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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 structure–function 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: 172–191
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 (5–9, 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 (1–4)). mediated by direct or indirect binding to collagen and
Substrates of the enzyme DPPIV that have relevance to im- fibronectin (19–21) 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 (5–11). 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 (12–17). 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 (31–38), 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. Kähne, G. W. McCaughan

Immunological Reviews 174/2000 173

higher magnification (inset) shows the basolateral pathogenesis of chronic liver injury and extracellular matrix expression of DPPIV seen in some patients (B). Interestingly. some potential substrates can be predicted. we have observed dramatically increased serum concentrations of DPPIV during liver regeneration (40) but downregulation of cell surface DPPIV in hepatoma cell lines (45). Altered expression by hepatocytes occurs in atitis C fibrosis (87).e. answered question is whether the role of FAP in cirrhosis is nase activity (23. and made as pro-enzymes requiring activation. to hepatocyte regulation of adenosine levels has not been fully considered but clearly requires further exam- ination (46). 55). gene family. In addition. The other natural substrates of FAP are unknown. ´200 (C). However. which is surprising because all other beneficial or detrimental. neovascular structures and infiltrat- ing leucocytes (44). i. 2C) (23. 43). This contrasts FAP with the matrix metalloproteinases. mononuclear leucocytes (A). 83). the sources of serum DPPIV are unknown (40. ´100 (B). related to include erythropoietin. where all hepato- cytes are DPPIV positive. In human cirrhosis we have described intense expression of DPPIV on proliferating bile ductules. the (ECM)-digesting enzymes are primary mediators of this role. and weaker immunoreactivity FAP is unique in the stellate cell arsenal of ECM-digesting is seen in the fibrous septa (C). Unlike DPPIV. Small substrate size is consistent with our understanding of the tertiary struc. Immunoperoxidase stain. 2.McCaughan et al · Molecular pathogenesis of liver disease unpublished). haematoxylin counterstain. we have noticed a correla. periseptal area of the regenerative nodule. B) or FAP (C). CD26 174 Immunological Reviews 174/2000 . with its ability to bind ADA at the bile canalicular surface. 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. capillary endothelium and activated tion between levels of FAP expression and tissue injury in hep. The contribu- tion of CD26/DPPIV. The DPPIV-like investigation. Structure of DPPIV-like peptidases ture of this protein family (see below). Furthermore. C) human livers were stained for DPPIV (A. Stellate cells have a central role in the cirrhotic liver. small. 49. In contrast. which are metal dependent. basolateral expression of DPPIV is often seen in severe liver injury (Fig. This question requires revisitation once inhibi- tors that are quite specific for DPPIV are developed (see below). hepatocyte expression is nor- mally confined to zones 2 and 3 of the acinus but this restricted expression is lost in regenerating nodules. ther preactivation nor metal ions. 48. Role of fibroblast activation protein in liver fibrosis Fibroblast activation protein (FAP) is a post-proline cleaving enzyme very closely related to DPPIV (23. FAP-specific immunoreactivity occurs at the tissue-remodelling interface. 55) (Table 1). DPPIV is normally expressed on the bile canalicular domain of the hepatocyte cell surface. These for enzyme activity (88). Expression of DPPIV and FAP in human liver. but distinct from prolyl endopeptidase (PEP) (family S9a) in kines melanoma growth-stimulating activity (MGSA/GRO) b the prolyl oligopeptidase (S9) enzyme family (89–91). containing less than 100 amino acids. FAP expression is limited to activated fibroblasts Fig. (Fig. so this paradox requires CD26/DPPIV is the prototypic type IV DPP. Magnifications: approximately ´600 (A). The central un- Both recombinant and natural human FAP exhibit gelati. This question could be addressed us- known substrates of prolyl oligopeptidase family members are ing FAP-deficient mice or a specific enzyme inhibitor. In humans. pancreatic polypeptide and the chemo. 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 (40–42). Cryostat sections of and hepatic stellate cells at sites of tissue injury and remodelling normal (A) and cirrhotic (B. enzymes in that it is on the cell surface and dependent on nei. and macrophage inhibitory peptide (MIP)-1b. nearly all secreted. forms enzyme family S9b. 2B).

