Journal of Biological Regulators and Homeostatic Agents

Pathogenesis of viral hepatitis

L.G. GUIDOTTI

Immunopathogenesis of Viral Hepatitis Unit, DIBIT - San Raffaele Scientific Institute, Milan, Italy and
The Scripps Research Institute Department of Molecular and Experimental Medicine, La Jolla, CA, USA

ABSTRACT: The aim of our research is to use animal models to elucidate the molecular basis for viral clearance and
liver disease in the pathogenesis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The results herein
discussed provide insight into immunological and virological processes that may lead to the development of new
therapeutic strategies to terminate chronic HBV and HCV infections. (J Biol Regul Homeost Agents 2003; 17: 115-9)

KEY WORDS: HBV, HCV, Transgenic mice, Chimpanzees, Cytokines, Chemokines, Cytotoxic T cells, Neutrophils, Matrix
metalloproteinases

The hepatitis B virus (HBV) and the hepatitis C virus
(HCV) cause acute and chronic liver disease,
cirrhosis, and hepatocellular carcinoma. HBV and
HCV are transmitted sexually, parenterally, and from
mother to infant at birth, like HIV. Over 500 million
people world-wide are chronically infected by these
viruses and HBV alone causes approximately 1 million
deaths each year. Since these viruses are not directly
cytopathic for the hepatocyte, the cellular immune
response to viral antigens is thought to be responsible
for both liver disease and viral clearance following
HBV and HCV infections. Indeed, patients with acute
viral hepatitis, who successfully clear these viruses,
mount a multispecific polyclonal cytotoxic T cell (CTL)
response to several viral-encoded antigens. In
contrast, this response is absent or much weaker in
chronically infected patients who do not clear these
viruses and thus, it is thought that the outcome of
HBV and HCV infections (viral clearance versus viral
persistence) is determined primarily by the vigor and
quality of the cellular immune response.
The experimental approaches to HBV and HCV
pathogenesis have been difficult because the host
range of these viruses is limited to man and
chimpanzees, and because in vitro systems for their
propagation do not exist. Studies of HBV and HCV
immunopathogenesis require the development of an
inbred animal model with a well defined immune
system. Thus, over the last several years, we have
developed and characterised HBV transgenic mice that
express all the viral gene products and replicate HBV
at high levels in the primary hepatocyte in vivo.
Contrary to HBV, to this date, no suitable mouse
models of HCV replication are available, and therefore,
much less is known about HCV pathogenesis.
HBV pathogenesis
Two lineages (1.3.32 and 1.3.46) of HBV transgenic
mice have been produced whose hepatocytes
replicate the virus at high levels without any evidence
of cytopathology (1). These mice were produced by
microinjection of a terminally redundant viral DNA
construct 1.3 HBV genomes in length, containing only
viral regulatory elements and no cellular promoters.
Out of all four HBV RNAs produced in the liver of
these animals, the two most abundant transcriptional
products of the transgene are the 3.5- and 2.1-kb
RNA, easily detectable by Northern blot analysis (1).
The 3.5 kb RNA (or pregenomic) RNA is reverse
transcribed by the viral polymerase into the HBV DNA
replicative intermediates which appear as a smear
when analyzed by Southern Blot (1). HBV replication
occurs inside of viral nucleocapsid particles more
abundant in centrilobular hepatocytes. As a
consequence of efficient viral replication, ultra-
structurally complete and infectious (2) viral particles
that are mor phologically indistinguishable from
human-derived virions are detected at high levels in
the transgenic mouse serum (between 107 and 108
viral particles per ml), further indicating that the HBV
life cycle can be efficiently completed in the transgenic
mouse hepatocyte (1).
We have examined in this model the antiviral and
immunopathological consequences of antigen
recognition by administration of virus-specific CTL.
Initially, an H2d-restricted CTL clone (designated 6C2)
that recognizes a dominant CTL epitope located
between residues 28-39 of HBsAg was used. In
subsequent experiments, we studied the cytopathic
and antiviral effector functions of many additional CTL
clones that were specific either for this epitope or for
independent epitopes in the HBV envelope, core and
polymerase proteins.
Antiviral mechanisms: Surprisingly, the antiviral
potential of the CTL in our transgenic mouse system
was shown to be primarily mediated by noncytolytic
mechanisms that involve the intrahepatic production of
type 1 inflammatory cytokines by the CTL (3-5). These
cytokines activate two functionally independent

