- Email: [email protected]
To kill or to cure: options in host defense against viral infection
478
To kill or to cure: options in host defense against viral infection Luca G Guidotti It is generally primarily
thought
and Francis V Chisari*
that viral clearance
by antigen-specific
infected
cells. This assumption
Recent
studies
virus-specific
have shown
cytotoxic
the hepatocytes.
This effect
cytotoxic occur
Intracellular greatly
following
during
other models
destructive
B virus killing
by interferon-y by the
recognition.
‘curative’
effects
has also been carried versus
hepatitis
antigen
mechanisms
the protective
of hepatitis
transferred,
are secreted
an unrelated
viral inactivation
amplify
Research curative
which
cytokine-dependent
in this model
for all viruses.
in the liver without
is mediated
factor-a,
T lymphocytes
noncytopathic
model
T cells can abolish
and replication
necrosis
mouse
that adoptively
gene expression and tumor
that destroy
may not be true
using a transgenic
B virus infection
is mediated
T cell responses
Similar
processes
infection
also
of the liver.
such as these
of the immune
could
response.
out to clarify the relevance
mechanisms
of viral clearance
of in
Hepatitis
of viral infection.
Address Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA; *e-mail: [email protected] Correspondence: Francis V Chisari Current Opinion
in immunology
recognition and destruction is required for viral clearance by CTLs. Considerable evidence has appeared in recent years, however, suggesting that certain viruses are also subject to control by the noncytopathic action of inflammatory cytokines produced by antigen-specific T cells and by non-specific inflammatory cells in infected tissue (see below). In those studies it has been difficult to determine the relative extents to which the immunostimulatory activity versus the antiviral potential of the cytokines contributed to viral clearance. In our review, we describe recent studies that dissect the relative contributions of the cytolytic and antiviral potential of the CTL response, using a hepatitis B virus transgenic mouse model, and we reexamine the literature on this subject from the perspective gained by these experiments.
1996, 8:478-483
0 Current Biology Ltd ISSN 0952-7915 Abbreviations cytotoxic T lymphocyte CTL HBV hepatitis B virus IFN interferon LCMV lymphocyhc chpriomeningitis virus TNF tumor nedrosis factor WHV Woodchuck hepatitis virus
Introduction As a result of the exquisite specificity of the T cell response, cognate recognition of viral antigens expressed at the surface of infected cells, followed by their destruction, is thought to be required for viral clearance by cytotoxic T lymphocytes (CTLs). The classical paper by Lukacher et al. [l] provided indirect evidence that CTLs exert their antiviral activity in &JO by killing the infected cells. In mice simultaneously infected with two subtypes of influenza virus type A, adoptive transfer of a CTL clone specific for one subtype controlled the replication of that particular subtype, but not the replication of the other subtype. D Kagi et a/. [Z] and CM Walsh et ai. [3] have demonstrated that lymphocyte choriomeningitis virus (LCMV) clearance did not occur in perforin knockout mice despite the fact that the number of CD8+ T cells and natural killer (NK) cells was similar to that in LCMV-infected wild-type mice, in which viral clearance did occur. Together, these three papers provide strong evidence to support the hypothesis that antigen
B virus pathogenesis
The hepatitis B virus (HBV) is a noncytopathic, enveloped virus with a circular double stranded DNA genome that causes acute and chronic necroinflammatory liver disease and hepatocellular carcinoma (reviewed in [4*]). Because the disease spectrum associated with HBV is extraordinarily variable, it is widely believed that the host response plays a critical role in the pathogenesis of the associated liver disease. Based on extensive studies of HBV pathogenesis in man and animal models, there is considerable evidence that viral hepatitis is initiated by an antigen-specific antiviral cellular immune response that sets in motion a cascade of antigen non-specific effector systems that actually cause most of the damage to the liver and eliminate the virus (reviewed in [4*]). The extent to which clearance of HBV depends on the destruction of infected cells by the CTL response to viral antigens, as opposed to the ability of the CTL response to cure infected cells without killing them, is the subject of this review. Is it possible to clear a virus from the liver simply by killing the infected cells? It is generally acknowledged that the CTL response clears viral infections by killing infected cells. While this may be the principal mechanism for clearance of viruses that infect relatively few cells, it may not be possible for the CTL response to eradicate all infected cells when the virus outnumbers the CTLs by several orders of magnitude. This is the case for many viruses, especially hepatotropic viruses like HBV in view of the large number of cells in the liver. Nonetheless, HBV is cleared in more than 95% of acutely infected adults [4’]. How does this occur? Does the CTL response kill all of the infected cells? In this paper we argue that it doesn’t, Consider
to kill
that many
a target
indeed
that it can’t.
more functions
cell in viwo than
are required
in
oitro:
for CTLs they must
Host defense against viral infection Guidotti and Chisari
be induced, commonly in the lymphoid compartment, which is not usually the site of viral replication. The cells must then enter the blood and reach the infected tissue, where they must receive and respond to a stop signal; it is necessary to migrate past any tissue barriers (e.g. endotheli’al cells, basement membranes, uninfected cells etc.) that separate them from their target, reach and recognize their cognate antigen, bind and kill the infected cell and then move on to find and kill the next target cell, which may be distant from the first one. Finally, the CTLs themselves undergo activation-induced cell death [S]. Thus, viral elimination by direct one-on-one CTL-mediated killing is not nearly as simple in ait~o as one might assume from the ease with which CTLs can kill target cells in vitro when the CTLs are usually contiguous with the target cells, and often outnumber them by a significant margin, factors that usually do not exist in &IO. Furthermore, the only functions the CTLs need to perform in vitro are antigen recognition and the delivery of one or more death signals; the target cells routinely employed in these assays have been specifically selected for their exquisite sensitivity to those signals and are not likely to be representative of primary cells infected in vivo.
it is, if clearance infected cells.