histidine ü 82 ü 83 Intron near catalytic serine ü 77 ü 49 Potential N-linked glycosylation sites 9 71. 55 ✓ 23. 84. 62 Expression by foetal mesenchymal cells ✓ 63 ✓ 48. 94) and most anti-CD26 mAbs stimulate T-cell prolifera- same overall topology. B cells. macrophages ✓ 64–70 ✗ 58. 62 Expression on activated hepatic stellate cells ? ✓ 23 Expression on activated T cells. McCaughan et al · Molecular pathogenesis of liver disease Table 1. 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. 67. 93–97). 50 Predicted 7-blade b propeller domain ü 54. MAb epitopes on b pro- face of the b propeller. the enzyme surface that is furthest from pellers depend on tertiary structure (98). discontinuous and involves propeller blades 4 and 6 (54. 48. inside have shown that the ADA-blocking epitope of CD26/DPPIV is this hollow enzyme and about 20 Å from the entrance. The All other mAbs except 2F9 bind extracellular residues N-termi- Immunological Reviews 174/2000 175 . 48. Sequence homologies the nature of epitopes on CD26. 3) (54. Concordantly. 48. They have lack of covalent bonding between blades 1 and 7 of the propel- polypeptide chains of about 80 kDa that produce two extracel. 86). 85 180 kDa 23. aspartate. ably restricts substrates to those capable of gaining access. 86).3 77 2q 23 80 a/b hydrolase domain ✓ 81 ✓ 55 Catalytic triad serine. 85 ü 48. not monomer ü 84 ü 23. 50 Diethyl pyrocarbonate produces monomer ? ü 50 Heating produces monomer ü 67. 86 ü 86 and FAP have very similar structures (Table 1). CD26 has at least five epitopes indicate that all members of the S9a and S9b families have the (93. 50 Binding to adenosine deaminase ✓ 51–53 ✗ 23 Anti-DPPIV MAb bind ✓ 54 ✗ 54 Anti-FAP MAb bind ✗ 23. 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. 49 Cleavage of chemokines ✓ 5–9 ? Gelatinase activity ✗ 23 ✓ 23. 50 Monomer mobility on SDS-PAGE 110 kDa 95 kDa 62 Dimer mobility on SDS-PAGE 150 kDa 21. 58. presum. 61 ✓ 48. NK cells. 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. 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. 59 Expression on activated fibroblasts in healing wounds and tumours ✓ 60. 48. 72 6 55 Cysteines 12 71. 72 8 55 Catalytically active as dimer. 78 26 49 Gene size 90 kb 79 90 kb 49 Gene location 2q 24. The position of the catalytic site. we the cell surface (Fig. 4). 55 Expression in normal adult tissues ubiquitous 56. Such a structure places the substrate tion (1). 84. 57 nil 58. 59 Physical characteristic Number of amino acids 766 71–75 760 55.