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Pathogenesis of viral hepatitis
virocidal pathways: an early pathway that eliminates
HBV nucleocapsid par ticles and their cargo of
replicating viral genomes from the hepatocyte (3, 6);
and a later pathway that post-transcriptionally
downregulates the viral RNA (7). In recent studies, we
showed that IFN- mediates most of the antiviral effect
of the CTL (8) and nitric oxide (NO) mediates most of
the antiviral activity of IFN- (9).
One might predict from the foregoing that HBV-
nonspecific inflammatory responses of the liver could
facilitate the clearance of HBV if they induce the local
production of antiviral cytokines (such as IFN- and
IFN-/) to which HBV is susceptible. Precisely these
events have been shown to occur in the HBV
transgenic mice during unrelated hepatotropic
infections of the liver which include lymphocytic
choriomeningitis virus (LCMV), adenovirus and mouse
cytomegalovirus (MCMV) (8, 10, 11) or after
administration of recombinant murine interleukin (IL)-
12, a cytokine produced by antigen presenting cells
that has the ability to induce IFN- secretion by T cells,
natural killer (NK) and NKT cells (12). Along these
lines, we also showed that a single injection of -
galactosylceramide -GalCer), a glycolipid antigen
presented to V14+, NK1.1+ T cells by the non-classical
MHC class I-like molecule CD1d, inhibits HBV
replication by directly activating NKT cells to produce
IFN- in the liver (13, 14). Furthermore, we have shown
that HBV replication is inhibited in these animals by
systemic administration of only very high doses of IFN-
or IFN- (8). Indeed, the minimal effective IFN- dose
required to inhibit hepatic HBV replication in this model
is between 1 x 105 and 5 x 105 units/mouse, while the
standard IFN- regimen for chronically infected patients
is about 3 x 106 units/day for ten days. A dose of 3 x
106 units in humans corresponds to a dose of about 1 x
103 units in a 25-gram mouse, which would have no
antiviral effect as a single dose in our model. These
results suggest that new therapeutic approaches aimed
to induce antiviral cytokines at the site of the infection
should be considered for the treatment of chronic HBV
infection in man.
Impor tantly, we have also recently produced
evidence suggesting that noncytopathic antiviral
mechanisms may contribute to viral clearance during
acute viral hepatitis in chimpanzees, thus validating
the transgenic mouse studies in a natural infection
model (2). Furthermore, recent experiments were
chimpanzees acutely infected with HBV were
depleted of CD8+ cells demonstrate that CD8+ cells
are the main effector cells responsible for viral
clearance and disease pathogenesis during acute
HBV infection, and they suggest that viral clearance is
mediated by both noncytolytic and cytolytic effector
functions of the CD8+-T-cell response (15).
In summary, the cytokine-mediated, noncytopathic
antiviral mechanisms described herein may represent
an important host survival strategy to control viral
infections, especially when vital organs are massively
infected. Future studies to define cytokine-induced
116
intracellular molecular events that control viral
infections will not only improve our understanding of
the host-virus interactions that determine the outcome
of infection, but they may also lead to the discovery of
new approaches for the treatment of persistent
viruses such as HBV, HCV and HIV.
Immunopathological mechanisms: Liver disease in
the CTL transfer model begins with antigen
recognition by the CTL and delivery of signals that
trigger the death of the hepatocyte by apoptosis (16).
Following antigen recognition, the CTL recruit many
host derived inflammatory cells into the liver, thereby
contributing to the formation of necroinflammatory foci
in which apoptotic hepatocytes and CTL are
outnumbered by host derived lymphomononuclear
(such as lymphocytes, NK cells and macrophages)
and polymorphonuclear (such as neutrophils and
eosinophils) inflammatory cells (17). These necro-
inflammatory foci are scattered throughout the liver
parenchyma and cause a focal lesion (16) histo-
logically identical to classical viral hepatitis in man.
Recruitment of host-derived antigen non-specific
inflammatory cells into the liver is a process that is
associated with the intrahepatic production of
chemokines and it is likely to contribute to the
pathogenesis of liver disease. Indeed, we recently
showed that blocking the chemokines CXCL9 and
CXCL10 reduces the intrahepatic recruitment of host-
derived lymphomononuclear cells and the severity of
liver disease (18). In those studied, we showed that
CXCL9 and CXCL10 are rapidly and strongly induced
in the liver after CTL transfer. The transferred CTL
produce neither chemokine; rather, they activate
(via the secretion of IFN-) hepatocytes and
nonparenchymal cells of the liver to produce them
(18). Based on these results, we concluded that target
organ production of CXCL9 and CXCL10 leads to an
exaggerated recruitment of host-derived inflammatory
cells to the liver which play an important role in the
pathogenesis of liver disease.
We also recently showed that the CTL-initiated
liver disease is ameliorated by the depletion of
neutrophils, indicating that this cell subset
contributes to the pathogenetic process as well (19).
Interestingly, depletion of neutrophils does not affect
the intrahepatic migration and antiviral activity of CTL
but profoundly inhibits the recruitment of all antigen
non-specific cells, including lymphomononuclear
cells. This effect occurs in face of high intrahepatic
levels of chemokine gene expression, suggesting
that neutrophil-dependent functions other than
chemokine production are necessar y for the
recruitment process to occur (19). These functions
may include the production of matrix-degrading
proteinases (MMP) that are known to facilitate
leukocyte trafficking through the endothelial barrier
and the extracellular matrix. In keeping with this, a
variety of MMP are strongly and rapidly induced in
the liver of mice after CTL transfer and future
exper iments will be perfor med to define their
Guidotti

