 (B), 200 (C). Immunoperoxidase stain, haematoxylin
counterstain.
and macrophage inhibitory peptide (MIP)-1b. The central un-
answered question is whether the role of FAP in cirrhosis is
beneficial or detrimental. This question could be addressed us-
ing FAP-deficient mice or a specific enzyme inhibitor.
Structure of DPPIV-like peptidases
CD26/DPPIV is the prototypic type IV DPP. The DPPIV-like
gene family, distinguished by a pair of glutamates that are about
430 residues N-terminal to the catalytic serine and are essential
for enzyme activity (88), forms enzyme family S9b, related to
but distinct from prolyl endopeptidase (PEP) (family S9a) in
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
polypeptide chains of about 80 kDa that produce two extracel-
lular domains: an a/b hydrolase domain similar to that of PEP
and a non-catalytic domain which we have predicted to be a
seven-blade b propeller (Fig. 3) (54, 86). Sequence homologies
indicate that all members of the S9a and S9b families have the
same overall topology. Such a structure places the substrate
entrance and antibody and ligand-binding sites on the lower
face of the b propeller, the enzyme surface that is furthest from
the cell surface (Fig. 4). The position of the catalytic site, inside
this hollow enzyme and about 20 from the entrance, presum-
ably restricts substrates to those capable of gaining access. The
lack of covalent bonding between blades 1 and 7 of the propel-
ler is predicted to allow some structural flexibility that may be
involved in enlarging the central pore for substrate entry (92).
Consideration of this structure provides some insights into
the nature of epitopes on CD26. CD26 has at least five epitopes
(93, 94) and most anti-CD26 mAbs stimulate T-cell prolifera-
tion (1). The most interesting epitope is that of the six mAbs
that inhibit ADA binding (54, 9397). MAb epitopes on b pro-
pellers depend on tertiary structure (98). Concordantly, we
have shown that the ADA-blocking epitope of CD26/DPPIV is
discontinuous and involves propeller blades 4 and 6 (54, 86).
All other mAbs except 2F9 bind extracellular residues N-termi-

Immunological Reviews 174/2000 175

McCaughan et al Molecular pathogenesis of liver disease
Enzyme activity
ADA binding
Upper face Antibody binding
ADA & Ab binding
C 6
N 1
L294
E205 L340
E206 V341
K441
R343 T440
Central
Lower face
pore
Fig. 3. Structurefunction relationships in the ADA-binding domain of
CD26/DPPIV. Ribbon diagram of the predicted b propeller domain with
spheres showing positions of point mutations of interest. Mutations
ablating enzyme activity are blue, a mutation ablating ADA binding is
yellow, mutations that diminish binding by mAbs that inhibit ADA
binding are red and the mutation that affects both mAb and ADA binding
is orange (54, 88). Each propeller blade consists of four b-strands,
nal to the ADA binding site (54, 94, 99). MAb 2F9 binds near
the C-terminus (94). The other epitopes have been localised,
using chimaeric and truncated molecules, to long sections of
sequence (54, 94, 99), suggesting that these epitopes are
dependent on tertiary structure. Our model of CD26/DPPIV
(Fig. 3) predicts that these epitopes involve propeller blades 1
and/or 2, but this requires confirmation.
Other post-proline dipeptidyl aminopeptidases
The target of DPPIV enzyme inhibitors is not always DPPIV (36,
38). Searches for other enzymes that cleave GlyPro or AlaPro
amino-terminal dipeptides have discovered DPP8 (C. A. Abbott,
D. Yu, G. W. McCaughan, M. D. Gorrell, unpublished); quies-
cent peptidyl peptidase (QPP) (101); attractin (102); a
glutamate carboxypeptidase, N-acetylated a-linked acidic
dipeptidase (NAALADase) II (103); and DPPIVb (104). Curi-
ously, despite their common catalytic activity of hydrolysing
176 Immunological Reviews 174/2000
C
7 N
1
2 3
4
K441 E206
E205
T440
2
5
L294
V341
L340
4 R343 3
numbered from the centre. Unlike WD repeat proteins (181), prolyl
oligopeptidase blades 1 and 7 are not bound covalently. The hydrolase
domain is anticipated to be above the propeller in the side view (left) and
behind the propeller in the view towards the lowerface (right). Substrate
entry into the central cavity is probably via the central pore of the propeller
(54, 88, 92). The figure was prepared using MOLSCRIPT (100) and
Canvas v. 5.03.
the prolyl bond, these enzymes have a mixture of characteris-
tics (105). T cells express DPP8, QPP and attractin. Like FAP
and CD26/DPPIV, DPPIVb and NAALADase II are cell surface
glycoproteins. In contrast, DPP8 and QPP are cytoplasmic.
Separate cDNAs encode surface and soluble forms of attractin.
Whereas DPP8, with 27% identity to DPPIV, and QPP, with
42% identity to prolyl carboxypeptidase, contain the a/b
hydrolase fold characteristic of the SC enzyme clan, NAALAD-
ase is structurally unrelated to DPPIV and attractin is unrelated
to any known enzyme. Indeed, it is not clear that attractin is a
protease (105), and it is possible that the reported low-level
hydrolysis of GlyPro substrates is indirect, perhaps via bind-
ing to a DPP. There is no structural data on DPPIVb. The dis-
coveries of carboxypeptidases and DPPIV-like enzymes that
cleave DPPIV substrates indicate that DPPIV inhibitors require
re-evaluation.

Fig. 4. A model of CD26/DPPIV action at the T-cell surface. The central
pore of the b-propeller domain, the likely site of substrate entry (see
Fig. 3), points away from the cell surface. The substrates of DPPIV include
the chemokines RANTES, MDC and eotaxin (see Fig. 1). The ADA-binding
activity of CD26 may contribute to the control of purine levels at the T-cell
surface, in addition to CD38, CD39, CD73 (5' nucleotidase) and adenosine
receptors (3, 106, 107). CD26-triggered signals are transmitted to the
The studies of type IV DPPs, which began as a fundamental
characterisation of an enzyme family expressed in normal liver
tissue, have led to a unique understanding of molecules that
play a significant role in inflammatory processes and fibrosis.
These studies now complement our more direct examination
of the intrahepatic expression of cytokines and growth factors
in liver disease.
Intrahepatic gene expression of inflammatory cytokines and
growth factors in human and experimental liver disease
Intrahepatic gene expression of inflammatory cytokines in
human chronic liver disease
Virtually all liver diseases are characterised by tissue inflamma-
tion, cellular injury, cellular regeneration and tissue fibrosis. In
our laboratory we have concentrated on examining the gene
expression of potential mediators of chronic inflammation and
McCaughan et al Molecular pathogenesis of liver disease
nucleus via the TCR/CD3 pathway (66, 108, 109), which includes
phosphorylation of p56lck, Fyn59, ZAP70 and c-Cbl (30, 37). The
cytoplasmic domain of CD26 is too short to transduce a signal, so it is
presumed that the transduction mechanism is indirect. CD45 is a candidate
intermediary molecule (110) and has two cytoplasmic phosphatase
domains. Our model of residues 133 to 766 of CD26/DPPIV is depicted
here (M. D. Gorrell, W. B. Church, unpublished).
regeneration using semi-quantitative polymerase chain reac-
tion (PCR). Our early studies in this area examined the tissue
expression of a large range of cytokines in human cirrhosis and
human liver allograft rejection (111, 112). Although the
results in allograft rejection were limited, probably due to
immunosuppressive therapy, the studies of human cirrhosis
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
cytokines, whilst primary biliary cirrhosis (PBC) had marked
upregulation only of IL-2, IL-8 and tumour necrosis factor-a
but not interferon-g (IFN-g).
These studies did not however examine earlier stage dis-
ease or the Th1/Th2 paradigm of tissue inflammation. In order
to do this we used hepatitis C virus (HCV) infection as a proto-
type disease where both of these parameters could be studied.
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
IL-2 and IFN-g (Fig. 5) (113). There was little IL-4 or IL-10
detected. Furthermore, IL-2 and IFN-g expression levels corre-
lated with each other, and both correlated with the degree of
tissue inflammation and fibrosis (Fig. 5). We have subsequently
shown marked upregulation of the IFN-g-inducing cytokine IL-
18 and documented an increase in an activated macrophage
population during HCV liver disease (Fig. 6) (114).
We documented that progressive HCV disease was not
associated with an accumulation of intrahepatic viral load
(115). These studies have been complementary to data indicat-
ing significant and persisting intrahepatic immune responses
including anti-HCV cytotoxic T-lymphocyte (CTL) responses in
chronic HCV disease (Table 2) (116118). This has allowed us
to put forward a model of HCV immune pathogenesis particu-
larly related to liver injury (117). The recent recognition of
CD81 as a potential HCV cell surface receptor and the known
autoimmune features of HCV infection have also been incorpo-
rated into the model (Fig. 7) (119).