In addition, consider the following numerical scenario. There are more than 1011 potentially infectible hepatocytes in the human liver and more than 90% of them are regularly infected by HBV [6]. Consider, also, that there are about 1012 lymphocytes in the body and that the HBV-specific CTL-precursor frequency at the height of the CTL response in a strongly reactive patient with acute hepatitis is rarely greater than 104 and usually it is much lower [7]. Hence, there should be no more than 108 HBV-specific CTLs in the body at any one time. Therefore, if every HBV-specific CTL in the entire body were to enter the liver at the same time, which is highly unlikely, and if most of the hepatocytes were infected, which is quite common, there would be one specific CTL in the liver for every 1000 infected hepatocytes. Under most circumstances, however, the effector: target cell ratio in the liver should be much lower.
This is because, in addition to killing some of the hepatocytes, the CTLs also downregulated the expression and replication of HBV by all of the hepatocytes in the liver without killing them. We have recently shown that all of the viral gene products, including the viral RNAs, their translation products and the episomal replicative DNA intermediates, are susceptible to this remarkable effect [lP]. Importantly, it is possible to block the noncytopathic antiviral effects of the CTLs by the prior administration of antibodies to interferon (1FN)y and tumor necrosis factor (TNF)ol. As IFNy is a powerful macrophage activator, it is likely that its effect is mediated by the production of TNFa by activated macrophages. This hypothesis is strengthened by the observation that recombinant TNFa also inhibits HBV gene expression and replication in these mice [13], as does interleukin (IL)-& and that the ability of IL-2 to suppress HBV RNA levels in the liver is mediated by TNFcx [14] via a post-transcriptional mechanism that accelerates the degradation of cytoplasmic HBV mRNA [15]. Similarly, the CTL-dependent downregulation of viral RNAs occurs at the post-transcriptional level [16*].
Even considering the temporally extended dynamics of the CTL response it is difficult to imagine that a single CTL, or even a succession of CTLs maintained at a constant and high frequency, could find, recognize and destroy more than 1000 infected hepatocytes, one at a time. Furthermore, consider that if only 10% of the hepatocytes were infected, the effector: target cell ratio would still be less than 1: 100 and the CTL would repeatedly have to survey many uninfected cells before finding a target. Another argument against this scenario is the fact that most infected patients clear the virus in a few weeks while experiencing only mild to moderate liver disease. Since most of the hepatocytes in the liver are usually infected by HBV, one would expect the incidence of severe or fatal acute hepatitis to be much higher than
were due entirely
to the destruction
479
of
For all of these reasons, we suggest that although the liver disease in viral hepatitis is certainly due to the destructive potential of the CTL response, viral clearance probably requires additional CTL functions in addition to their ability to kill infected cells.
CTLs can inhibit HBV gene expression and replication without killing the hepatocyte We have recently examined the cytopathic and curative antiviral properties of the CTL response in transgenic mice constructed to replicate HBV in the liver at high levels without any evidence of cytopathology [S]. Following adoptive transfer of CDB+, MHC class I restricted, hepatitis B surface antigen (HBsAg)-specific CTL lines and clones, however, the mice develop a necroinflammatory liver disease resembling acute viral hepatitis [9,10]. Interestingly, the CTL clones cannot induce a second episode of hepatitis if they are readministered to the mice within 3-4 weeks of the first CTL injection [ 111.
These results suggest that a strong intrahepatic immune response to HBV can suppress viral gene expression and replication and perhaps even ‘cure’ infected hepatocytes without killing them. Additionally, the results illustrate that the infected cells can become active participants in the antiviral response by responding to cytokine-induced signals and activating specific intracellular pathways that interrupt the viral life cycle. Indeed, we have identified two independent antiviral events that occur in the CTL-activated hepatocytes: one degrades the viral nucle-
480
Immunity to infection
ocapsids and their cargo of replicative and the other post-transcriptionally RNA. This is a significant which views the infected viral infection.
DNA intermediates, degrades the viral
departure from current dogma, cell merely as a victim of the
Additionally, the data suggest that a weak immune response or incomplete viral inactivation could contribute to viral persistence and chronic liver disease by reducing the expression of viral antigens just enough for some of the infected cells to escape immune recognition. If this scenario is accurate, the influence of CTL-derived cytokines on HBV gene expression and replication could be viewed either as a survival strategy by the virus, contributing to persistence, or as a tissue-sparing antiviral strategy by the host, contributing to viral elimination, depending on the strength and cytokine profile of the immune response.
