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Journal of Biological Regulators and Homeostatic Agents

Pathogenesis of viral hepatitis


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

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

Wichtig Editore, 2003 0393-974X/115-05 $02.50

Pathogenesis of viral hepatitis

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


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

Pathogenesis of viral hepatitis

exhaustion of an initially vigorous T cell response are the T cell response is greatly attenuated in subjects
associated with viral persistence (23, 24). Importantly, that become chronically infected many of whom also
CD8+ T cell-mediated destruction of infected cells is show evidence of mutational viral escape that can
probably not the only way to eliminate the virus occur early in the acute infection. In addition, a CD8+
because viral clearance can occur in the absence of T cell response can be detected in chronically infected
liver disease as long as antiviral CD8+ T cells are individuals, and it is often functionally impaired, both
present and produce antiviral cytokines such as IFN-. in terms of its ability to home or to accumulate in the
Lastly, the fact that the virus can persist in the face of liver and in its ability to produce IFN-. Future studies
a multispecific CD4+ and CD8+ T cell response by aimed at defining the mechanisms whereby HCV can
progressive mutational escape from the T cell evade or suppress the immune response and
response further attests to the importance of the establish chronic infection are clearly warranted.
immune response in viral clearance and disease
pathogenesis during HCV infection.
The virological and immunological basis for the ACKNOWLEDGEMENTS
survival of HCV in the face of a CD8+ CTL response is
unclear at the moment. Recent results obtained in This work was supported by grant AI40696 from the
HCV-infected chimpanzees demonstrate, however, National Institutes of Health. This is manuscript number
several impor tant aspects of the host-virus 15647-MEM from the Scripps Research Institute.
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 Reprint requests to:
results suggest that although the infection can be Luca G. Guidotti, MD
controlled to varying degrees by the destruction of Unit di Immunopatogenesi dellEpatite Virale
infected cells, full control of the infection may also DIBIT - Istituto Scientifico San Raffaele
Via Olgettina, 58
involve noncytolytic T cell effector functions such as 20132 Milano, Italy
the production of antiviral cytokines.
In summary, viral clearance during HCV infection is and The Scripps Research Institute
Department of Molecular and Experimental Medicine
associated with the induction of a vigorous and 10550 North Torrey Pines Road
diversely targeted T cell response that effectively La Jolla, CA 92037, USA
homes to the liver and produces IFN-. In contrast,

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