The model proposed above needs to be examined in light
of the data suggesting that anti-HCV CD4 T-cell responses in
chronic HCV may be attenuated (120). We have recently dis-
cussed the apparent paradox of impaired anti-HCV immunity
in the setting of an aggressive intrahepatic cell-mediated Th1
response (117). Perhaps the paradox can be resolved by under-
standing that much of the hepatic inflammation in chronic
HCV disease may not be antigen specific. This is supported by
the apparent recognition of non-HCV-infected cells by anti-
HCV-specific CTLs in vitro (121). If this is the case in vivo the
enigma of what drives the non-specific inflammatory response
remains to be addressed.
Intrahepatic gene expression of growth factors
in chronic liver injury
Although the pathogenesis of liver fibrosis and aspects of
hepatic inflammation are well understood, the molecular trig-
gers of chronic liver regeneration during the cirrhotic process
are unclear. Our studies of human cirrhosis showed significant
upregulation only of epidermal growth factor (EGF) (122).
However, a sequential analysis of experimental cirrhosis indi-
cated early but unsustained upregulation of the intrahepatic
expression of hepatocyte growth factor which was followed by

178 Immunological Reviews 174/2000


a marked increase in the expression of EGF and fibroblast
growth factor (FGF) (123). There was little increase in trans-
forming growth factor-a. At the protein level there was marked
expression of FGF in areas surrounding proliferating bile
ductules. Although the molecular pathways of acute liver regen-
eration are being increasingly well understood, the important
players in the chronic regenerative process, which is a hallmark
of the cirrhotic process, have received insufficient attention.
Although the above approaches have given us insights into
disease pathogenesis, they have been limited by an examination
of a restricted set of molecules and known or potential disease
pathways. New molecular techniques, such as suppression sub-
tractive hybridisation and gene array analysis, have the poten-
tial to find new and interesting molecules and novel pathogenic
processes that may be implicated in liver tissue injury. We have
recently used both of these techniques to explore human cir-
rhosis.
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).
Recent advances in differential gene expression analysis
techniques applied to human cirrhosis
Differential gene expression: an overview
Differential gene expression is a common approach to facilitate
understanding of gene function. This approach correlates gene
expression differences with cell or tissue phenotypes. Differen-
tial gene expression can be examined using sequence intensive
methods such as serial analysis of gene expression (SAGE)
(124), electronic subtraction (125) and DNA sequencing
chips, but these methods are limited to well-resourced institu-
tions (126). The more common and accessible differential
gene expression methods compare mRNA pools using cDNA
arrays or PCR-based methods, such as representational differ-
ences analysis (RDA) (127) and differential display (128).
cDNA array analysis is a powerful technique for examining
expression profiles of hundreds to thousands of genes simulta-
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
Immunological Reviews 174/2000 179
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
pools using PCR-based methods with subsequent differential
expression screening or by resolution of differentially
expressed products on a gel (129). These techniques are more
labour intensive than array analysis but enable identification of
novel genes.
In liver disease most differential expression experiments
identify and examine individual candidate genes, as described
above. Approaches aimed at profiling multiple genes or identi-
fying novel genes in human liver are limited (130), with a sig-
nificant portion of genes identified being tumour related (131,
132). Liver cDNA array analyses have appeared as abstracts, but
few are published. Additionally, most differential expression
experiments using these techniques compare cell lines rather
than tissue samples. The liver, with its genetic diversity, pleth-
ora of functions and well-characterised disease phenotypes,
represents an ideal tissue to examine differential gene expres-
sion.
Differential gene expression in human cirrhosis
Intrahepatic differential gene expression in human cirrhosis
was examined using cDNA array analysis and a modification of
the classical RDA technique, suppression subtractive hybridisa-
tion (133). Our aim was to identify new genes or pathways
involved in the pathogenesis of cirrhosis, specifically genes
involved in immune-mediated liver injury. Comparisons were
made between hepatic liver injury secondary to HCV, AIH, PBC
and primary sclerosing cholangitis (PSC) and non-diseased
liver (donor liver biopsies and normal liver). The diseased tis-
sue was taken at the time of liver transplantation from patients
with end-stage cirrhosis. Initially, mRNA pools were compared
using suppression subtractive hybridisation, which enriches
for differentially expressed genes by using adapter ligation and
subsequent PCR amplification of differentially expressed spe-
cies (CLONTECH PCR Select, Palo Alto, CA, USA). Table 3 sum-
marises the results of the subtraction analysis. Having identified
candidate genes, further characterisation involved confirma-
tion of differential expression with real-time reverse trans-
criptase (RT)-PCR using an ABI Prism 7700 sequence detector
(Perkin Elmer, Roche, New Jersey, USA) and Sybr Green 1. Sybr
Green 1 is a double-stranded DNA-specific dye which enables
PCR product to be quantified as it is produced (134). One
sequence identified as upregulated using subtraction hybridis-
ation was the tumour antigen CO-029 (135, 136), which has
not been examined in non-tumour tissue. The real time quan-
titative RT-PCR supported the subtraction result, confirming
significant upregulation, greater than 1.8-fold, of the CO-029
mRNA in HCV compared to normal liver. Subsequent CO-029

180 Immunological Reviews 174/2000


Table 3. Suppression subtractive hybridisation
Tester vs driver Clones screened Differentially expressed clones
AIH vs PBC 81 >4
PBC vs AIH 81 >10
HCV vs AIH 182 >8
AIH vs HCV 182 >25
HCV vs normal 84 > 49
Normal vs HCV 84 > 33
Suppression subtractive hybridisation utilises paired forward and reverse subtrac-
tions enriching for differentially expressed mRNA in a tester sample compared to a
driver sample. Differential expression screening was performed using dot-blot
hybridisation with 32P-2'-deoxycytidine 5'-triphosphate (dCTP)-labelled first strand
mAb immunostaining localised immunopositivity to hepato-
cytes and biliary epithelium without significant stromal stain-
ing in both normal and diseased tissue. CO-029 belongs to the
tetraspanin family of molecules, which includes CD81, a puta-
tive receptor for HCV. Initial identification of genes such as CO-
029 using these techniques is now more rapid than subsequent
characterisation.
Array analysis used two cDNA arrays (Human Gene Array
1.0 and Cytokine/Receptor Array, CLONTECH) representing a
total of 664 unique genes. Probes were made from pooled
mRNA (each pool was derived from four patients) from nor-
mal, donor, AIH, PBC, HCV and PSC liver tissue. Genes on each
array were quantified using a phosphoimage analyser and
adjusted for ubiquitin housekeeper gene expression. Table 4 lists
some preliminary data on genes identified in the inflammatory
and fibrotic pathways in chronic HCV cirrhosis. Fig. 8 gives an
example of a cDNA array hybridisation. A proportion of the
array results was consistent with previous semi-quantitative
PCR results generated by other researchers and by us. For exam-
ple, inflammatory mediators such as IL-8, IFN-g receptor b sub-
unit and IL-2 receptor were found to be upregulated in HCV
liver on array analysis, concordant with studies that used other
methods (111, 137). In addition, a 1.7 to 5.1-fold upregula-
tion of gene expression in hepatitis C liver demonstrated by
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
found to be upregulated in HCV compared to normal liver for
the first time (data not shown). Molecules such as MHC Class I
C-4 subunit were demonstrated to be upregulated in HCV cir-
rhosis compared to normal, an increase previously demon-
strated in cholestasis (138), which is an effect of cirrhosis, and
McCaughan et al Molecular pathogenesis of liver disease
Novel clones Examples of interesting clones
9 Human NK-cell enhancing factor
Endothelial monocyte-activating polypeptide II
2 NK-cell-activating factor
1 Oligoadenylate synthetase
2 Interleukin-8
Matrin-3 homologue
1 Interferon-induced 15 kDa/17 kDa protein
CO-029 tetraspanin
1 a-1-antichymotrypsin mRNA
Protein disulphide isomerase
cDNA following reverse transcription or by labelling the subtracted sample with
32P-dCTP. Differential expression was considered to be a 10-fold or greater differ-
ence in signal intensity. The 145 clones successfully sequenced represented 89
unique clones.
in non-cirrhotic HCV liver injury compared to other forms of
chronic hepatitis (139).
The differential expression of two genes was identified by
both suppression subtractive hybridisation and cDNA array
analysis. The upregulation of IL-8 and endothelial monocyte-
activating polypeptide II in cirrhotic liver was detected by sup-
pression subtractive hybridisation. Subsequent cDNA array
analysis found greater than 3.5-fold upregulation of these
inflammation-associated molecules in both HCV and AIH liver.
These preliminary results indicate that differential gene
expression using the techniques of cDNA array analysis and
subtractive hybridisation has the potential to rapidly increase
our knowledge of pathogenic pathways in experimental and
human liver disease. We are currently completing the analysis
of our gene array data and pursuing new genes identified by
suppression subtractive hybridisation. We believe these results
will reveal novel insights into HCV infection, PBC, AIH, PSC
and the cirrhotic process in general.
The third area of interest in our laboratory has been to
investigate the mechanisms of spontaneous liver transplant tol-
erance. These investigations arose from our initial studies of
human liver allograft rejection, which identified a role for CD8-
positive lymphocytes in the destruction of the biliary epithe-
lium and the microvasculature in chronic rejection (140, 141).