HBV gene expression and replication are abolished during an unrelated infection of the liver, even in the complete absence of liver disease One might predict from the foregoing evidence that coinfection or superinfection of the HBV-infected liver by other pathogens could facilitate HBV clearance if they induce the local production of antiviral cytokines such as IFNy or TNFa to which HBV is susceptible. This prediction would be consistent with isolated case reports that chronic HBV infection resolved during an intermittent infection of the liver by hepatitis A or hepatitis C [17,18]. To investigate this hypothesis, we infected adult and newborn HBV-transgenic mice with lymphocytic choriomeningitis virus (LCMV) which, like HBV, is known to infect the liver and cause an inflammatory liver disease that is initiated by virus-specific CTLs [4*,19]. In experimentally infected immunocompetent adult mice LCMV is cleared by a MHC class I restricted, CD8+ CTL response [20,21] that is absent when newborn mice are infected [22,23]. In newborns, the lack of a CTL response results in a lifelong persistent noncytopathic infection of many organs, including the liver [24,25]. In these studies we demonstrated that within two days of LCMV infection of adult HBV-transgenic mice virtually all of the intrahepatic macrophages but less than 1% of the hepatocytes were infected by LCMV. At the same time TNFa and IFNc# were induced in the liver and hepatic HBV replication was noncytopathically abolished. It appears, therefore, that LCMV activates the production of TNFcl and IFNcx//P in the liver by the infected macrophages and that these cytokines activate the hepatocytes to reduce their burden of HBV While all these events occur before the appearance of an inflammatory infiltrate in the liver, this antiviral effect is amplified by the subsequent LCMV-induced cellular immune response in acutely infected animals due to the production of increased amounts of TNFcx and the late induction of
IFNy by the LCMV-specific inflammatory cells that they
T cells and the non-specific activate [26*].
Importantly, HBV gene expression and replication were totally and persistently abolished, without any accompanying liver disease, when the HBV-transgenic mice were persistently infected by LCMV and their intrahepatic macrophages expressed high levels of TNFa and IFNo$ [26’]. This demonstrates, for the first time, that activated hepatic macrophages can persistently abolish HBV gene expression and replication from the hepatocytes in the absence of an inflammatory liver disease, strongly suggesting that this cell type, as well as HBV-specific CTLs, plays a key role in the control of HBV infection. Indeed, hepatic macrophages are likely to be activated during HBV infection because they are characteristically hypertrophic and hyperplastic during human viral hepatitis [27] as well as in mammalian [28] and avian [29] models of hepadnavirus infection. Rapid clearance of Woodchuck hepatitis virus (WHV) is also the rule after experimental infection of immunocompetent adult animals even when 100% of the hepatocytes are infected. Viral clearance occurs in the absence of massive destruction or regeneration of hepatocytes and in the presence of inflammatory cells and Kupffer cell hyperplasia [27], suggesting that cytokine-dependent pathways like those described in the transgenic mouse model may also play an important role in viral clearance during WHV infection. Collectively, these results suggest, that during acute HBV infection in man, viral clearance is probably achieved by the cooperation of at least two mechanisms, as illustrated in Fig. 1. First, HBV-specific CTLs recognize and kill a small fraction of infected hepatocytes. Second, they secrete inflammatory cytokines that directly or indirectly (via macrophage activation) potentially cure most of the infected hepatocytes by the intracellular inactivation pathways described above.
What about other viral infections? The current observations raise the possibility that cytokine-activated pathways may be operative in other viral infections. Indeed, Martz and Howell have previously introduced the concept of cytokine-dependent ‘intracellular inactivation’ for viruses such as the herpes simplex virus [30]. If cytokines do play an effector role in the antiviral immune response, each virus can be expected to display its own individual cytokine sensitivity profile, and some viruses (including different subtypes of the same virus) may simply be insensitive to cytokine-mediated control. For example, the ability of LCMV to establish a persistent infection in aiuo in the presence of concentrations of TNFa and IFNo@ that abolished HBV replication correlates with the relative resistance of this virus to control by these cytokines. Additionally, perforin-deficient mice are unable to clear certain LCMV strains [2,3], suggesting that the specific destruction of infected cells by CTLs is required for viral clearance, although these experiments
Host defense
Figure
against
viral infection
Guidotti and Chisari
481
1
Mechanisms
of viral clearance
from
the hepatocyte. (a) Upon antigen activation, CTLs deliver an apoptotic signal mediated by both Fas ligand (FasL) and perforin to their target hepatocytes (Hc), killing them. (b) They also secrete IFNy and TNFcq cytokines that have been shown to abolish HBV gene expression and viral replication in viva, infection. TNFa the intrahepatic
curing the
can also be produced by macrophages (Kupffer
cells, KC) following activation by IFNy. The curative effect of the CTL response is much more efficient than its destructive effect (1 O-l OO-fold more cells are cured
Antigen recognition
& Hc
-
Perforin
than destroyed). The outcome of an infection may depend on the relative balance of these two effects, with a predominantly curative response leading to viral clearance, and a predominantly destructive response leading to viral persistence and chronic liver disease. +, infected with virus; -, not infected with virus.
