- Email: [email protected]
The role of cytotoxic T cells and cytokines in the control of hepatitis B virus infection
Vaccine 20 (2002) A80–A82
The role of cytotoxic T cells and cytokines in the control of hepatitis B virus infection Luca G. Guidotti∗ Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Abstract The aim of this study was to elucidate the molecular basis for viral clearance and liver disease in the pathogenesis of hepatitis B virus (HBV) infection. Using transgenic mice that replicate HBV at high levels in the liver as recipients of HBV-specific cytotoxic T cells (CTL), we have shown that the antiviral potential of the CTL is primarily mediated by noncytolytic mechanisms that involve the intra-hepatic production of IFN-␥ by these cells. We also showed that, following antigen recognition, HBV-specific CTL recruit antigen non-specific inflammatory cells that contribute to amplify the liver disease initiated by CTL. These results provided insight into immunological and virological processes that may lead to the development of new therapeutic strategies to terminate chronic HBV infection. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: HBV; CTL; IFN-␥
1. Introduction The hepatitis B virus (HBV) is an enveloped DNA that causes acute and chronic liver disease characterized by a necroinflammatory lymphomononuclear cell infiltrate and Kupffer cell hyperplasia [1,2]. HBV infection in immunocompetent adults usually results in a self-limited, transient liver disease and viral clearance. A small percentage of these patients (5–10%) develop chronic hepatitis associated with viral persistence. When neonates are infected, however, over 90% of them will become persistently infected, suffering different degrees of chronic liver disease. Unfortunately, cirrhosis and hepatocellular carcinoma are frequent complications of chronic HBV infection. According to the Centers for Disease Control and Prevention (CDC), over 350 million people are chronically infected with this virus and HBV alone is responsible for about 1 million deaths each year worldwide. About 300,000 people are infected yearly in the United States and more than 5000 patients die each year from the complications of the infection. Since HBV is not directly cytopathic for the hepatocyte, the immune response to viral antigens is thought to be responsible for both liver disease and viral clearance following HBV infection. Indeed, patients with acute viral hepatitis, who successfully clear the virus, mount a multispecific polyclonal CTL response to several HBV-encoded ∗
Tel.: +1-858-784-2758; fax: +1-858-784-2960. E-mail address: [email protected] (L.G. Guidotti).
antigens [1–3]. In contrast, this response is absent or extremely weak in chronically infected patients who do not clear the virus [1–3] and thus, it is believed that the outcome of HBV infection (viral clearance versus viral persistence) is determined primarily by the vigor and quality of the cellular immune response [1,2,4]. The experimental approaches to HBV pathogenesis have been difficult because the host range of HBV is limited to man and chimpanzees, and because in vitro systems for the propagation of HBV do not exist. Studies of HBV immunopathogenesis using models of HBV-related hepadnavirus infections in the woodchuck, ground squirrel and Pekin duck have also been difficult because the immune systems of these outbred species have not been characterized. Definitive analysis of the immunological mechanisms involved in HBV pathogenesis required the development of an inbred animal model with a well defined immune system, i.e. the HBV transgenic mouse. Thus, during the last several years, we have developed and characterized HBV transgenic mice that express all the viral gene products and replicate HBV at high levels in the primary hepatocyte in vivo [5]. 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 [5]. 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
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 3 9 2 - 4
L.G. Guidotti / Vaccine 20 (2002) A80–A82
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 [5]. 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 [5]. HBV replication occurs inside of viral nucleocapsid particles more abundant in centrilobular hepatocytes. As a consequence of efficient viral replication, ultrastructurally complete and infectious [6] viral particles that are morphologically 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 [5]. We have examined in this model the antiviral and immunopathological consequences of antigen recognition by administration of CTL. Initially, an H2d -restricted CTL clone (designated 6C2) that recognizes a dominant CTL epitope located between residues 28 and 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.