these enzymes have a mixture of characteris- the C-terminus (94). Unlike WD repeat proteins (181). Abbott. Each propeller blade consists of four b-strands. with and/or 2. 92). 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. ing to a DPP. T cells express DPP8. attractin (102). despite their common catalytic activity of hydrolysing 176 Immunological Reviews 174/2000 . unpublished). G. suggesting that these epitopes are glycoproteins. Mutations domain is anticipated to be above the propeller in the side view (left) and ablating enzyme activity are blue. Indeed. re-evaluation. MAb 2F9 binds near the prolyl bond. The hydrolase spheres showing positions of point mutations of interest. N-acetylated a-linked acidic cleave DPPIV substrates indicate that DPPIV inhibitors require dipeptidase (NAALADase) II (103). Curi. There is no structural data on DPPIVb.03. DPP8 and QPP are cytoplasmic. 94. perhaps via bind- D. with 27% identity to DPPIV. Like FAP using chimaeric and truncated molecules. 3. a coveries of carboxypeptidases and DPPIV-like enzymes that glutamate carboxypeptidase. McCaughan. M. 88). 3) predicts that these epitopes involve propeller blades 1 Whereas DPP8. tics (105). it is not clear that attractin is a 38). DPPIVb and NAALADase II are cell surface sequence (54. hydrolysis of GlyPro substrates is indirect. W. and DPPIVb (104). nal to the ADA binding site (54. (Fig. dependent on tertiary structure. Structure–function relationships in the ADA-binding domain of numbered from the centre. Yu. 99). The other epitopes have been localised. and QPP. D.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. 42% identity to prolyl carboxypeptidase. The dis- cent peptidyl peptidase (QPP) (101). Ribbon diagram of the predicted b propeller domain with oligopeptidase blades 1 and 7 are not bound covalently. QPP and attractin. Canvas v. Gorrell. 88. quies. 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. a mutation ablating ADA binding is behind the propeller in the view towards the lowerface (right). but this requires confirmation. 5. and it is possible that the reported low-level amino-terminal dipeptides have discovered DPP8 (C. The figure was prepared using MOLSCRIPT (100) and is orange (54. prolyl CD26/DPPIV. ously. Substrate yellow. contain the a/b hydrolase fold characteristic of the SC enzyme clan. 94. Searches for other enzymes that cleave GlyPro or AlaPro protease (105). In contrast. 99). A. to any known enzyme. Our model of CD26/DPPIV Separate cDNAs encode surface and soluble forms of attractin. to long sections of and CD26/DPPIV.

109). 4. IL-8 and tumour necrosis factor-a Intrahepatic gene expression of inflammatory cytokines in but not interferon-g (IFN-g). D. The central nucleus via the TCR/CD3 pathway (66. cellular regeneration and tissue fibrosis. 106. Our model of residues 133 to 766 of CD26/DPPIV is depicted receptors (3. which includes pore of the b-propeller domain. The substrates of DPPIV include cytoplasmic domain of CD26 is too short to transduce a signal. Church. which began as a fundamental regeneration using semi-quantitative polymerase chain reac- characterisation of an enzyme family expressed in normal liver tion (PCR). Fyn59. expression of potential mediators of chronic inflammation and Our work clearly showed that HCV liver injury was associated Immunological Reviews 174/2000 177 . unpublished). B. Our early studies in this area examined the tissue tissue. A model of CD26/DPPIV action at the T-cell surface. 112). W. Autoimmune hepatitis (AIH) was characterised by increases in these inflammatory Intrahepatic gene expression of inflammatory cytokines and cytokines. MDC and eotaxin (see Fig. 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. human chronic liver disease These studies did not however examine earlier stage dis- Virtually all liver diseases are characterised by tissue inflamma. whilst primary biliary cirrhosis (PBC) had marked growth factors in human and experimental liver disease upregulation only of IL-2. 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. revealed marked upregulation of mRNA of various cytokines. The ADA-binding presumed that the transduction mechanism is indirect. In order tion. The studies of type IV DPPs. 3). the studies of human cirrhosis in liver disease. IL-6 and IL-8 (111). The Fig. ease or the Th1/Th2 paradigm of tissue inflammation. so it is the chemokines RANTES. in addition to CD38. 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. 37). Although the These studies now complement our more direct examination results in allograft rejection were limited. 108. CD39. human liver allograft rejection (111. CD73 (5' nucleotidase) and adenosine domains. 107). CD26-triggered signals are transmitted to the here (M. Gorrell. particularly IL-2. points away from the cell surface. McCaughan et al · Molecular pathogenesis of liver disease Fig. the likely site of substrate entry (see phosphorylation of p56lck. ZAP70 and c-Cbl (30. probably due to of the intrahepatic expression of cytokines and growth factors immunosuppressive therapy. cellular injury. 1).