Destruction
Inactivation of virus
of cell
0 1996 Current Oprlon in lmmunolog~
do not rule out the possibility that a threshold of cell destruction is needed to induce an effective concentration of antiviral cytokines in an infected organ. The fact that some strains of LCMV replicate uncontrollably in mice that lack IFNy receptors [31], or that have been treated with IFNy-specific antibodies [32] might suggest that this cytokine either directly participates in the control of LCMV replication or contributes to viral clearance by inducing an effective cytolytic T cell response. In support of the former hypothesis, it has been shown previously that LCMV infection in mice can be cleared, without evidence of cytopathology [33], before the onset of detectable cytolytic activity and by extremely small numbers of adoptively transferred CTLs [34]. Thus, the hypothesis that T-cell-derived cytokines may control viral infections noncytopathically was already formulated by Lehmann-Grube and co-workers more than ten years ago [34]. In addition to this indirect evidence in the LChlV model, control of viral replication if/ cie,o by IFNa/P, IFNy and TNFcl has also been suggested for several other viral infections, including vaccinia virus [35,36,37’], measles virus [38*], herpes simplex virus (HSV) [39,40*], influenza virus [41*], Semliki Forest virus (SFV) [31,42’] and Theiler’s virus [43’]. Furthermore, a variety of cellular antiviral proteins are induced in response to IFN activation, some of which have been shown to impair influenza and vesicular stomatitis virus transcription i/l ah-o [44,45] while others lead to cleavage of single-stranded picornaviral RNA [46]. Additionally, TNFcx and/or TNFP have been reported to inhibit the replication of HSV, adenovirus and vesicular stomatitis virus ia oh-o [47,48], and HIV replication has
been shown to be inhibited in vitro by non-cytolytic factors produced by HIV-specific class I restricted CTLs upon antigen recognition [49]. Although the precise nature of the antiviral factors produced by the CDS+ T cells is a matter of some conjecture at present [50]. Finally, a number of viruses encode proteins that block the transcriptional activation of the IFN-activatable genes [51,52], downregulate TNFa production in vitro [53], or represent receptor analogues for IFNc@, IFNy and TNFa [54-57,58*], apparently as a strategy to blunt the antiviral activity of these cytokines. However, these viral functions may be more important for protecting the virus against cytokines produced by the infected cells rather than cytokines produced by antiviral CTLs.
Conclusion Viral clearance might depend heavily on noncytopathic curative mechanisms, especially in massive infections of vital organs. If this is correct, the dual destructive/curative process could explain the co-evolution of cytopathic and noncytopathic antiviral functions in the effector limb of the immune response and could influence the mutational threshold required for cytokine-sensitive viruses to escape immunological control. It could also explain the spectrum of outcomes observed during certain noncytopathic viral infections in different individuals, with dominance of the curative process leading to viral clearance and recovery, and dominance of the destructive pathway leading either to low-grade chronic inflammation and viral persistence, or massive tissue destruction and the death of the host. hluch more attention to the curative potential of antiviral cytokines produced by antigen-specific and antigen-non-specific responses to viral infections, as well
482
Immunity
to infection
as the relative susceptibility of individual viruses to control by these cytokines, is certainly warranted.
Acknowledgements We thank Bonnie W&r for help with manuscript preparation. This work was supported by grants ROIAI20001 and R37CA40489 from the National Institutes of Health. This is manuscript number 10086-MEM from the Scripps Research Institute.
References
and recommended
1 7.
Davis GL, Hoofnagle JH, Waggoner JG: Acute type A hepatitis during chronic hepatitis B virus infection: association of depressed hepatitis B virus replication with appearance of endogenous alpha interferon. J Med Viral 1984, 14:141-l 47.
18.
Sheen I-S, Liaw Y-F, Lin D-Y, Chu C-M: Role of hepatitis C and delta viruses in the termination of chronic hepatitis B surface antigen carrier state: a multivariate analysis in a longitudinal follow-up study. J Infect Dis 1994, 170:1358-l 361.
19.
Lohler J, Gossman J, Kratzberg T, Lehmann-Grube F: Murine hepatitis caused by lymphocytic choriomeningitis virus. I. The hepatic lesions. Lab Invest 1994, 70~263-277.
reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
. ..
Tsui, LV, Guidotti LG, lshikawa T, Chisari FV: Post-transcriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyteactivated hepetocytes. Proc Nat/ Acad Sci USA 1995, 92:12396-l 2402. HBV-specific CTLs noncyiopathically downregulate most HBV RNAs in the hepatocyte nucleus by a cytokine-dependent post-transcriptional mechanism that involves recognition of virus-specific sequences contained in the longest viral transcripts. These results suggest that noncytopathic cytokine-dependent clearance of a virus may occur only if the virus genome (or its products) carries appropriate responsive elements. 16. .
of special interest of outstanding interest
1.
Lukacher AE, Braciale VL, Braciale TJ: In viva effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific. I Exp Med 1984, 160:814-826.