2. 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 [7,8]. These cytokines activate two functionally independent virocidal pathways: an early pathway that eliminates HBV nucleocapsid particles and their cargo of replicating viral genomes from the hepatocyte [9,10]; and a later pathway that post-transcriptionally downregulates the viral RNA [11]. In recent studies, we showed that IFN-␥ mediates most of the antiviral effect of the CTL [12] and nitric oxide (NO) mediates most of the antiviral activity of IFN-␥ [13]. 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) [12,14,15] 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 [16]. Along these lines, we also showed that a single injection of ␣-galactosylceramide (␣-GalCer), a gly-
A81
colipid antigen presented to V␣14+ , 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 [17,18]. Furthermore, we have shown that HBV replication is inhibited in these animals by systemic administration of only very high doses of IFN-␣ or IFN-␥ [12]. Indeed, the minimal effective IFN-␣ dose required to inhibit hepatic HBV replication in this model is between 1 × 105 and 5 × 105 units per mouse, while the standard IFN-␣ regimen for chronically infected patients is about 3 × 106 units per day for 10 days. A dose of 3 × 106 units in humans corresponds to a dose of about 1 × 103 units in a 25-g 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. Importantly, we also recently produced evidence suggesting that non-cytopathic antiviral mechanisms may contribute to viral clearance during acute viral hepatitis in chimpanzees, thus validating the transgenic mouse studies in a natural infection model [6]. Moreover, we showed that cytokines known to abolish HBV replication from the hepatocyte also clear a persistent LCMV infection from the hepatocyte non-cytopathically, indicating that, like HBV, LCMV is also susceptible to intracellular inactivation by cytokine-induced antiviral mechanisms that are operative in the hepatocyte [19]. In contrast, other events, presumably killing, are needed to eliminate LCMV from non-parenchymal cells of the liver and from the spleen [19]. These observations suggest that the relative sensitivity of viruses to curative mechanisms may depend on the capacity of the infected cell to produce the appropriate intracellular antiviral factors. This may be particularly important for viruses that infect a large number of parenchymal cells in vital organs, like the liver or the brain. In summary, the cytokine-mediated, non-cytopathic 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 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.
3. 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 [20]. Following antigen recognition, the CTL recruit many host-derived inflammatory cells into the liver, thereby contributing to the formation of necroinflammatory foci in
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L.G. Guidotti / Vaccine 20 (2002) A80–A82
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 [21]. These necroinflammatory foci are scattered throughout the liver parenchyma and cause a focal lesion [20] histologically 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 [22]. 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 non-parenchymal cells of the liver to produce them [22]. 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. In preliminary experiments, we also 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. 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 necessary for the recruitment process to occur. 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 experiments will be performed to define their 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 the design of potential immunotherapeutic approaches for the treatment of HBV infection in chronically infected patients. Acknowledgements This work was supported by grant AI40696 from the National Institutes of Health. This is manuscript number 15047-MEM from the Scripps Research Institute.
References [1] Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29–60. [2] Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest 1997;99:1472–7. [3] Maini MK, Boni C, Lee CK, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000;191:1269–80. [4] Bertoletti A, Maini MK. Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection? Curr Opin Immunol 2000;12:403–8. [5] Guidotti LG, Matzke B, Schaller H, Chisari FV. High level hepatitis B virus replication in transgenic mice. J Virol 1995;69:6158–69. [6] Guidotti LG, Rochford R, Chung L, Shapiro M, Purcell R, Chisari FV. Viral clearance without destruction of infected cells during acute HBV infection. Science 1999;284:825–9. [7] Guidotti LG, Chisari FV. Cytokine-induced viral purging—role in viral pathogenesis. Curr Opin Microbiol 1999;2:388–91. [8] Guidotti LG, Chisari FV. Non-cytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 2001;19:65–91. [9] Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 1996;4:25–36. [10] Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of IFN-␣/ eliminates viral RNA-containing capsids in HBV transgenic mice. J Virol 2000;74:4165–73. [11] Tsui LV, Guidotti LG, Ishikawa T, Chisari FV. Post-transcriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes. Proc Natl Acad Sci USA 1995;92:12398–402. [12] McClary H, Koch R, Chisari FV, Guidotti LG. Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J Virol 2000;74:2255–64. [13] Guidotti LG, McClary H, Moorhead Loudis J, Chisari FV. Nitric oxide inhibits hepatitis B virus replication in the liver of transgenic mice. J Exp Med 2000;191:1247–52. [14] Guidotti LG, Borrow P, Hobbs MV, et al. Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Natl Acad Sci USA 1996;93:4589–94. [15] Cavanaugh VJ, Guidotti LG, Chisari FV. Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in HBV transgenic mice. J Virol 1998;72:2630–7. [16] Cavanaugh VJ, Guidotti LG, Chisari FV. Interleukin-12 inhibits hepatitis B virus replication in HBV transgenic mice. J Virol 1997;71:3236–43. [17] Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo (in process citation). J Exp Med 2000;192:921–30. [18] Kakimi K, Lane TE, Chisari FV, Guidotti LG. Cutting edge: inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver. J Immunol 2001;167:6701–5. [19] Guidotti LG, Borrow P, Brown A, McClary H, Koch R, Chisari FV. Non-cytopathic clearance of lymphocytic choriomeningitis virus from the hepatocyte. J Exp Med 1999;189:1555–64. [20] Ando K, Guidotti LG, Wirth S, et al. Class I restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J Immunol 1994;152:3245–53. [21] Ando K, Moriyama T, Guidotti LG, et al. Mechanisms of class I restricted immunopathology: a transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541–54. [22] Kakimi K, Lane TE, Wieland S, et al. Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 2001;194:1755–66.