6) (114). 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-γ specimens. 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. 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) (116–118). B. hepatic inflammation are well understood. 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 178 Immunological Reviews 174/2000 . 5). C. Portal inflammation component of SS versus IFN-␥. We have recently dis- IL-2 and IFN-g (Fig. in the setting of an aggressive intrahepatic cell-mediated Th1 lated with each other.McCaughan et al · Molecular pathogenesis of liver disease Fig. 7) (119). We have subsequently standing that much of the hepatic inflammation in chronic shown marked upregulation of the IFN-g-inducing cytokine IL. Fibrosis component of SS versus IFN-␥. (115). Inflammation and fibrosis correlate with T1 cytokine content in HCV liver. Furthermore. 5) (113). the molecular trig- larly related to liver injury (117). rated into the model (Fig. [From (113)] Scheuer score (total) B IFN-γ Scheuer score (fibrosis) C IFN-γ Scheuer score (portal) with an increase in the hepatic expression of the Th1 cytokines chronic HCV may be attenuated (120). upregulation only of epidermal growth factor (EGF) (122). Total Scheuer score (SS) versus IFN-␥. 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. HCV disease may not be antigen specific. However. There was little IL-4 or IL-10 cussed the apparent paradox of impaired anti-HCV immunity detected. 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. This has allowed us Although the pathogenesis of liver fibrosis and aspects of to put forward a model of HCV immune pathogenesis particu. Our studies of human cirrhosis showed significant autoimmune features of HCV infection have also been incorpo. HCV-specific CTLs in vitro (121). Perhaps the paradox can be resolved by under- tissue inflammation and fibrosis (Fig. 5. and both correlated with the degree of response (117). A. IL-2 and IFN-g expression levels corre.

The more common and accessible differential pathways. 6. Chronic hepatitis tissue stained with MAC387. but these methods are limited to well-resourced institu- of a restricted set of molecules and known or potential disease tions (126). have received insufficient attention. G. which uses high-density arrays of oligonucleotides. expression profiles of hundreds to thousands of genes simulta- rhosis. a marked increase in the expression of EGF and fibroblast Recent advances in differential gene expression analysis growth factor (FGF) (123). Chronic hepatitis tissue stained with CD68. methods such as serial analysis of gene expression (SAGE) Although the above approaches have given us insights into (124). gene expression methods compare mRNA pools using cDNA tractive hybridisation and gene array analysis. HCV-negative normal tissue stained with CD68. 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. A. Differen- players in the chronic regenerative process. E. and (3) gene-chip® technology. Chronic hepatitis tissue stained with CD68. HCV-negative normal tissue stained with MAC387. B. which is a hallmark tial gene expression can be examined using sequence intensive of the cirrhotic process. Although the molecular pathways of acute liver regen. Representational Immunological Reviews 174/2000 179 . F. such as representational differ- tial to find new and interesting molecules and novel pathogenic ences analysis (RDA) (127) and differential display (128). D. they have been limited by an examination chips. There was little increase in trans. techniques applied to human cirrhosis forming growth factor-a. McCaughan et al · Molecular pathogenesis of liver disease Fig. H. 2) high-density microarrays using dual fluoro- chrome-labelled probes. Cirrhotic tissue stained with MAC387. Immunostaining of HCV-positive human liver tissues with CD68 and MAC387 monoclonal antibodies. We have cDNA array analysis is a powerful technique for examining recently used both of these techniques to explore human cir. Cirrhotic tissue stained with CD6. New molecular techniques. electronic subtraction (125) and DNA sequencing disease pathogenesis. such as suppression sub. have the poten. This approach correlates gene eration are being increasingly well understood. arrays or PCR-based methods. Chronic hepatitis tissue stained with MAC387. Macrophages in HCV liver. understanding of gene function. the important expression differences with cell or tissue phenotypes. Immunoperoxidase method with haematoxylin counterstain (approximately ´200 magnification). Three types of arrays are commonly used: 1) mem- brane-based arrays that are typically hybridised with radiola- belled probes. processes that may be implicated in liver tissue injury. C. neously.