An analysis of liver transplant tolerance
Liver passenger leucocytes are important
in liver transplant tolerance
The spontaneous acceptance, without evidence of rejection, of
liver allografts has been well documented in many animal spe-
cies. A number of mechanisms have been proposed for the
Immunological Reviews 174/2000 181
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


resistance of the liver to rejection. These include production of
protective factors by the liver such as soluble MHC class I mol-
ecules that can neutralise cytotoxic T cells directed against the
liver donor MHC (142) or a soluble form of Fas that blocks
apoptotic death of hepatocytes mediated by Fas-ligand-express-
ing effector T cells (143). There is also a soluble factor, appar-
ently unrelated to either of these proteins, which appears in the
serum of tolerant liver-transplanted rats and which can specif-
ically suppress in vitro alloreactivity (144). However, efforts to
induce transplant tolerance with soluble factors have met with
little success (145).
In contrast to the relative ineffectiveness of soluble factors
in prolonging allograft survival, there is considerable evidence
that mobile cellular elements of the transplanted liver are cen-
tral to the liver tolerance process. One line of evidence that
donor cells played a role came from studies in which donor liv-
ers were parked in intermediate hosts to replace passenger leu-
cocytes of donor strain with those of the intermediate host
(146). Livers of PVG rat strains were parked in DA rat strain
intermediate hosts. Although the DA strain is unrelated, the
liver is not rejected, and liver leucocytes are exchanged with
the recipient. The livers thus contained PVG parenchyma and
DA passenger leucocytes, while DA livers parked in PVG inter-
mediate hosts contained DA parenchyma and PVG passenger
leucocytes. Transplantation of these livers to PVG strain recipi-
ents showed that tolerance was markedly reduced in those liv-
ers in which the donor leucocytes matched the recipient.
Direct evidence that donor passenger leucocytes were
important in liver transplant tolerance came from experiments
in our laboratory in which these cells were reduced by irradia-
tion of the liver donor prior to transplantation of the liver
(147). Recipients of passenger leucocyte-depleted livers rap-
idly rejected the liver in strain combinations in which non-irra-
diated livers are normally accepted (147). Reconstitution of
donor passenger leucocytes by parking of the leucocyte-
depleted liver in a normal donor for 48 h (147) or by injection
of donor leucocytes (148) restored acceptance of the liver.
These experiments show the importance of passenger leuco-
cytes of donor origin in promoting liver acceptance and fulfil
Kochs postulates for demonstrating the requirement for these
cells for acceptance. The role of liver leucocytes in liver accep-
tance has been confirmed by different groups using other strain
combinations (149, 150).
Characterisation of rat liver leucocytes
Given this potential role of passenger liver leucocytes in the
generation of spontaneous tolerance to liver transplants, as well
as data suggesting that liver leucocytes may contribute to the
McCaughan et al Molecular pathogenesis of liver disease
generation of oral tolerance (151153), we have sought to
characterise liver leucocytes in the rat. The liver has a large pop-
ulation of passenger leucocytes such as B cells, T cells and mye-
loid cells such as monocytes and MHC class II-expressing cells
with dendritic morphology (147). It also contains populations
of atypical lymphocytes such as NK T cells (154). Liver leuco-
cyte phenotypes, however, still remain poorly characterised in
the rat, despite the prominence of this species in studies of liver
transplant tolerance. Interestingly, we have been unable to iden-
tify NK T cells in the rat by the standard phenotypic markers
used in mice. In mouse strains expressing the NK R-P1 subfam-
ily of NK markers, NK T cells can be identified as bearing both
this marker and the ab T-cell receptor (TCR). NK T cells in the
mouse can also be identified by intermediate ab TCR expres-
sion, or the phenotype TCRpos CD4/CD8 double negative or
TCRpos CD4low (155). None of these characteristic phenotypes
could be demonstrated in the rat (D. G. Bowen, G. W. McCaughan,
G. A. Bishop, unpublished data). It is feasible that the pheno-
type of the NK T cell differs markedly in the rat from that
seen in mice or humans; however, the possibility exists that this
leucocyte subset is truly absent in the rat, and its putative im-
munomodulatory functions (156, 157) are subsumed by other
cell subtypes.
The role of the liver in immune function remains unclear.
It has been postulated that the liver functions as a graveyard
for activated T cells, with activated T cells congregating in the
liver to undergo apoptosis (158). However, we have studied
recirculation of liver leucocytes in the rat and have demon-
strated that the liver-derived T cells remain capable of recircu-
lation, tending to home to the lymph nodes and spleen, and are
still readily identifiable 1 week after transfer. Whilst liver-
derived T cells do not recirculate selectively to the liver, we
found that liver-derived NK cells (pit cells) home specifically
to the liver. This probably relates to differences in expression of
adhesion molecules such as CD11a and CD 18 (159), as well as
CD 11b/c (D. G. Bowen, G. W. McCaughan, G. A. Bishop,
unpublished). The specific homing of pit cells to the liver is
unsurprising, given the differences in morphology and cyto-
toxicity that exist between pit cells and NK cells found in other
tissues (160). Further phenotypic examination of this leuco-
cyte subset may lead to hypotheses on function. A broader
range of phenotypic markers would facilitate studies of the rat.
Despite the differences in phenotype between liver leuco-
cytes and leucocytes found in other organs, we have shown that
it is unlikely that leucocytes unique to the liver have a unique
ability to induce tolerance as spleen cells are as efficient as liver
leucocytes in their ability to reconstitute acceptance of leuco-
cyte-depleted livers (148, 149).
Immunological Reviews 174/2000 183
McCaughan et al Molecular pathogenesis of liver disease
Paradoxical immune activation in lymphoid tissues
in liver transplant tolerance
We have observed that when the lymphoid tissues of allograft
recipients are compared there is a surprising increase of IL-2
and IFN-g mRNA in the spleen and draining lymph nodes of
liver-tolerant animals which is reduced or absent in the corre-
sponding lymphoid tissues of recipients of liver, kidney or skin
allografts in the process of rejection (161164). This finding is
unusual because IL-2 and IFN-g are associated with rejection
and their level of expression in the rejecting organ can increase
over 1,000-fold at the mRNA level (162, 163). There is, how-
ever, a major difference between tolerance and rejection in the
site and timing of IL-2 and IFN-g expression. We have shown
that they reach peak expression 1 day after transplantation in
the lymphoid tissues of tolerant animals, while they appear
later and their expression is confined principally to the graft in
animals rejecting the transplanted organ (162, 163) (reviewed
in (165)).
Even more surprising is the finding that exogenous IL-2
prevents liver transplant tolerance when given at the time of
liver transplantation (150, 166). The paradox thus arises that
early expression of IL-2 and IFN-g mRNA in lymphoid tissues
is associated with liver graft acceptance but at the same time
administration of exogenous IL-2 prevents acceptance. This is a
central issue in understanding the process involved in the out-
come of the immune response to the liver, and one that has
ramifications outside of liver transplantation. The immune
response must apparently proceed through steps of activation
before it subsequently matures to either reject or accept the
liver. This concept is novel and challenges our established view
of the ways in which tolerance to transplanted organs is
achieved. Almost all clinical and experimental methods devel-
oped to obtain long-term survival of transplanted organs in
humans or in animal models are based on the assumption that
the immune response must be inhibited by all means possible.
All immunosuppressive drugs in current clinical use have been
selected for their ability to block immune activation in T cells
or to remove components of the T-cell repertoire.
Immune activation leading to tolerance is not unique to
liver transplantation and the process is well documented in
many models of immune tolerance. In a landmark paper
describing tolerance to minor lymphocyte-stimulating (Mls)
superantigen, Webb & Sprent described immune activation of
Mls-specific transgenic T cells preceding their clonal deletion
(167). Subsequently, many studies have shown that under cer-
tain circumstances T-cell activation is followed by death of the
responding T cells, and that IL-2 is involved in this deletional
tolerance mechanism (168, 169). Recently, evidence has
184 Immunological Reviews 174/2000
emerged that the involvement of IL-2 and IFN-g in transplant
tolerance is not confined to liver transplantation alone as toler-
ance induced by co-stimulatory blockade using anti-CD40
ligand antibody and CTLA4-Ig cannot be induced in IL-2 or
IFN-g knockout recipients (170, 171). This is clear evidence
that at least some forms of transplantation tolerance are depen-
dent on immune activation and the expression of IL-2 and
IFN-g.
Apoptotic cell death of activated T cells in liver transplant
tolerance
Apoptosis of antigen-reactive T cells is one means by which
immune responses are limited, and mice that are deficient in
molecules such as Fas or Fas-ligand, which are involved in apop-
tosis, die of autoimmune disease (reviewed in (172)). A con-
sistent liver transplantation tolerance finding (from our labora-
tory and others) is that in the early stages after transplantation
there are more apoptotic cells in transplanted organs in the pro-
cess of tolerance than in those undergoing irreversible rejection
(173175). This paradoxical situation of greater cell death in
tolerant grafts than in those that are rejected is partly explained
by the observation that these dying cells are concentrated in the
areas of leucocyte infiltrate, indicating that they may be recipi-
ent leucocytes which have infiltrated the graft and then died
(173, 174). Thus there are two processes that lead to the
appearance of apoptotic cells within the graft. One involves
death of graft parenchymal cells during rejection (176) and the
other involves death of alloreactive graft-infiltrating leucocytes
during development of tolerance.