2.
Kagi D, Ledermann B, Burki K, Seiler P, Odermatt B, Olsen J, Podack ER, Zinkemagel R, Hengartner H: Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 1994, 369:31-37.
20.
Oldstone MBA, Blount P, Southern PJ, Lampert PW: Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system. Nature 1986, 3211239-243.
3.
Walsh CM, Matloubian M, Liu C, Ueda R, Kurahara CG, Christensen JL, Huang MTF, Young JD-E, Ahmed R, Clark WR: Immune function in mice lacking the perforin gene. Proc Nat/ Acad Sci USA 1994, 91 :I 0854-I 0858.
21.
Ahmed R, Jamieson BD, Porter DD: Immune therapy of a persistent and disseminated viral infection. J Viral 1987, 61:3920-3929.
22.
Fung-Leung W-P, Kunding TM, Zinkernagel RM, Mak TW: Immune response against lymphocytic choriomeningitis virus infection in mice without CD6 expression. J fxp Med 1991, 174:1425-1429.
23.
Moskophidis D, Cobbold SP, Waldmann H, Lehmann-Grube F: Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response. J Viral 1987, 61 :1867-l 874.
4. Chisari FV, Ferrari C: Hepatitis 6 virus immunopathogenesis. . Annu Rev lmmunoll995, 13:29-60. A comprehensive review of HBV immunobiology and pathogenesis in humans and transgenic mice. The immunological basis for viral clearance and viral persistence is critically discussed. 5.
Razvi ES, Jiang Z, Woda BA, Welsh RM: Lymphocyte apoptosis during the silencing of the immune response to acute viral infections in normal, Ipr and Bcl-2 mice. Am J Pathol 1995, 147:79-91.
6.
Yoo JY, Howard R, Waggoner JG, Hoofnagle JH: Peroxidase-anti-peroxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis. J Med Viral 1987, 23:273-281.
24.
Pircher H, Bijrki K, Lang R, Hengartner H, Zinkemagel RM: Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 1969, 342:559-561.
7.
Rehermann B, Lau D, Hoofnagle JH, Chisari FV: Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection. J C/in invest 1996, 97:1655-l 665.
25.
King CC, Jamieson BD, Reddy K, Bali N, Conception RJ, Ahmed R: Viral infection of the thymus. J Viral 1992, 66:3155-3160.
0.
Guidotti LG, Matzke B, Schaller H, Chisari FV: High level hepatitis B virus replication in transgenic mice. J Viral 1995, 69:6158-6169.
9.
Ando K, Guidotti LG, Wirth S, lshikawa T, Missale G, Moriyama T, Schreiber RD, Schlicht HJ, Huang S, Chisari FV: Class I restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in viva. J lmmunol 1994, 152:3245-3253.
10.
Moriyama T, Guilhot S, Klopchin K, Moss 8, Pinked CA, Palmiter RD, Brinster RL, Kanagawa 0, Chisari FV: lmmunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 1990, 248:361-364.
11.
Wirth S, Guidotti LG, Ando K, Schlicht HJ, Chisari FV: Breaking tolerance leads to autoantibody production but not autoimmune liver disease in HBV envelope transgenic mice. J lmmunol 1995, 154:2504-2515.
12. ..
Guidotti LG, lshikawa 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. The first direct evidence that physiologic concentrations of antiviral cytokines induced by antigen-specific CTLs can noncytopathically abolish viral gene expression and replication. 13.
14.
15.
Gilles PN, Fey G, Chisari FV: Tumor necrosis factor-alpha negatively regulates hepatitis B virus gene expression in transgenic mice. J Viral 1992, 66:3955-3960. Guidotti LG, Guilhot S, Chisari FV: Interleukin-2 and interferon alpha/beta downregulate hepatitis B virus gene expression in viva by tumor necrosis factor dependent and independent pathways. J Viral 1994, 68:1265-l 270. Guilhot S, Guidotti LG, Chisari RI: Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mica by a post-transcriptional mechanism. J Viral 1993, 67:7444-7449.
26. .
Guidotti LG, Borrow P, Hobbs MV, Matzke B, Gresser I, Oldstone MBA, Chisari FV: Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Nat/ Acad Sci USA 1996, 93:4569-4594. HBV replication in the hepatocytes of HBV-transgenic mice is noncytopathically abolished by cytokines (particularly TNFa and IFNaIP) produced by LCMV-infected macrophages in the absence of an LCMV-specific T-cell response. Evidence is provided of an important role for cytokines produced by macrophages in viral clearance, and that LCMV itself is resistant to the antiviral cytokines it induces even though they readily control HBV replication. 27.
Uchida T, Kronborg I, Peters RL: Acute viral hepatitis: morphological and functional correlations in human livers. Hum Pathol 1984, 15:267-277.
28.
Kajino K, Jilbert AR, Saputelli J, Aldrich C, Cullen J, Mason WS: Woodchuck heDatitis virus infections: verv raoid recoverv after a prolonged viiemia and infection of virt&lly’every hepetocyte. J Viral 1994, 68:5792-5803.
29.