PBC expressed products on a gel (129). The liver. Palo Alto. of the CO-029 tion (133). Liver cDNA array analyses have appeared as abstracts. Green 1 is a double-stranded DNA-specific dye which enables sion. sue was taken at the time of liver transplantation from patients In liver disease most differential expression experiments with end-stage cirrhosis. New Jersey. Table 3 sum- 132). 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 (114–116) differences analysis and differential display compare mRNA involved in the pathogenesis of cirrhosis. Approaches aimed at profiling multiple genes or identi. The real time quan- was examined using cDNA array analysis and a modification of titative RT-PCR supported the subtraction result. mRNA pools were compared identify and examine individual candidate genes. Autoantibodies and associated immune complex disease are possibly induced by T1 direct CD81 B-cell stimulation (117). lymph DC nodes and peripheral blood leads to T2 cytokine production and associated persistent anti-HCV Liver antibody production. CA. Having identified few are published. suppression subtractive hybridisa. Subsequent CO-029 180 Immunological Reviews 174/2000 . Roche. (Perkin Elmer. specifically genes pools using PCR-based methods with subsequent differential involved in immune-mediated liver injury. with its genetic diversity. Continued liver injury is associated T1 T2 with a persistent T1 response within the liver. Initially. Compartmentalisation model of chronic HCV infection. with a sig. Additionally.8-fold. for differentially expressed genes by using adapter ligation and fying novel genes in human liver are limited (130). which has Intrahepatic differential gene expression in human cirrhosis not been examined in non-tumour tissue. further characterisation involved confirma- experiments using these techniques compare cell lines rather tion of differential expression with real-time reverse trans- than tissue samples. AIH. Our aim was to identify new genes or pathways mRNA in HCV compared to normal liver. 136). significant upregulation. Evidence for intrahepatic Th1 HCV liver damage ? response in chronic HCVa HCV ? T2 response CTL/macrophage activation Increased intrahepatic IL-2. most differential expression candidate genes. USA) and Sybr Green 1. cies (CLONTECH PCR Select. but marises the results of the subtraction analysis. as described using suppression subtractive hybridisation. Comparisons were expression screening or by resolution of differentially made between hepatic liver injury secondary to HCV. The diseased tis- novel genes. greater than 1. HCV Immune recognition events within the spleen.McCaughan et al · Molecular pathogenesis of liver disease Fig. 7. pleth. USA). 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). which enriches above. T1/T2 Lymph nodes Spleen Blood Table 2. One sequence identified as upregulated using subtraction hybridis- Differential gene expression in human cirrhosis ation was the tumour antigen CO-029 (135. criptase (RT)-PCR using an ABI Prism 7700 sequence detector ora of functions and well-characterised disease phenotypes. subsequent PCR amplification of differentially expressed spe- nificant portion of genes identified being tumour related (131. PCR product to be quantified as it is produced (134). IFN-g. confirming the classical RDA technique. Sybr represents an ideal tissue to examine differential gene expres.