Proof that many of the graft-infiltrating cells in tolerant liv-
ers are apoptotic leucocytes came from studies in which sec-
tions were double stained for co-expression of surface CD8 and
markers of apoptosis (177) or by purification of graft-infiltrat-
ing cells from the transplanted organ and analysis by multico-
lour flow cytometry (162). These experiments showed that
there were many apoptotic leucocytes in tolerant livers in the
first 2 weeks after transplantation. Examination of the subsets
of apoptotic leucocytes showed that both CD8 (162, 177) and
CD4 T cells (162) were apoptotic, and that greatest apoptosis
was in the activated T-cell population (162).
The close link between apoptosis of alloreactive leucocytes
and subsequent allograft tolerance has been confirmed by a
number of studies (162, 173, 174). These show that high lev-
els of leucocyte apoptosis were observed in liver tolerance but
not rejection of kidney allografts, and that induction of liver
rejection by administration of exogenous IL-2, by irradiation of
the liver donor or by peri-transplant corticosteroid therapy
markedly reduced the level of apoptosis. This close association
 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
T cells, especially activated T cells, is further evidence that liver
transplant tolerance is akin to activation-induced cell death
(AICD).
Mechanisms of AICD in recipient lymphoid tissues
Possible immune pathways by which donor leucocytes might
induce recipient T-cell activation then apoptotic death in recip-
ient lymphoid tissues are shown in Fig. 9. Recipient T cells are
normally activated in recipient lymphoid tissues by donor den-
dritic cells (178, 179) and then migrate to the graft and
increase their expression of IL-2 and IFN-g at that site. One pos-
sible mechanism by which liver passenger leucocytes induce
tolerance is that immunomodulatory cells of the donor form a
complex with the donor dendritic cell and alloreactive T cells
of the recipient in the recipient lymphoid tissues (Fig. 9A). The
modified stimulatory complex is postulated to deliver an aber-
rant stimulatory signal to the responding recipient T cell. This
aberrant signal, instead of stimulating the responder T cell to
migrate to the graft and increase its cytokine expression there,
induces immediate activation, cytokine expression in recipient
lymphoid tissues and consequent apoptotic death.
Another possible mechanism by which recipient alloreac-
tive T cells are neutralised is that the donor dendritic cells
deliver an activating signal to recipient T cells as normal. These
activated T cells subsequently encounter donor immunomodu-
latory cells in recipient lymphoid tissues, causing them to
increase their expression of IL-2 and IFN-g mRNA and subse-
quently die (Fig. 9B). These events could be similar to the
sequential stimulation involved in AICD, where stimulation via
the TCR leads to activation of the T cell and progression into
cell cycle. Subsequent re-engagement of the TCR of these acti-
vated T cells results in apoptotic cell death (180).
A third possibility is that dendritic leucocytes and immu-
nomodulatory cells compete to stimulate alloreactive T cells of
the recipient. Immunomodulatory donor leucocytes might
operate by delivering signal 1 through their engagement of the
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
cells but are lacking on many leucocyte populations. In this
case, the pool of alloreactive precursor T cells can either be
stimulated by dendritic cells, after which they effect graft
destruction, or by co-stimulatory molecule-deficient donor
leucocytes, after which they increase expression of IL-2 and
IFN-g and then die. In this case, if the numbers of donor immu-
nomodulatory leucocytes are sufficiently large, then the pro-
portion of alloreactive T cells that receive an apoptotic signal
will greatly outnumber those that receive an activating signal
from dendritic cells and the alloreactive T-cell pool will be
depleted (Fig. 9C).
In summary, liver transplant tolerance, one of the most
powerful models of peripheral tolerance to transplanted
organs, is associated with a process that superficially resembles
AICD. Although the basic processes involved in AICD have been
demonstrated during liver transplant tolerance, there is still
much evidence required to firmly establish AICD as the princi-
pal tolerising mechanism. It is of primary importance to estab-
lish which cells in recipient lymphoid tissues are stimulated to
increase expression of IL-2 and IFN-g following liver transplan-
tation. It is also of great interest to know whether this is due to
the presence of donor leucocytes in lymphoid tissues and, if so,
what population of donor leucocytes can most efficiently
induce AICD. Finally, there are many potential applications of
these findings to clinical transplantation. It may be possible to
induce tolerance to liver or other organ grafts by AICD-like
mechanisms using donor leucocytes purified from the spleen
or from the liver perfusate of the organ donor. An unresolved
question is to what degree current immunosupression inhibits
AICD mechanisms that might otherwise be occurring during
clinical liver transplantation.
Conclusions
Our laboratory has examined three overlapping themes in the
study of chronic liver injury using molecular and cellular tech-
niques. These approaches have provided unique insights into
the role of the prolyl oligopeptidases and inflammatory cyto-
kines in the pathogenesis of liver injury. Current work has
delineated new members of the prolyl oligopeptidase gene
family, described novel pathways in the pathogenesis of liver
disease and defined new approaches to liver allograft tolerance.

References
1. De Meester I, Korom S, Van Damme J,
Scharp S. CD26, let it cut or cut it down.
Immunol Today 1999;20:367375.
2. Morimoto C, Schlossman SF. The structure
and function of CD26 in the T cell immune
response.
Immunol Rev 1998;161:5570.
3. Franco R, Valenzuela A, Lluis C, Blanco J.
Enzymatic and extraenzymatic role of ecto-
adenosine deaminase in lymphocytes.
Immunol Rev 1998;161:2742.
4. Von Bonin A, Hhn J, Fleischer B. Dipeptidyl-
peptidase IV/CD26 on T cells: analysis of an
alternative T cell activation pathway.
Immunol Rev 1998;161:4353.
5. Oravecz T, et al. Regulation of the receptor
specificity and function of the chemokine
RANTES (regulated on activation normal
T cell expressed and activated) by dipeptidyl
peptidase IV (CD26)-mediated cleavage.
J Exp Med 1997;186:18651872.
6. Proost P, et al. Amino-terminal truncation of
chemokines by CD26/dipeptidyl-peptidase
IV. Conversion of RANTES into a potent
inhibitor of monocyte chemotaxis and
HIV-1-infection.
J Biol Chem 1998;273:72227227.
7. Struyf S, et al. Enhanced anti-HIV-1 activity
and altered chemotactic potency of NH2-
terminally processed macrophage-derived
chemokine (MDC) imply an additional MDC
receptor.
J Immunol 1998;161:26722675.
8. Struyf S, et al. CD26/dipeptidyl-peptidase IV
down-regulates the eosinophil chemotactic
potency, but not the anti-HIV activity of
human eotaxin by affecting its interaction
with CC chemokine receptor 3.
J Immunol 1999;162:49034909.
9. Shioda T, et al. Anti-HIV-1 and chemotactic
activities of human stromal cell-derived
factor 1a (SDF-1a) and SDF-1b are abolished
by CD26/dipeptidyl peptidase IV-mediated
cleavage.
Proc Natl Acad Sci USA 1998;95:63316336.
10. Iwata S, et al. CD26/dipeptidyl peptidase IV
differentially regulates the chemotaxis of
T cells and monocytes toward RANTES:
possible mechanism for the switch from
innate to acquired immune response.
Int Immunol 1999;11:417426.
11. Oravecz T, Guan E, Boykins R, Gorrell M,
Norcross M. Inhibition of Th2-type
chemokine signalling by dipeptidyl peptidase
IV (CD26)-mediated enzymatic cleavage. In:
Keystone Symposium. Chemokines and
Chemokine Receptors; 1999 Jan 1823;
Keystone (CO). 1999. p. 54.
12. Galli G, et al. Enhanced HIV expression
during Th2-oriented responses explained by
the opposite regulatory effect of IL-4 and
IFN-g of fusin/CXCR4.
Eur J Immunol 1998;28:32803290.
13. Bonecchi R, et al. Differential expression of
chemokine receptors and chemotactic
responsiveness of type 1 T helper cells (Th1s)
and Th2s.
J Exp Med 1998;187:129134.
14. Wang J, et al. IL-4 and a glucocorticoid up-
regulate CXCR4 expression on human CD4+ T
lymphocytes and enhance HIV-1 replication.
J Leukoc Biol 1998;64:642649.
15. Sallusto F, Lanzavecchia A, Mackay CR.
Chemokines and chemokine receptors in
T-cell priming and Th1/Th2-mediated
responses.
Immunol Today 1998;19:568574.
16. Sallusto F, Lenig D, Mackay CR,
Lanzavecchia A. Flexible programs of
chemokine receptor expression on human
polarized T helper 1 and 2 lymphocytes.
J Exp Med 1998;187:875883.
17. Loetscher P, et al. CCR5 is characteristic of
Th1 lymphocytes.