Fig. 1 - Depletion of neutrophils diminishes the severity of the CTL-initiated liver disease and blocks the recruitment of antigen non-
specific cells into the liver: histological features. Histological analysis of the necroinflammatory foci detected in livers from HBV
transgenic mice treated with control irrelevant Abs (Irr Ab + CTL, left) or anti-Gr-1 Abs (-Gr-1 + CTL, right) that were sacrificed 1 day
after CTL transfer. Anti-Gr-1 Abs quantitatively depleted neutrophils from the animals. Cells displaying the histological features of
apoptotic hepatocytes (asterisks), lymphocytes (long arrows), macrophages (arrowheads) and PMNs (short arrows) are indicated.
Note that depletion of neutrophils did not significantly reduce the extent of hepatocellular apoptosis and drop out, or the number of
lymphomononuclear cells resembling CTLs in the liver (right panel). However, depletion of neutrophils dramatically reduced the
recruitment of antigen non-specific cells into the liver, so that cells morphologically resembling the transferred CTLs and
macrophages were the only inflammatory cell subsets detectable in these livers (right panel).
Abstracted from Sitia, et al Proc Natl Acad Sci USA 2002; 99: 13717-22

pathogenetic role in our system. In conclusion, the
notion that a reduced recruitment of host-derived
inflammatory into the liver can be associated with
maintenance of CTL-dependent antiviral effects but
diminished tissue damage may help in the design of
potential immunotherapeutic approaches for the
treatment of HBV infection in chronically infected
patients.
HCV pathogenesis
HCV is primarily transmitted by percutaneous and
mucosal routes. The liver is the primary target organ
and the hepatocyte is the primary target cell of the
virus, although various lymphoid populations,
especially B cells and dendritic cells might also be
infected (20, 21) at much lower levels. Over 170
million people are currently infected by HCV (22).
Acute HCV infection is usually asymptomatic, making
diagnosis of the early stages of infection very difficult.
A striking feature of HCV infection is its tendency
towards chronicity; at least 70% of acute infections
become persistent. Chronic infection is often
associated with significant liver disease, especially
chronic active hepatitis, cirrhosis and hepatocellular
carcinoma (22).
Cellular immune responses are also likely to
determine the outcome of HCV infection, but the
dynamics of such responses and their relationship to
viral clearance and persistence are poorly
understood. It is widely believed, however, that HCV
is noncytopathic and that immunologically mediated
events play an important role in HCV pathogenesis,
although the mechanisms whereby HCV causes
acute and chronic hepatitis are still unclear.
Nonetheless, it has been shown that the onset of viral
clearance and liver disease coincide with the onset of
the CD8 + T cell response and the entry of virus-
specific CD8+ T cells into the liver, and that primary
failure to induce a T cell response or functional

117
Pathogenesis of viral hepatitis
exhaustion of an initially vigorous T cell response are
associated with viral persistence (23, 24). Importantly,
CD8+ T cell-mediated destruction of infected cells is
probably not the only way to eliminate the virus
because viral clearance can occur in the absence of
liver disease as long as antiviral CD8+ T cells are
present and produce antiviral cytokines such as IFN-.
Lastly, the fact that the virus can persist in the face of
a multispecific CD4+ and CD8+ T cell response by
progressive mutational escape from the T cell
response further attests to the importance of the
immune response in viral clearance and disease
pathogenesis during HCV infection.
The virological and immunological basis for the
survival of HCV in the face of a CD8+ CTL response is
unclear at the moment. Recent results obtained in
HCV-infected chimpanzees demonstrate, however,
several impor tant aspects of the host-virus
relationship during acute HCV infection (23). First,
HCV outpaces the immune response by several
weeks. Second, HCV is a strong inducer of type 1
interferon but it is relatively resistant to its antiviral
activity. Third, the intrahepatic CD4+ and CD8+ T cell
responses to the virus and the production of IFN-
mRNA in the liver correlate with the outcome of
infection. Fourth, viral clearance and control occur in
response to relatively high titer infections that trigger
an early, vigorous, multispecific, IFN--producing
intrahepatic T cell response. Fifth, uncontrolled
persistent infection occurs in the setting of relatively
low titer viremia and the absence of an intrahepatic
virus-specific T cell response or IFN-. Finally, the
results suggest that although the infection can be
controlled to varying degrees by the destruction of
infected cells, full control of the infection may also
involve noncytolytic T cell effector functions such as
the production of antiviral cytokines.
In summary, viral clearance during HCV infection is
associated with the induction of a vigorous and
diversely targeted T cell response that effectively
homes to the liver and produces IFN-. In contrast,
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ACKNOWLEDGEMENTS
This work was supported by grant AI40696 from the
National Institutes of Health. This is manuscript number
15647-MEM from the Scripps Research Institute.
Reprint requests to:
Luca G. Guidotti, MD
Unit di Immunopatogenesi dellEpatite Virale
DIBIT - Istituto Scientifico San Raffaele
Via Olgettina, 58
20132 Milano, Italy
guidotti.luca@hsr.it
and The Scripps Research Institute
Department of Molecular and Experimental Medicine
10550 North Torrey Pines Road
La Jolla, CA 92037, USA
guidotti@scripps.edu
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