The role of cytotoxic T cells and cytokines in the control of hepatitis B virus infection Luca G. Guidotti∗ Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Abstract The aim of this study was to elucidate the molecular basis for viral clearance and liver disease in the pathogenesis of hepatitis B virus (HBV) infection. Using transgenic mice that replicate HBV at high levels in the liver as recipients of HBV-specific cytotoxic T cells (CTL), we have shown that the antiviral potential of the CTL is primarily mediated by noncytolytic mechanisms that involve the intra-hepatic production of IFN-␥ by these cells. We also showed that, following antigen recognition, HBV-specific CTL recruit antigen non-specific inflammatory cells that contribute to amplify the liver disease initiated by CTL. These results provided insight into immunological and virological processes that may lead to the development of new therapeutic strategies to terminate chronic HBV infection. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: HBV; CTL; IFN-␥
1. Introduction The hepatitis B virus (HBV) is an enveloped DNA that causes acute and chronic liver disease characterized by a necroinflammatory lymphomononuclear cell infiltrate and Kupffer cell hyperplasia [1,2]. HBV infection in immunocompetent adults usually results in a self-limited, transient liver disease and viral clearance. A small percentage of these patients (5–10%) develop chronic hepatitis associated with viral persistence. When neonates are infected, however, over 90% of them will become persistently infected, suffering different degrees of chronic liver disease. Unfortunately, cirrhosis and hepatocellular carcinoma are frequent complications of chronic HBV infection. According to the Centers for Disease Control and Prevention (CDC), over 350 million people are chronically infected with this virus and HBV alone is responsible for about 1 million deaths each year worldwide. About 300,000 people are infected yearly in the United States and more than 5000 patients die each year from the complications of the infection. Since HBV is not directly cytopathic for the hepatocyte, the immune response to viral antigens is thought to be responsible for both liver disease and viral clearance following HBV infection. Indeed, patients with acute viral hepatitis, who successfully clear the virus, mount a multispecific polyclonal CTL response to several HBV-encoded ∗
Tel.: +1-858-784-2758; fax: +1-858-784-2960. E-mail address: [email protected] (L.G. Guidotti).
antigens [1–3]. In contrast, this response is absent or extremely weak in chronically infected patients who do not clear the virus [1–3] and thus, it is believed that the outcome of HBV infection (viral clearance versus viral persistence) is determined primarily by the vigor and quality of the cellular immune response [1,2,4]. The experimental approaches to HBV pathogenesis have been difficult because the host range of HBV is limited to man and chimpanzees, and because in vitro systems for the propagation of HBV do not exist. Studies of HBV immunopathogenesis using models of HBV-related hepadnavirus infections in the woodchuck, ground squirrel and Pekin duck have also been difficult because the immune systems of these outbred species have not been characterized. Definitive analysis of the immunological mechanisms involved in HBV pathogenesis required the development of an inbred animal model with a well defined immune system, i.e. the HBV transgenic mouse. Thus, during the last several years, we have developed and characterized HBV transgenic mice that express all the viral gene products and replicate HBV at high levels in the primary hepatocyte in vivo [5]. 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 [5]. 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
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 3 9 2 - 4
L.G. Guidotti / Vaccine 20 (2002) A80–A82
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 [5]. 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 [5]. HBV replication occurs inside of viral nucleocapsid particles more abundant in centrilobular hepatocytes. As a consequence of efficient viral replication, ultrastructurally complete and infectious [6] viral particles that are morphologically 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 [5]. We have examined in this model the antiviral and immunopathological consequences of antigen recognition by administration of CTL. Initially, an H2d -restricted CTL clone (designated 6C2) that recognizes a dominant CTL epitope located between residues 28 and 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.