Nature 1998;391:344345.

186 Immunological Reviews 174/2000


18. Scheel-Toellner D, et al. CD26 expression in
leprosy and other granulomatous diseases
correlates with the production of
interferon-g.
Lab Invest 1995;73:685690.
19. Hanski C, Huhle T, Reutter W. Involvement of
plasma membrane dipeptidyl peptidase IV in
fibronectin-mediated adhesion of cells on
collagen.
Biol Chem Hoppe Seyler
1985;366:11691176.
20. Gonzalez-Gronow M, Weber MR, Gawdi G,
Pizzo SV. Dipeptidyl peptidase IV (CD26) is a
receptor for streptokinase on rheumatoid
synovial fibroblasts.
Fibrinol Proteol 1998;12:129135.
21. Piazza GA, Callanan HM, Mowery J,
Hixson DC. Evidence for a role of dipeptidyl
peptidase IV in fibronectin-mediated
interactions of hepatocytes with extracellular
matrix.
Biochem J 1989;262:327334.
22. Bermpohl F, Loster K, Reutter W, Baum O. Rat
dipeptidyl peptidase IV (DPP IV) exhibitis
endopeptidase activity with specificity for
denatured fibrillar collagens.
FEBS Lett 1998;428:152156.
23. Levy MT, McCaughan GW, Abbott CA, Park JE,
Rettig WJ, Gorrell MD. Fibroblast activation
protein: a cell surface dipeptidyl peptidase
and gelatinase expressed by stellate cells at the
tissue remodelling interface in human
cirrhosis.
Hepatology 1999;29:17681778.
24. Trugnan G, et al. Dipeptidyl peptidase IV
(DPPIV/CD26): biochemistry and control of
cell-surface expression. In: Kenny AJ,
Boustead CM, eds. Cell-surface peptidases in
health and disease. Oxford: BIOS Scientific
Publishers; 1997. p. 203217.
25. Willheim M, et al. Cell surface
characterization of T lymphocytes and
allergen-specific T cell clones correlation of
CD26 expression with T-H1 subsets.
J Allergy Clin Immunol 1997;100:348355.
26. Proost P, et al. Truncation of macrophage-
derived chemokine by CD26/ dipeptidyl-
peptidase IV beyond its predicted cleavage site
affects chemotactic activity and CC
chemokine receptor 4 interaction.
J Biol Chem 1999;274:39883993.
27. Dong RP, et al. Characterization of adenosine
deaminase binding to human CD26 on T cells
and its biologic role in immune response.
J Immunol 1996;156:13491355.
28. Martin M, Huguet J, Centelles JJ, Franco R.
Expression of ecto-adenosine deaminase and
CD26 in human T cells triggered by the
TCR-CD3 complex possible role of
adenosine deaminase as costimulatory
molecule.
J Immunol 1995;155:46304643.
McCaughan et al Molecular pathogenesis of liver disease
29. Dong RP, et al. Determination of adenosine
deaminase binding domain on CD26 and its
immunoregulatory effect on T cell activation.
J Immunol 1997;159:60706076.
30. Hegen M, et al. Enzymatic activity of CD26
(dipeptidylpeptidase IV) is not required for
its signalling function in T cells.
Immunobiology 1993;189:483493.
31. Schn E, et al. The role of dipeptidyl
peptidase IV in human T lymphocyte
activation. Inhibitors and antibodies against
dipeptidyl peptidase IV suppress lymphocyte
proliferation and immunoglobulin synthesis
in vitro.
Eur J Immunol 1987;17:18211826.
32. Reinhold D, et al. Inhibitors of dipeptidyl
peptidase IV induce secretion of transforming
growth factor-b(1) in PWM-stimulated
PBMC and T cells.
Immunology 1997;91:354360.
33. Reinhold D, et al. Transforming growth
factor-b 1 (TGF-b 1) inhibits DNA synthesis
of PWM-stimulated PBMC via suppression of
IL-2 and IL-6 production.
Cytokine 1994;6:382388.
34. Flentke GR, Munoz E, Huber BT, Plaut AG,
Kettner CA, Bachovchin WW. Inhibition of
dipeptidyl aminopeptidase IV (DP-IV) by
Xaa-boroPro dipeptides and use of these
inhibitors to examine the role of DP-IV in
T-cell function.
Proc Natl Acad Sci USA 1991;88:15561559.
35. Tanaka T, et al. Enhancement of antigen-
induced T-cell proliferation by soluble
CD26/dipeptidyl peptidase IV.
Proc Natl Acad Sci USA 1994;91:30823086.
36. Tanaka S, Murakami T, Horikawa H,
Sugiura M, Kawashima K, Sugita T.
Suppression of arthritis by the inhibitors of
dipeptidyl peptidase IV.
Int J Immunopharmacol 1997;19:1524.
37. Khne T, Neubert K, Faust J, Ansorge S. Early
phosphorylation events induced by
DPIV/CD26-specific inhibitors.
Cell Immunol 1998;189:6066.
38. Chiravuri M, Schmitz T, Yardley K,
Underwood R, Dayal Y, Huber BT. A novel
apoptotic pathway in quiescent lymphocytes
identified by inhibition of a post-proline
cleaving aminodipeptidase: a candidate target
protease, quiescent cell proline dipeptidase.
J Immunol 1999;163:30923099.
39. Steeg C, Hartwig U, Fleischer B. Unchanged
signaling capacity of mutant
CD26/dipeptidylpeptidase IV molecules
devoid of enzymatic activity.
Cell Immunol 1995;164:311315.
40. Abbott CA, et al. Increased serum levels of
dipeptidyl peptidase IV (CD26) in rats
undergoing liver regeneration.
Int Hepatol Commun 1995;4:165174.
Immunological Reviews 174/2000
41. Hanski C, Zimmer T, Gossrau R, Reutter W.
Increased activity of dipeptidyl peptidase IV
in serum of hepatoma-bearing rats coincides
with the loss of the enzyme from the
hepatoma plasma membrane.
Experientia 1986;42:826828.
42. Ivanov S, Kolinska J, Kraml J,
Chelibonova-Lorer H.
g-Glutamyltranspeptidase and dipeptidyl
peptidase IV activity in the serum of normal
and hepatoma-bearing chickens and in the
plasma membranes from liver and hepatoma
MC-29.
Cancer Biochem Biophys 1992;13:4349.
43. Iwaki-Egawa S, Watanabe Y, Kikuya Y,
Fujimoto Y. Dipeptidyl peptidase IV from
human serum: purification, characterization,
and N-terminal amino acid sequence.
J Biochem (Tokyo) 1998;124:428433.
44. Matsumoto Y, Bishop GA, McCaughan GW.
Altered zonal expression of the CD26 antigen
(dipeptidyl peptidase IV) in human cirrhotic
liver.
Hepatology 1992;15:10481053.
45. McCaughan GW, Siah CL, Abbott C,
Wickson J, Ballesteros M, Bishop GA.
Dipeptidyl peptidase IV is down-regulated in
rat hepatoma cells at the mRNA level.
J Gastroenterol Hepatol 1993;8:142145.
46. Roman RM, Fitz JG. Emerging roles of
purinergic signaling in gastrointestinal
epithelial secretion and hepatobiliary
function.
Gastroenterology 1999;116:964979.
47. Yoshimoto T, Fischl M, Orlowski RC,
Walter R. Post-proline cleaving enzyme and
post-proline dipeptidylpeptidase.
J Biol Chem 1978;253:37083716.
48. Rettig WJ, et al. Fibroblast activation protein:
purification, epitope mapping and induction
by growth factors.
Int J Cancer 1994;58:385392.
49. Niedermeyer J, et al. Mouse fibroblast-
activation protein: conserved Fap gene
organization and biochemical function as a
serine protease.
Eur J Biochem 1998;254:650654.
50. Pineiro-Sanchez ML, Goldstein LA, Dodt J,
Howard L, Yeh Y, Chen WT. Identification of
the 170-kDa melanoma membrane-bound
gelatinase (seprase) as a serine integral
membrane protease.
J Biol Chem 1997;272:75957601.
51. Morrison ME, Vijayasaradhi S, Engelstein D,
Albino AP, Houghton AN. A marker for
neoplastic progression of human
melanocytes is a cell surface ectopeptidase.
J Exp Med 1993;177:11351143.
52. Kameoka J, Tanaka T, Nojima Y,
Schlossman SF, Morimoto C. Direct
association of adenosine deaminase with a
T cell activation antigen, CD26.
Science 1993;261:466469.
187
McCaughan et al Molecular pathogenesis of liver disease
53. De Meester I, et al. Binding of adenosine
deaminase to the lymphocyte surface via
CD26.
Eur J Immunol 1994;24:566570.
54. Abbott CA, McCaughan GW, Levy MT,
Church WB, Gorrell MD. Binding to human
dipeptidyl peptidase IV by adenosine
deaminase and antibodies that inhibit ligand
binding involves overlapping, discontinuous
sites on a predicted b propeller domain.
Eur J Biochem 1999;266:798810.