Jilbert AR, Wu T-T, England JM, De La Hall PM, Carp NZ, O’Connell AP, Mason WS: Rapid resolution of duck hepetitis B virus infections occurs after massive hepatocellular involvement. J Viral 1992, 66:1377-l 388.
30.
Martz E, Howell DM: CTL: virus control cells first and cytolytic cells second? lmmunol Today 1989, 10:79-86.
31.
Mijller U, Steinhoff U, Reis LFL, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M: Functional role of type I and type II interferons in antiviral defense. Science 1994, 264:1916-l 921.
32.
Moskophidis D, Battegay M, Bruendler M-A, Laine E, Gresser I, Zinkemagel R: Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and gamma interferon. J Viral 1994, 68:1951-i 955.
33.
Gegin C, Lehmann-Grube F: Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present
Host defense
an immunodominant viral cytotoxic T lymphocyte lmmunol1992,149:3331-3338.
epitope. I
34.
Lehmann-Grube F, Assmann U, Loliger C, Moskophidis D, Lohler J: Mechanism of recovery from acute virus infection. I. Role of T lymphocytes in the clearance of lymphocytic choriomeningitis virus from spleens of mice. J lmmunol 1985, 134:608-615.
35.
Sambhi SK, Kohonen-Corish MRJ, Ramshaw IA: Local production of tumor necrosis factor encoded by recombinant vaccinia virus is effective in controlling viral replication in ho. Proc Nat/ Acad Sci USA 1991, 88:4025-4029.
36.
Kohonen-Corish MRJ, King NJC, Woodhams CE, Ramshaw IA: lmmunodeficient mice recover from infection with vaccinia virus expressing interferon-y. Eur J lmmunol 1990, 20:157-l 61.
Van Den Broek MF, Mijller U, Huang S, Aguet M, Zinkernagel RM: Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors. J Viral 1995, 69:4792-4796. LCMV and vaccinia virus replicate much faster in mice that lack both IFN u/p and y receptors. Finke D, Brinckmann UG, Ter Meulen V, Liebert UG: Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis. J Viral 1995, 695469-5474. Measles virus is not cleared from the central nervous system in mice depleted of IFNy. 39.
Rossol-Voth R, Ross01 S, Schijtt KH, Corridori S, De Cian W, Falke D: In viva protective effect of tumor necrosis factor a against experimental infection with herpes simplex virus type I. J Gen Viral 1991, 72:143-147.
Bouley DM, Kanangat S, Wire W, Rouse BT: Characterization of herpes simplex virus type-l infection and herpetic stromal keratitis development in IFN-y knockout mice. J lmmunol 1995, 165:3964-3971. IFNy is required to clear herpes simplex virus from the eye.
Topham DJ, Tripp RA, Sarawar SR, Sangster MY, Doherty PC: Immune CD4+ T cells oromote the clearance of influenza virus from major histocompatibility complex class II -I- respiratory epithelium. J Viral 1996, 70:1288-i 291. CD4+ effector T cells can eliminate influenza virus in respiratory epithelial cells that MHC lack class II expression. The involvement of cytokines and/or antibody is discussed. Hwang SY, Hertzog PJ, Holland KA, Sumarsono SH, Tymms MJ, Hamilton JA, Whiny G, Bertoncello I, Kola I: A null mutation in the gene encoding a type I interferon receptor component eliminates anti-proliferative responses to interferons a and p and alters macrophage responses. Proc Nat/ Acad Sci USA 1995, 92:11284-l 1288. Semliki Forest virus replicates uncontrollably in several organs of mice that lack a component of the IFN a@ receptor.
42. .
Fiette L, Aubert C, Miller U, Huang S, Aguet M, Brahic M, Bureau J-F: Theiler’s virus infection of 129Sv mice that lack the interferon a/p or interferon y receptors. J Exp Med 1995, 181:2069-2076. IFN a@ and y play a non-redundant role in the control of Theiler’s virus infection. 43. .
Guidotti and Chisari
483
Horisberger M, Staeheli P, Hailer 0: Interferon induces unique protein in mouse cells bearing a gene for resistance to influenza virus. Proc Nat/ Acad Sci USA 1983, 60:191 O-l 914.
45.
Staeheli P, Pavlovic J: Inhibition of vesicular stomatiiis virus mRNA synthesis by human MxA protein. J Viral 1991, 66:4498-4501.
46.
Kumar R, Choubey D, Lengyel P, Sen GC: Studies on the role of 2’-6’-oligoadenylate synthetase-RNase L pathway in beta interferon-mediated inhibition of encephalomyocarditis virus replication. J Viral 1988, 62:3175-3181.
47.
Mestan J, Digel W, Mittnacht S, Hillen H, Blohm D, Moller A, Jacobsen H, Kirchner H: Antiviral effect of recombinant tumor necrosis factor in vitro. Nature 1986, 323:816-819.
48.
Wong GHW, Goeddel DV: Tumor necrosis factor a and p inhibit virus replication and synergize with interferons. Nafure 1986, 323:819-822.
49.
Walker CM, Erickson AL, Hsueh FC, Levy JA: Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD6+ cells involves a non-cytotoxic mechanism. J Virol 1991, 65:5921-5927.