mAb immunostaining localised immunopositivity to hepato. a puta. an increase previously demon. 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. both suppression subtractive hybridisation and cDNA array tive receptor for HCV. which identified a role for CD8- methods (111. Molecules such as MHC Class I in liver transplant tolerance C-4 subunit were demonstrated to be upregulated in HCV cir. The 145 clones successfully sequenced represented 89 hybridisation with 32P-2'-deoxycytidine 5'-triphosphate (dCTP)-labelled first strand unique clones. A number of mechanisms have been proposed for the Immunological Reviews 174/2000 181 . 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. Subsequent cDNA array Array analysis used two cDNA arrays (Human Gene Array analysis found greater than 3. and cies. 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. concordant with studies that used other human liver allograft rejection.7 to 5. PCR results generated by other researchers and by us. 8 gives an suppression subtractive hybridisation. In addition. PBC. Differential expression was considered to be a 10-fold or greater differ- driver sample. without evidence of rejection. donor. of rhosis compared to normal. These investigations arose from our initial studies of liver on array analysis. a 1. A proportion of the will reveal novel insights into HCV infection.5-fold upregulation of these 1. The spontaneous acceptance. Fig. pression subtractive hybridisation. PBC.1-fold upregula. The third area of interest in our laboratory has been to ple. expression using the techniques of cDNA array analysis and mal. total of 664 unique genes. in non-cirrhotic HCV liver injury compared to other forms of cytes and biliary epithelium without significant stromal stain. McCaughan et al · Molecular pathogenesis of liver disease Table 3. For exam. Initial identification of genes such as CO. ing in both normal and diseased tissue. analysis. 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. PSC array results was consistent with previous semi-quantitative and the cirrhotic process in general. inflammatory mediators such as IL-8. cDNA array analysis was confirmed by quantitative real time RT-PCR with Sybr Green 1. AIH. We believe these results example of a cDNA array hybridisation. Probes were made from pooled These preliminary results indicate that differential gene mRNA (each pool was derived from four patients) from nor. CO-029 belongs to the The differential expression of two genes was identified by tetraspanin family of molecules.0 and Cytokine/Receptor Array. which is an effect of cirrhosis. 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). 141). liver allografts has been well documented in many animal spe- strated in cholestasis (138). 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. Other interesting genes involved in liver regeneration. which includes CD81. HCV and PSC liver tissue. Table 4 lists human liver disease. chronic hepatitis (139). Differential expression screening was performed using dot-blot ence in signal intensity. CLONTECH) representing a inflammation-associated molecules in both HCV and AIH liver. AIH. investigate the mechanisms of spontaneous liver transplant tol- unit and IL-2 receptor were found to be upregulated in HCV erance. 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. 137). IFN-g receptor b sub.

This array contains cDNAs for 265 genes spotted in duplicate. An ATLAS cytokine/receptor array probed with 32P-labelled cDNA made from HCV cirrhotic liver mRNA (four patients).2 Inflammation Fibronectin receptor >-subunit 5.6 Signalling Glutathione-S-transferase M1 3.2 Signalling Tristetraproline 2. The ratios compare signals from individual arrays adjusted for background and housekeeping gene signals. A cDNA array analysis of HCV liver.7 Fibrosis Fibroblast growth factor receptor-1 precursor 2. Upregulation of genes 2 and 3 was seen in HCV compared to normal using a housekeeper gene (gene 1) signal as a reference.2 Stress response Proto-oncogene c-jun 6.McCaughan et al · Molecular pathogenesis of liver disease Fig.4 Signalling Ephrin A3 precursor 2.2 Fibrosis Transforming growth factor->-3 4. Normal liver was non-diseased tissue from transplant donor biopsies or non-tumour tissue from patients having hepatic metastases resected. Table 4.8 Fibrosis Epithelial discoidin domain receptor-1 2. 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. 182 Immunological Reviews 174/2000 .8 Inflammation Lymphotoxin > 2. 8.7 Inflammation Monocyte chemotactic protein-1 precursor 2.0 Fibrosis Helix-loop-helix protein 2.2 Oncogene Hybridisation signals from the cDNA arrays were quantified by phosphoimage anal- ysis. cDNA array analysis grouping genes with pathogenic processes Gene HCV:normal ratio Pathogenic process Interleukin-8 4.6 Inflammation MHC Class I C-4 antigen a subunit 2.