55. Scanlan MJ, et al. Molecular cloning of
fibroblast activation protein a, a member of
the serine protease family selectively
expressed in stromal fibroblasts of epithelial
cancers.
Proc Natl Acad Sci USA 1994;91:56575661.
56. Gossrau R. Histochemical and biochemical
distribution of dipeptidyl peptidase IV (DPP
IV).
Histochemistry 1979;60:231248.
57. McCaughan GW, Wickson JE, Creswick PF,
Gorrell MD. Identification of the bile
canalicular cell surface molecule GP110 as the
ectopeptidase dipeptidyl peptidase IV: an
analysis by tissue distribution, purification
and N-terminal amino acid sequence.
Hepatology 1990;11:534544.
58. Garin-Chesa P, Old LJ, Rettig WJ. Cell surface
glycoprotein of reactive stromal fibroblasts as
a potential antibody target in human
epithelial cancers.
Proc Natl Acad Sci USA 1990;87:72357239.
59. Rettig WJ, Garin-Chesa P, Beresford HR,
Oettgen HF, Melamed MR, Old LJ. Cell-
surface glycoproteins of human sarcomas:
differential expression in normal and
malignant tissues and cultured cells.
Proc Natl Acad Sci USA 1988;85:31103114.
60. Ten Kate J, et al. Quantitative changes in
adenosine deaminase isoenzymes in human
colorectal adenocarcinomas.
Cancer Res 1984;44:46884692.
61. Andy RJ, Kornfeld R. Biosynthesis of the
adenosine deaminase-binding protein in
human fibroblasts and hepatoma cells.
J Biol Chem 1984;259:98329839.
62. Rettig WJ, et al. Regulation and heteromeric
structure of the fibroblast activation protein
in normal and transformed cells of
mesenchymal and neuroectodermal origin.
Cancer Res 1993;53:33273335.
63. Finstad CL, Cordon-Cardo C, Bander NH,
Whitmore WF, Melamed MR, Old LJ.
Specificity analysis of mouse monoclonal
antibodies defining cell surface antigens of
human renal cancer.
Proc Natl Acad Sci USA 1985;82:29552959.
64. Schn E, Mansfeld HW, Demuth HU, Barth A,
Ansorge S. The dipeptidyl peptidase IV, a
membrane enzyme involved in the
proliferation of T lymphocytes.
Biomed Biochim Acta 1985;44:K9K15.
188 Immunological Reviews 174/2000
65. Sannes PL. Subcellular localization of
dipeptidyl peptidases II and IV in rat and
rabbit alveolar macrophages.
J Histochem Cytochem 1983;31:684690.
66. Hegen M, Niedobitek G, Klein CE, Stein H,
Fleischer B. The T cell triggering molecule
Tp103 is associated with dipeptidyl
aminopeptidase IV activity.
J Immunol 1990;144:29082914.
67. Gorrell MD, Wickson J, McCaughan GW.
Expression of the rat CD26 antigen
(dipeptidyl peptidase IV) on subpopulations
of rat lymphocytes.
Cell Immunol 1991;134:205215.
68. Bristol LA, Sakaguchi K, Appella E, Doyle D,
Takacs L. Thymocyte costimulating antigen is
CD26 (dipeptidyl-peptidase IV).
Costimulation of granulocyte, macrophage,
and T lineage cell proliferation via CD26.
J Immunol 1992;149:367372.
69. Fleischer B, Sturm E, De-Vries JE, Spits H.
Triggering of cytotoxic T lymphocytes and
NK cells via the Tp103 pathway is dependent
on the expression of the T cell receptor/CD3
complex.
J Immunol 1988;141:11031107.
70. Bhling F, Junker U, Reinhold D, Neubert K,
Jager L, Ansorge S. Functional role of CD26
on human B lymphocytes.
Immunol Lett 1995;45:4751.
71. Misumi Y, Hayashi Y, Arakawa F, Ikehara Y.
Molecular cloning and sequence analysis of
human dipeptidyl peptidase IV, a serine
proteinase on the cell surface.
Biochim Biophys Acta 1992;1131:333336.
72. Tanaka T, et al. Cloning and functional
expression of the T cell activation antigen
CD26.
J Immunol 1992;149:481486.
73. Hong W, Doyle D. cDNA cloning for a bile
canaliculus domain-specific membrane
glycoprotein of rat hepatocytes.
Proc Natl Acad Sci USA 1987;84:79627966.
74. Ogata S, Misumi Y, Ikehara Y. Primary
structure of rat liver dipeptidyl peptidase IV
deduced from its cDNA and identification of
the NH2-terminal signal sequence as the
membrane-anchoring domain.
J Biol Chem 1989;264:35963601.
75. Marguet D, Bernard AM, Vivier I, Darmoul D,
Naquet P, Pierres M. cDNA cloning for mouse
thymocyte-activating molecule. A
multifunctional ecto-dipeptidyl peptidase IV
(CD26) included in a subgroup of serine
proteases.
J Biol Chem 1992;267:22002208.
76. Niedermeyer J, et al. Mouse fibroblast
activation protein: molecular cloning,
alternative splicing and expression in the
reactive stroma of epithelial cancers.
Int J Cancer 1997;71:383389.
77. Abbott CA, Baker E, Sutherland GR,
McCaughan GW. Genomic organisation, exact
localization, and tissue expression of the
human CD26 (dipeptidyl peptidase IV) gene.
Immunogenetics 1994;40:331338.
78. Bernard AM, Mattei MG, Pierres M,
Marguet D. Structure of the mouse dipeptidyl
peptidase IV (CD26) gene.
Biochemistry 1994;33:1520415214.
79. Abbott CA, Gorrell MD, Levy MT,
McCaughan GW. Molecular analyses of
human and rat dipeptidyl peptidase IV.
Adv Exp Med Biol 1997;421:161170.
80. Mathew S, et al. The gene for fibroblast
activation protein a (FAP), a putative cell
surface-bound serine protease expressed in
cancer stroma and wound healing, maps to
chromosome band 2q23.
Genomics 1995;25:335337.
81. Ollis DL, et al. The a/b hydrolase fold.
Protein Eng 1992;5:197211.
82. Ogata S, Misumi Y, Tsuji E, Takami N, Oda K,
Ikehara Y. Identification of the active site
residues in dipeptidyl peptidase IV by affinity
labeling and site-directed mutagenesis.
Biochemistry 1992;31:25822587.
83. Park JE, Lenter MC, Zimmermann RN,
Garin-Chesa P, Old LJ, Rettig WJ. Fibroblast
activation protein: a dual-specificity serine
protease expressed in reactive human tumor
stromal fibroblasts.
J Biol Chem 1999;274:3650536512.
84. Bednarczyk JL, Carroll SM, Marin C,
McIntyre BW. Triggering of the proteinase
dipeptidyl peptidase IV (CD26) amplifies
human T lymphocyte proliferation.
J Cell Biochem 1991;46:206218.
85. De Meester I, Vanhoof G, Hendriks D,
Demuth HU, Yaron A, Scharp S.
Characterization of dipeptidyl peptidase IV
(CD26) from human lymphocytes.
Clin Chim Acta 1992;210:2334.
86. Gorrell MD, Abbott CA, Khne T, Levy MT,
Church WB, McCaughan GW. Relating
structure to function in the b-propeller
domain of dipeptidyl peptidase IV: point
mutations that ablate adenosine deaminase
binding, antibody binding or DPP IV enzyme
activity.
Adv Exp Med Biol (In press).
87. Levy MT, McCaughan GW, Marinos G,
Gorrell MD. Chronic hepatitis C: fibroblast
activation protein expression correlates with
hepatic fibrosis.
Hepatology 1999;30:323A.
88. Abbott CA, McCaughan GW, Gorrell MD. Two
highly conserved glutamic acid residues in
the predicted b-propeller domain of
dipeptidyl peptidase IV are required for its
enzyme activity.
FEBS Lett 1999;458:278284.

89. Barrett AJ. Nomenclature Committee of the
International Union of Biochemistry and
Molecular Biology (NC-IUBMB). Enzyme
nomenclature. Recommendations 1992.
Supplement 4: corrections and additions
(1997).
Eur J Biochem 1997;250:16.
90. Rawlings ND, Barrett AJ. MEROPS: the
peptidase database.
Nucleic Acids Res 1999;27:325331.
91. Barrett AJ, Rawlings ND, Woess JF. Handbook
of proteolytic enzymes. London: Academic
Press; 1998.
92. Flop V, Bocskei Z, Polgar L. Prolyl
oligopeptidase an unusual b-propeller
domain regulates proteolysis.
Cell 1998;94:161170.
93. Gorrell MD, Levy MT, Abbott CA,
Washington EA, McCaughan GW. CD26:
Adenosine deaminase binding,
immunoblotting and species cross reactivity
examined using recombinant proteins. In:
Kishimoto T, ed. Leucocyte typing VI. Vol VI.
New York: Garland Publishing Inc;1997. p.
484485.
94. Dong RP, et al. Correlation of the epitopes
defined by anti-CD26 mAbs and CD26
function.
Mol Immunol 1998;35:1321.