50.
Baiter M: Elusive HIV-suppressor 270:1560-1561.
51.
Foster GR, Ackrill AM, Goldin RD, Kerr IM, Thomas HC, Stark GR: Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA. Proc Nat/ Acad Sci USA 1991,88:2888-2892.
52.
Kalvakolanu DVR, Bandyopadhyay SK, Harter ML, Sen GC: Inhibition of interferon-inducible gene expression by adenovirus EIA proteins; block in transcriptional complex formation. froc Nat/ Acad Sci USA 1991, 88:7459-7463.
53.
Adler H, Jungi TW, Pfister H, Strasser M, Sileghem M, Peterhans E: Cytokine regulation by virus infection: bovine viral diarrhea virus, a flavivirus, downregulates production of tumor necrosis factor alpha in macrophages in vitro. J Viral 1996, 70:2650-2653.
54.
Symons JA, Alcami A, Smith GL: Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 1995, 81:551-560.
55.
Upton C, Mossman K, McFadden G: Encoding a homolog of the interferon-y receptor by myxoma virus. Science 1992, 268:1369-i 372.
56.
Alcami A, Smith GL: Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity. J Viral 1995, 69:4633-4639.
57.
Schreiber M, McFadden G: The myxoma virus TNF-receptor homologue (T2) inhibits tumor necrosis factor-a in a speciesspecific fashion. Virology 1994, 204:692-705.
40. .
41. .
viral infection
44.
37. .
38. .
against
factors found. Science
1995,
McFadden G, Graham K, Ellison K, Barry M, Macen J, Schreiber M, Mossman K, Nash P, Lalani A, Everett H: Interruption of cytokine networks by poxviruses: lessons from myxoma virus. J Leukoc Biol 1995, 571731-738. A very detailed review describing the virokines and viroreceptors produced during myxoma virus infection and their interactions with the cytokine network. 58. .
To kill or to cure: options in host defense against viral infection Luca G Guidotti It is generally primarily
thought
and Francis V Chisari*
that viral clearance
by antigen-specific
infected
cells. This assumption
Recent
studies
virus-specific
have shown
cytotoxic
the hepatocytes.
This effect
cytotoxic occur
Intracellular greatly
following
during
other models
destructive
B virus killing
by interferon-y by the
recognition.
‘curative’
effects
has also been carried versus
hepatitis
antigen
mechanisms
the protective
of hepatitis
transferred,
are secreted
an unrelated
viral inactivation
amplify
Research curative
which
cytokine-dependent
in this model
for all viruses.
in the liver without
is mediated
factor-a,
T lymphocytes
noncytopathic
model
T cells can abolish
and replication
necrosis
mouse
that adoptively
gene expression and tumor
that destroy
may not be true
using a transgenic
B virus infection
is mediated
T cell responses
Similar
processes
infection
also
of the liver.
such as these
of the immune
could
response.
out to clarify the relevance
mechanisms
of viral clearance
of in
Hepatitis
of viral infection.
Address Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA; *e-mail: [email protected] Correspondence: Francis V Chisari Current Opinion
in immunology
recognition and destruction is required for viral clearance by CTLs. Considerable evidence has appeared in recent years, however, suggesting that certain viruses are also subject to control by the noncytopathic action of inflammatory cytokines produced by antigen-specific T cells and by non-specific inflammatory cells in infected tissue (see below). In those studies it has been difficult to determine the relative extents to which the immunostimulatory activity versus the antiviral potential of the cytokines contributed to viral clearance. In our review, we describe recent studies that dissect the relative contributions of the cytolytic and antiviral potential of the CTL response, using a hepatitis B virus transgenic mouse model, and we reexamine the literature on this subject from the perspective gained by these experiments.