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). Further phenotypic examination of this leuco- cells for acceptance. Reconstitution of to the liver. unpublished data). tending to home to the lymph nodes and spleen. McCaughan et al · Molecular pathogenesis of liver disease resistance of the liver to rejection. mouse can also be identified by intermediate ab TCR expres- tral to the liver tolerance process. 157) are subsumed by other DA passenger leucocytes. 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. However. Despite the differences in phenotype between liver leuco- cytes and leucocytes found in other organs. G. However. liver to undergo apoptosis (158). A broader tance has been confirmed by different groups using other strain range of phenotypic markers would facilitate studies of the rat. 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. NK T cells in the that mobile cellular elements of the transplanted liver are cen. and its putative im- the recipient. used in mice. unpublished). G. the seen in mice or humans. combinations (149. leucocytes. characterise liver leucocytes in the rat. Whilst liver- (147). 150). In mouse strains expressing the NK R-P1 subfam- In contrast to the relative ineffectiveness of soluble factors ily of NK markers. efforts to transplant tolerance. mediate hosts contained DA parenchyma and PVG passenger The role of the liver in immune function remains unclear. 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 Koch’s postulates for demonstrating the requirement for these tissues (160). Bishop. It is feasible that the pheno- (146). The livers thus contained PVG parenchyma and munomodulatory functions (156. 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. McCaughan. Interestingly. cocytes of donor strain with those of the intermediate host G. of atypical lymphocytes such as NK T cells (154). derived T cells do not recirculate selectively to the liver. The specific homing of pit cells to the liver is These experiments show the importance of passenger leuco. with dendritic morphology (147). while DA livers parked in PVG inter. Liver leuco- ently unrelated to either of these proteins. G. This probably relates to differences in expression of donor passenger leucocytes by parking of the leucocyte. G. of donor leucocytes (148) restored acceptance of the liver. cyte subset may lead to hypotheses on function. which appears in the cyte phenotypes. we idly rejected the liver in strain combinations in which non-irra. A. Recipients of passenger leucocyte-depleted livers rap. Bowen. the possibility exists that this liver is not rejected. The role of liver leucocytes in liver accep. One line of evidence that sion. appar. A. 149). Bishop. Although the DA strain is unrelated. however. Immunological Reviews 174/2000 183 . 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. or the phenotype TCRpos CD4/CD8 double negative or donor cells played a role came from studies in which donor liv. There is also a soluble factor. 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. TCRpos CD4low (155). It also contains populations ing effector T cells (143). None of these characteristic phenotypes ers were parked in intermediate hosts to replace passenger leu. could be demonstrated in the rat (D. the rat. McCaughan. found that liver-derived NK cells (“pit cells”) home specifically diated livers are normally accepted (147). adhesion molecules such as CD11a and CD 18 (159). W. despite the prominence of this species in studies of liver ically suppress in vitro alloreactivity (144). unsurprising. G. we have sought to protective factors by the liver such as soluble MHC class I mol. Bowen. there is considerable evidence this marker and the ab T-cell receptor (TCR). and liver leucocytes are exchanged with leucocyte subset is truly absent in the rat. It has been postulated that the liver functions as a “graveyard” ents showed that tolerance was markedly reduced in those liv. NK T cells can be identified as bearing both in prolonging allograft survival. W. with activated T cells congregating in the ers in which the donor leucocytes matched the recipient. cell subtypes. lation. however. for activated T cells. still remain poorly characterised in serum of tolerant liver-transplanted rats and which can specif. These include production of generation of oral tolerance (151–153). Transplantation of these livers to PVG strain recipi. as well as depleted liver in a normal donor for 48 h (147) or by injection CD 11b/c (D. and are tion of the liver donor prior to transplantation of the liver still readily identifiable 1 week after transfer. The liver has a large pop- ecules that can neutralise cytotoxic T cells directed against the ulation of passenger leucocytes such as B cells.