95. Plana M, et al. Induction of interleukin 2
(IL 2) and interferon-g and enhancement of
IL 2 receptor expression by a CD26
monoclonal antibody.
Eur J Immunol 1991;21:10851088.
96. Torimoto Y, Dang NH, Tanaka T, Prado C,
Schlossman SF, Morimoto C. Biochemical
characterization of CD26 (dipeptidyl
peptidase IV): functional comparison of
distinct epitopes recognized by various anti-
CD26 monoclonal antibodies.
Mol Immunol 1992;29:183192.
97. De Meester IA, et al. Costimulation of CD4+
and CD8+ T cells through CD26: the ADA-
binding epitope is not essential for complete
signaling.
J Leukoc Biol 1995;58:325330.
98. Oxvig C, Springer TA. Experimental support
for a b-propeller domain in integrin
a-subunits and a calcium binding site on its
lower surface.
Proc Natl Acad Sci USA 1998;95:48704875.
99. Hhn J, Olek S, Fleischer B, von Bonin A. The
adenosine deaminase-binding region is
distinct from major anti-CD26 mAb epitopes
on the human dipeptidyl peptidase IV(CD26)
molecule.
Cell Immunol 1999;192:3340.
100. Kraulis P. MOLSCRIPT: a program to produce
both detailed and schematic plots of protein
structures.
J Appl Crystallogr 1991;24:946950.
101. Underwood R, Chiravuri M, Schmitz T,
Yardley K, Lee H, Huber BT. Sequence,
purification and cloning of an intracellular
serine protease, quiescent cell proline
dipeptidase (QPP).
J Biol Chem 1999;274:3405334058.
102. Duke-Cohan JS, Gu J, McLaughlin DF, Xu Y,
Freeman GJ, Schlossman SF. Attractin
(DPPT-L), a member of the CUB family of cell
adhesion and guidance proteins, is secreted
by activated human T lymphocytes and
modulates immune cell interactions.
Proc Natl Acad Sci USA
1998;95:1133611341.
103. Pangalos MN, et al. Isolation and expression
of novel human glutamate carboxypeptidases
with N-acetylated a-linked acidic dipeptidase
and dipeptidyl peptidase IV activity.
J Biol Chem 1999;274:84708483.
104. Jacotot E, et al. Dipeptidyl-peptidase IV-b, a
novel form of cell-surface-expressed protein
with dipeptidyl-peptidase IV activity.
Eur J Biochem 1996;239:248258.
105. Abbott CA, Yu D, McCaughan GW,
Gorrell MD. Post proline cleaving peptidases
having DP IV like enzyme activity.
Adv Exp Med Biol (In press).
106. Franco R, et al. Cell surface adenosine
deaminase: much more than an ectoenzyme.
Prog Neurobiol 1997;52:283294.
107. Lund FE, Cockayne DA, Randall TD,
Solvason N, Schuber F, Howard MC. CD38: a
new paradigm in lymphocyte activation and
signal transduction.
Immunol Rev 1998;161:7993.
108. Fox DA, et al. Ta1, a novel 105 KD human
T cell activation antigen defined by a
monoclonal antibody.
J Immunol 1984;133:12501256.
109. Mittrucker HW, Steeg C, Malissen B,
Fleischer B. The cytoplasmic tail of the T cell
receptor z chain is required for signaling via
CD26.
Eur J Immunol 1995;25:295297.
110. Torimoto Y, Dang NH, Vivier E, Tanaka T,
Schlossman SF, Morimoto C. Coassociation of
CD26 (dipeptidyl peptidase IV) with CD45
on the surface of human T lymphocytes.
J Immunol 1991;147:25142517.
111. Napoli J, Bishop GA, McCaughan GW.
Increased intrahepatic messenger RNA
expression of interleukins 2, 6, and 8 in
human cirrhosis.
Gastroenterology 1994;107:789798.
112. Bishop GA, Rokahr KL, Napoli J,
McCaughan GW. Intragraft cytokine mRNA
levels in human liver allograft rejection
analysed by reverse transcription and
semiquantitative polymerase chain reaction
amplification.
Transpl Immunol 1993;1:253261.
Immunological Reviews 174/2000
McCaughan et al Molecular pathogenesis of liver disease
113. Napoli J, Bishop GA, McGuinness PH,
Painter DM, McCaughan GW. Progressive
liver injury in chronic hepatitis C infection
correlates with increased intrahepatic
expression of Th1-associated cytokines.
Hepatology 1996;24:759765.
114. McGuinness PH, Painter D, Davies S,
McCaughan GW. Increases in intrahepatic
CD68 positive cells, MAC387 positive cells
and pro-inflammatory cytokines (particularly
interleukin 18) in chronic hepatitis C
infection.
Gut 2000;45:260269.
115. McGuinness PH, Bishop GA, Painter DM,
Chan R, McCaughan GW. Intrahepatic
hepatitis C RNA levels do not correlate with
degree of liver injury in patients with chronic
hepatitis C.
Hepatology 1996;23:676687.
116. Cerny A, Chisari FV. Pathogenesis of chronic
hepatitis C: immunological features of
hepatic injury and viral persistence.
Hepatology 1999;30:595601.
117. McCaughan GW, Napoli J, McGuinness PH,
Bishop GA. T1 vs T2 cytokine response in
chronic HCV: implications for mechanisms of
liver injury.
Viral Hepat Rev 1997;3:129142.
118. Koziel MJ, et al. Intrahepatic cytotoxic T
lymphocytes specific for hepatitis C virus in
persons with chronic hepatitis.
J Immunol 1992;149:33393344.
119. Pileri P, et al. Binding of hepatitis C virus to
CD81.
Science 1998;282:938941.
120. Gerlach JT, et al. Recurrence of hepatitis C
virus after loss of virus-specific CD4+ T-cell
response in acute hepatitis C.
Gastroenterology 1999;117:933941.
121. Ando K, et al. Perforin, Fas/Fas Ligand, and
TNF-a pathways as specific and bystander
killing mechanisms of hepatitis C virus-
specific human CTL.
J Immunol 1997;158:52835291.
122. Napoli J. Intrahepatic cytokines in chronic
liver disease [PhD thesis]. Sydney: University
of Sydney; 1996.
123. Napoli J, Prentice D, Niinami C, Bishop GA,
Desmond P, McCaughan GW. Sequential
increases in the intrahepatic expression of
epidermal growth factor, basic fibroblast
growth factor, and transforming growth
factor b in a bile duct ligated rat model of
cirrhosis.
Hepatology 1997;26:624633.
124. Velculescu VE, Zhang L, Vogelstein B,
Kinzler KW. Serial analysis of gene
expression.
Science 1995;270:484487.
125. Wan JS, et al. Cloning differentially expressed
mRNAs.
Nat Biotechnol 1996;14:16851691.
189
McCaughan et al Molecular pathogenesis of liver disease
126. Carulli JP, et al. High throughput analysis of
differential gene expression.
J Cell Biochem Suppl 1998;31:286296.
127. Wang Z, Brown DD. A gene expression
screen.
Proc Natl Acad Sci USA
1991;88:1150511509.
128. Liang P, Pardee AB. Differential display of
eukaryotic messenger RNA by means of the
polymerase chain reaction.
Science 1992;257:967971.
129. Kozian DH, Kirchbaum BJ. Comparitive gene-
expression analysis.
Trends Biotechnol 1999;17:7378.
130. Kedar V, Fletcher L, Powell L. Molecular
cloning of a novel mRNA highly expressed in
haemochromatotic human liver and
proliferating cells.
Biochem Biophys Res Commun
1996;226:461466.
131. Wang KK, Wan DF, Qiu XK, Lu PX, Gu JR.
Differential expression of a cDNA clone in
human liver versus hepatic cancerhighly
homologous to aryl-dialkyl-phosphatase.
Cell Res 1997;7:7990.
132. Huang LR, Hsu HC. Cloning and expression
of CD24 gene in human hepatocellular
carcinoma: a potential early tumor marker
gene correlates with p53 mutation and tumor
differentiation.
Cancer Res 1995;55:47174721.
133. Diachenko LB, Ledesma J, Chenchik AA,
Siebert PD. Combining the technique of RNA
fingerprinting and differential display to
obtain differentially expressed mRNA.
Biochem Biophys Res Commun
1996;219:824828.
134. Morrison TB, Weis JJ, Wittwer CT.
Quantification of low-copy transcripts by
continuous SYBR Green I monitoring during
amplification.
Biotechniques 1998;24:954958, 960, 962.
135. Szala S, Kasai Y, Steplewski Z, Rodeck U,
Koprowski H, Linnenbach AJ. Molecular
cloning of cDNA for the human tumor-
associated antigen CO-029 and identification
of related transmembrane antigens.
Proc Natl Acad Sci USA 1990;87:68336837.
136. Sela BA, Steplewski Z, Koprowski H. Colon
carcinoma-associated glycoproteins
recognized by monoclonal antibodies
CO-029 and GA22-2.
Hybridoma 1989;8:481491.
137. Fukuda R, et al. Intrahepatic expression of
pro-inflammatory cytokine mRNAs and
interferon efficacy in chronic hepatitis C.