2. 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 [7,8]. These cytokines activate two functionally independent virocidal pathways: an early pathway that eliminates HBV nucleocapsid particles and their cargo of replicating viral genomes from the hepatocyte [9,10]; and a later pathway that post-transcriptionally downregulates the viral RNA [11]. In recent studies, we showed that IFN-␥ mediates most of the antiviral effect of the CTL [12] and nitric oxide (NO) mediates most of the antiviral activity of IFN-␥ [13]. 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) [12,14,15] 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 [16]. Along these lines, we also showed that a single injection of ␣-galactosylceramide (␣-GalCer), a gly-
A81
colipid antigen presented to V␣14+ , 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 [17,18]. Furthermore, we have shown that HBV replication is inhibited in these animals by systemic administration of only very high doses of IFN-␣ or IFN-␥ [12]. Indeed, the minimal effective IFN-␣ dose required to inhibit hepatic HBV replication in this model is between 1 × 105 and 5 × 105 units per mouse, while the standard IFN-␣ regimen for chronically infected patients is about 3 × 106 units per day for 10 days. A dose of 3 × 106 units in humans corresponds to a dose of about 1 × 103 units in a 25-g 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. Importantly, we also recently produced evidence suggesting that non-cytopathic antiviral mechanisms may contribute to viral clearance during acute viral hepatitis in chimpanzees, thus validating the transgenic mouse studies in a natural infection model [6]. Moreover, we showed that cytokines known to abolish HBV replication from the hepatocyte also clear a persistent LCMV infection from the hepatocyte non-cytopathically, indicating that, like HBV, LCMV is also susceptible to intracellular inactivation by cytokine-induced antiviral mechanisms that are operative in the hepatocyte [19]. In contrast, other events, presumably killing, are needed to eliminate LCMV from non-parenchymal cells of the liver and from the spleen [19]. These observations suggest that the relative sensitivity of viruses to curative mechanisms may depend on the capacity of the infected cell to produce the appropriate intracellular antiviral factors. This may be particularly important for viruses that infect a large number of parenchymal cells in vital organs, like the liver or the brain. In summary, the cytokine-mediated, non-cytopathic 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 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.
3. 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 [20]. Following antigen recognition, the CTL recruit many host-derived inflammatory cells into the liver, thereby contributing to the formation of necroinflammatory foci in
A82
L.G. Guidotti / Vaccine 20 (2002) A80–A82
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 [21]. These necroinflammatory foci are scattered throughout the liver parenchyma and cause a focal lesion [20] histologically 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 [22]. 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 non-parenchymal cells of the liver to produce them [22]. 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. In preliminary experiments, we also 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. 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 necessary for the recruitment process to occur. 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 experiments will be performed to define their 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 the design of potential immunotherapeutic approaches for the treatment of HBV infection in chronically infected patients. Acknowledgements This work was supported by grant AI40696 from the National Institutes of Health. This is manuscript number 15047-MEM from the Scripps Research Institute.
References [1] Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29–60. [2] Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest 1997;99:1472–7. [3] Maini MK, Boni C, Lee CK, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000;191:1269–80. [4] Bertoletti A, Maini MK. Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection? Curr Opin Immunol 2000;12:403–8. [5] Guidotti LG, Matzke B, Schaller H, Chisari FV. High level hepatitis B virus replication in transgenic mice. J Virol 1995;69:6158–69. [6] Guidotti LG, Rochford R, Chung L, Shapiro M, Purcell R, Chisari FV. Viral clearance without destruction of infected cells during acute HBV infection. Science 1999;284:825–9. [7] Guidotti LG, Chisari FV. Cytokine-induced viral purging—role in viral pathogenesis. Curr Opin Microbiol 1999;2:388–91. [8] Guidotti LG, Chisari FV. Non-cytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 2001;19:65–91. [9] Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 1996;4:25–36. [10] Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of IFN-␣/ eliminates viral RNA-containing capsids in HBV transgenic mice. J Virol 2000;74:4165–73. [11] Tsui LV, Guidotti LG, Ishikawa T, Chisari FV. Post-transcriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes. Proc Natl Acad Sci USA 1995;92:12398–402. [12] McClary H, Koch R, Chisari FV, Guidotti LG. Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J Virol 2000;74:2255–64. [13] Guidotti LG, McClary H, Moorhead Loudis J, Chisari FV. Nitric oxide inhibits hepatitis B virus replication in the liver of transgenic mice. J Exp Med 2000;191:1247–52. [14] Guidotti LG, Borrow P, Hobbs MV, et al. Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Natl Acad Sci USA 1996;93:4589–94. [15] Cavanaugh VJ, Guidotti LG, Chisari FV. Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in HBV transgenic mice. J Virol 1998;72:2630–7. [16] Cavanaugh VJ, Guidotti LG, Chisari FV. Interleukin-12 inhibits hepatitis B virus replication in HBV transgenic mice. J Virol 1997;71:3236–43. [17] Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo (in process citation). J Exp Med 2000;192:921–30. [18] Kakimi K, Lane TE, Chisari FV, Guidotti LG. Cutting edge: inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver. J Immunol 2001;167:6701–5. [19] Guidotti LG, Borrow P, Brown A, McClary H, Koch R, Chisari FV. Non-cytopathic clearance of lymphocytic choriomeningitis virus from the hepatocyte. J Exp Med 1999;189:1555–64. [20] Ando K, Guidotti LG, Wirth S, et al. Class I restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J Immunol 1994;152:3245–53. [21] Ando K, Moriyama T, Guidotti LG, et al. Mechanisms of class I restricted immunopathology: a transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541–54. [22] Kakimi K, Lane TE, Wieland S, et al. Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 2001;194:1755–66.