tolerance ever. Thus there are two processes that lead to the come of the immune response to the liver. 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. by irradiation of responding T cells. lour flow cytometry (162). of apoptotic leucocytes showed that both CD8 (162. that at least some forms of transplantation tolerance are depen- sponding lymphoid tissues of recipients of liver.000-fold at the mRNA level (162. 169). One involves ramifications outside of liver transplantation. 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. Almost all clinical and experimental methods devel. 174). Examination of the subsets or to remove components of the T-cell repertoire. This paradoxical situation of greater cell death in liver transplantation (150. 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. and mice that are deficient in that they reach peak expression 1 day after transplantation in molecules such as Fas or Fas-ligand. Recently.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. liver. many studies have shown that under cer. 171). This is clear evidence liver-tolerant animals which is reduced or absent in the corre. 163). how. and one that has appearance of apoptotic cells within the graft. while they appear tosis. kidney or skin dent on immune activation and the expression of IL-2 and allografts in the process of rejection (161–164). and that IL-2 is involved in this deletional the liver donor or by peri-transplant corticosteroid therapy tolerance mechanism (168. 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. 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 (173–175). Webb & Sprent described immune activation of number of studies (162. evidence has markedly reduced the level of apoptosis. This close association 184 Immunological Reviews 174/2000 . This is a ent leucocytes which have infiltrated the graft and then died central issue in understanding the process involved in the out. This finding is IFN-g. 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. and that induction of liver tain circumstances T-cell activation is followed by death of the rejection by administration of exogenous IL-2. 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. (173. 174). which are involved in apop- the lymphoid tissues of tolerant animals. 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. 177) and Immune activation leading to tolerance is not unique to CD4 T cells (162) were apoptotic. 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. many models of immune tolerance. 166). not rejection of kidney allografts. and that greatest apoptosis liver transplantation and the process is well documented in was in the activated T-cell population (162). Subsequently. 173. 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. There is. 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). die of autoimmune disease (reviewed in (172)). indicating that they may be recipi- administration of exogenous IL-2 prevents acceptance. We have shown immune responses are limited. 163) (reviewed tory and others) is that in the early stages after transplantation in (165)).

and donor leucocytes. McCaughan et al · Molecular pathogenesis of liver disease Fig. the TCR leads to activation of the T cell and progression into sible mechanism by which liver passenger leucocytes induce cell cycle. is further evidence that liver induces immediate activation. the recipient. 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. These events could be similar to the dritic cells (178. 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. causing them to ient lymphoid tissues are shown in Fig. 9B). This operate by delivering signal 1 through their engagement of the aberrant signal. 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. 179) and then migrate to the graft and sequential stimulation involved in AICD. T cells. B) Activation of recipient T cells by donor antigen A. quently die (Fig. 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. 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. 9A). especially activated T cells. latory cells in recipient lymphoid tissues. which is delivered by co-stim- Immunological Reviews 174/2000 185 . 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. where stimulation via increase their expression of IL-2 and IFN-g at that site. leading to aberrant stimulation. Possible mechanisms of liver transplant tolerance induction by donor leucocytes. The nomodulatory cells compete to stimulate alloreactive T cells of modified stimulatory complex is postulated to deliver an aber. Immunomodulatory donor leucocytes might rant stimulatory signal to the responding recipient T cell. Sequential stimulation antigen-presenting cells. instead of stimulating the responder T cell to recipient TCR but not signal 2. Aberrant stimulation presenting cells occurs normally but the T activated T cells then encounter donor T DC IM leucocytes. 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. 9. Donor leucocytes transplanted with the liver play a major role in liver transplant tolerance. which deliver a T-cell signal for activation and survival of T recipient T cells. One pos. cytokine expression in recipient transplant tolerance is akin to activation-induced cell death lymphoid tissues and consequent apoptotic death. 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). (AICD). T C) There is competition between donor B. 9. 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.

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