Liver 1996;16:390399.
138. Calmus Y, et al. Cholestasis induces major
histocompatibility complex class I expression
in hepatocytes.
Gastroenterology 1992;102:13711377.
190 Immunological Reviews 174/2000
139. Chu CM, Liaw YF. Coexpression of
intercellular adhesion molecule-1 and class I
major histocompatibility complex antigens
on hepatocyte membrane in chronic viral
hepatitis.
J Clin Pathol 1993;46:10041008.
140. Matsumoto Y, McCaughan GW, Painter DM,
Bishop GA. Evidence that portal tract
microvascular destruction precedes bile duct
loss in human liver allograft rejection.
Transplantation 1993;56:6975.
141. McCaughan GW, et al. A quantitative analysis
of T lymphocyte populations in human liver
allografts undergoing rejection: the use of
monoclonal antibodies and double
immunolabeling.
Hepatology 1990;12:13051313.
142. Zavazava N, Kronke M. Soluble HLA class I
molecules induce apoptosis in alloreactive
cytotoxic T lymphocytes.
Nat Med 1996;2:10051010.
143. Krams SM, et al. Human hepatocytes produce
an isoform of Fas that inhibits apoptosis.
Transplantation 1998;65:713721.
144. Lord R, Kamada N, Kobayashi E, Goto S,
Sunagawa M. Isolation of a 40 kDa
immunoinhibitory protein induced by rat
liver transplantation.
Transpl Immunol 1995;3:174179.
145. Priestley CA, Dalchau R, Sawyer GJ, Fabre JW.
A detailed analysis of the potential of water-
soluble classical class I MHC molecules for the
suppression of kidney allograft rejection and
in vitro cytotoxic T cell responses.
Transplantation 1989;48:10311038.
146. Sriwatanawongsa V, Davies HS, Calne RY. The
essential roles of parenchymal tissues and
passenger leukocytes in the tolerance induced
by liver grafting in rats.
Nat Med 1995;1:428432.
147. Sun J, McCaughan GW, Gallagher ND,
Sheil AGR, Bishop GA. Deletion of
spontaneous rat liver allograft acceptance by
donor irradiation.
Transplantation 1995;60:233236.
148. Sun J, et al. Tolerance to rat liver allografts IV.
Tolerance depends on the quantity of donor
tissue and on donor leukocytes.
Transplantation 1996;62:17251730.
149. Shimizu Y, et al. Restoration of tolerance to
rat hepatic allografts by spleen-derived
passenger leukocytes.
Transpl Int 1996;9:593595.
150. Tu YZ, Arima T, Flye MW. Rejection of
spontaneously accepted rat liver allografts
with recipient interleukin-2 treatment or
donor irradiation.
Transplantation 1997;63:177181.
151. Cantor HM, Dumont AE. Hepatic suppression
of sensitization to antigen absorbed into the
portal system.
Nature 1967;215:744745.
152. Yang R, Liu Q, Grosfeld JL, Pescovitz MD.
Intestinal venous drainage through the liver is
a prerequisite for oral tolerance induction.
J Pediatr Surg 1994;29:11451148.
153. Trop S, Samsonov D, Gotsman I, Alper R,
Diment J, Ilan Y. Liver-associated lymphocytes
expressing NK1.1 are essential for oral
immune tolerance induction in a murine
model.
Hepatology 1999;29:746755.
154. Yoshimoto T, Bendelac A, Watson C, Hu-Li J,
Paul WE. Role of NK1.1+ T cells in a Th2
response and in immunoglobulin E
production.
Science 1995;270:18451847.
155. Bendelac A, Rivera MN, Park SH, Roark JH.
Mouse CD1-specific NK1 T cells:
development, specificity, and function.
Annu Rev Immunol 1997;15:535562.
156. Godfrey DI, Kinder SJ, Silvera P, Baxter AG.
Flow cytometric study of T cell development
in NOD mice reveals a deficiency in
abTCR+CDRCD8 thymocytes.
J Autoimmun 1997;10:279285.
157. Hammond KJL, Poulton LD, Palmisano LJ,
Silveira PA, Godfrey DI, Baxter AG. ab-T cell
receptor (TCR)+CD4CD8 (NKT)
thymocytes prevent insulin-dependent
diabetes mellitus in nonobese diabetic
(NOD)/Lt mice by the influence of
interleukin (IL)-4 and/or IL-10.
J Exp Med 1998;187:10471056.
158. Crispe IN, Mehal WZ. Strange brew: T cells in
the liver.
Immunol Today 1996;17:522525.
159. Luo D, et al. The role of adhesion molecules
in the recruitment of hepatic natural killer
cells (pit cells) in rat liver.
Hepatology 1996;24:14751480.
160. Vanderkerken K, Bouwens L, Wisse E.
Characterization of a phenotypically and
functionally distinct subset of large granular
lymphocytes (pit cells) in rat liver sinusoids.
Hepatology 1990;12:7075.
161. Bishop GA, et al. Tolerance to rat liver
allografts. III. Donor cell migration and
tolerance-associated cytokine production in
peripheral lymphoid tissues.
J Immunol 1996;156:49254931.
162. Sharland AF, et al. Evidence that apoptosis of
activated T cells occurs in spontaneous
tolerance of liver allografts and is blocked by
manipulations which break tolerance.
Transplantation 1999;68:17361745.
163. Rokahr KL, et al. Paradoxical early immune
activation during acceptance of liver
allografts compared with rejection of skin
grafts in a rat model of transplantation.
Immunology 1998;95:257263.
164. Bishop GA, McCaughan GW, Sun J, Sheil AGR.
Microchimerism and transplant tolerance.
Immunol Today 1997;18:455456.

165. Bishop GA, Sharland AF, McCaughan GW.
High-dose/activation-associated tolerance
model for allografts: lessons from
spontaneous tolerance of transplanted livers.
Curr Opin Organ Transplant 1999;4:5864.
166. Delriviere L, et al. Administration of
exogenous interleukin-2 abrogates
spontaneous rat liver allograft acceptance but
does not affect long-established graft survival.
Transplantation 1997;63:16981701.
167. Webb S, Morris C, Sprent J. Extrathymic
tolerance of mature T cells: clonal
elimination as a consequence of immunity.
Cell 1990;63:12491256.
168. Lenardo MJ. Interleukin-2 programs mouse
ab T lymphocytes for apoptosis.
Nature 1991;353:858861.
169. Critchfield J, et al. T cell deletion in high
antigen dose therapy of autoimmune
encephalomyelitis.
Science 1994;263:11391143.
170. Konieczny TT, et al. IFN-g is critical for long-
term allograft survival induced by blocking
the CD28 and CD40 ligand T cell
costimulation pathways.
J Immunol 1998;160:20592064.
McCaughan et al Molecular pathogenesis of liver disease
171. Dai ZH, Konieczny BT, Boddoura FK,
Lakkis FG. Impaired alloantigen-mediated
T cell apoptosis and failure to induce long-
term allograft survival in IL-2-deficient mice.
J Immunol 1998;161:16591663.
172. Nagata S, Golstein P. The Fas death factor.
Science 1995;267:1449.
173. Sharland A, et al. Kinetics of intragraft
cytokine expression, cellular infiltration and
cell death in rejection of renal allografts
compared with acceptance of liver allografts
in a rat model. Early activation and apoptosis
is associated with liver graft acceptance.
Transplantation 1998;65:13701377.
174. Qian S, et al. Apoptosis within spontaneously
accepted mouse liver allografts. Evidence for
deletion of cytotoxic T cells and implications
for tolerance induction.
J Immunol 1997;158:46544661.
175. Bergese SD, Klenotic SM, Wakely ME,
Sedmak DD, Orosz CG. Apoptosis in murine
cardiac grafts.
Transplantation 1997;63:320325.
176. Krams SM, Egawa H, Quinn MB,
Villanueva JC, Garcia-Kennedy R,
Martinez OM. Apoptosis as a mechanism of
cell death in liver allograft rejection.
Transplantation 1995;59:621625.
177. Meyer D, et al. Apoptosis of T lymphocytes in
liver and/or small bowel allografts during
tolerance induction.
Transplantation 1998;66:15301536.
Immunological Reviews 174/2000
178. Larsen CP, Austyn JM, Morris PJ. The role of
graft-derived dendritic leukocytes in the
rejection of vascularized organ allografts.
Recent findings on the migration and
function of dendritic leukocytes after
transplantation.
Ann Surg 1990;212:308315.
179. Larsen CP, Morris PJ, Austyn JM. Migration of
dendritic leukocytes from cardiac allografts
into host spleens. A novel pathway for
initiation of rejection.
J Exp Med 1990;171:307314.
180. Ucker DS, Hebshi LD, Blomquist JF,
Torbett BE. Physiological T-cell death:
susceptibility is modulated by activation,
aging, and transformation, but the
mechanism is constant.
Immunol Rev 1994;142:273299.
181. Gaitatzes C. The WD-repeat family of
proteins. Available from: URL: http://bmerc-
www.bu.edu/wdrepeat/.
191