1996, 8:478-483
0 Current Biology Ltd ISSN 0952-7915 Abbreviations cytotoxic T lymphocyte CTL HBV hepatitis B virus IFN interferon LCMV lymphocyhc chpriomeningitis virus TNF tumor nedrosis factor WHV Woodchuck hepatitis virus
Introduction As a result of the exquisite specificity of the T cell response, cognate recognition of viral antigens expressed at the surface of infected cells, followed by their destruction, is thought to be required for viral clearance by cytotoxic T lymphocytes (CTLs). The classical paper by Lukacher et al. [l] provided indirect evidence that CTLs exert their antiviral activity in &JO by killing the infected cells. In mice simultaneously infected with two subtypes of influenza virus type A, adoptive transfer of a CTL clone specific for one subtype controlled the replication of that particular subtype, but not the replication of the other subtype. D Kagi et a/. [Z] and CM Walsh et ai. [3] have demonstrated that lymphocyte choriomeningitis virus (LCMV) clearance did not occur in perforin knockout mice despite the fact that the number of CD8+ T cells and natural killer (NK) cells was similar to that in LCMV-infected wild-type mice, in which viral clearance did occur. Together, these three papers provide strong evidence to support the hypothesis that antigen
B virus pathogenesis
The hepatitis B virus (HBV) is a noncytopathic, enveloped virus with a circular double stranded DNA genome that causes acute and chronic necroinflammatory liver disease and hepatocellular carcinoma (reviewed in [4*]). Because the disease spectrum associated with HBV is extraordinarily variable, it is widely believed that the host response plays a critical role in the pathogenesis of the associated liver disease. Based on extensive studies of HBV pathogenesis in man and animal models, there is considerable evidence that viral hepatitis is initiated by an antigen-specific antiviral cellular immune response that sets in motion a cascade of antigen non-specific effector systems that actually cause most of the damage to the liver and eliminate the virus (reviewed in [4*]). The extent to which clearance of HBV depends on the destruction of infected cells by the CTL response to viral antigens, as opposed to the ability of the CTL response to cure infected cells without killing them, is the subject of this review. Is it possible to clear a virus from the liver simply by killing the infected cells? It is generally acknowledged that the CTL response clears viral infections by killing infected cells. While this may be the principal mechanism for clearance of viruses that infect relatively few cells, it may not be possible for the CTL response to eradicate all infected cells when the virus outnumbers the CTLs by several orders of magnitude. This is the case for many viruses, especially hepatotropic viruses like HBV in view of the large number of cells in the liver. Nonetheless, HBV is cleared in more than 95% of acutely infected adults [4’]. How does this occur? Does the CTL response kill all of the infected cells? In this paper we argue that it doesn’t, Consider
to kill
that many
a target
indeed
that it can’t.
more functions
cell in viwo than
are required
in
oitro:
for CTLs they must
Host defense against viral infection Guidotti and Chisari
be induced, commonly in the lymphoid compartment, which is not usually the site of viral replication. The cells must then enter the blood and reach the infected tissue, where they must receive and respond to a stop signal; it is necessary to migrate past any tissue barriers (e.g. endotheli’al cells, basement membranes, uninfected cells etc.) that separate them from their target, reach and recognize their cognate antigen, bind and kill the infected cell and then move on to find and kill the next target cell, which may be distant from the first one. Finally, the CTLs themselves undergo activation-induced cell death [S]. Thus, viral elimination by direct one-on-one CTL-mediated killing is not nearly as simple in ait~o as one might assume from the ease with which CTLs can kill target cells in vitro when the CTLs are usually contiguous with the target cells, and often outnumber them by a significant margin, factors that usually do not exist in &IO. Furthermore, the only functions the CTLs need to perform in vitro are antigen recognition and the delivery of one or more death signals; the target cells routinely employed in these assays have been specifically selected for their exquisite sensitivity to those signals and are not likely to be representative of primary cells infected in vivo.
it is, if clearance infected cells.
In addition, consider the following numerical scenario. There are more than 1011 potentially infectible hepatocytes in the human liver and more than 90% of them are regularly infected by HBV [6]. Consider, also, that there are about 1012 lymphocytes in the body and that the HBV-specific CTL-precursor frequency at the height of the CTL response in a strongly reactive patient with acute hepatitis is rarely greater than 104 and usually it is much lower [7]. Hence, there should be no more than 108 HBV-specific CTLs in the body at any one time. Therefore, if every HBV-specific CTL in the entire body were to enter the liver at the same time, which is highly unlikely, and if most of the hepatocytes were infected, which is quite common, there would be one specific CTL in the liver for every 1000 infected hepatocytes. Under most circumstances, however, the effector: target cell ratio in the liver should be much lower.
This is because, in addition to killing some of the hepatocytes, the CTLs also downregulated the expression and replication of HBV by all of the hepatocytes in the liver without killing them. We have recently shown that all of the viral gene products, including the viral RNAs, their translation products and the episomal replicative DNA intermediates, are susceptible to this remarkable effect [lP]. Importantly, it is possible to block the noncytopathic antiviral effects of the CTLs by the prior administration of antibodies to interferon (1FN)y and tumor necrosis factor (TNF)ol. As IFNy is a powerful macrophage activator, it is likely that its effect is mediated by the production of TNFa by activated macrophages. This hypothesis is strengthened by the observation that recombinant TNFa also inhibits HBV gene expression and replication in these mice [13], as does interleukin (IL)-& and that the ability of IL-2 to suppress HBV RNA levels in the liver is mediated by TNFcx [14] via a post-transcriptional mechanism that accelerates the degradation of cytoplasmic HBV mRNA [15]. Similarly, the CTL-dependent downregulation of viral RNAs occurs at the post-transcriptional level [16*].
Even considering the temporally extended dynamics of the CTL response it is difficult to imagine that a single CTL, or even a succession of CTLs maintained at a constant and high frequency, could find, recognize and destroy more than 1000 infected hepatocytes, one at a time. Furthermore, consider that if only 10% of the hepatocytes were infected, the effector: target cell ratio would still be less than 1: 100 and the CTL would repeatedly have to survey many uninfected cells before finding a target. Another argument against this scenario is the fact that most infected patients clear the virus in a few weeks while experiencing only mild to moderate liver disease. Since most of the hepatocytes in the liver are usually infected by HBV, one would expect the incidence of severe or fatal acute hepatitis to be much higher than
were due entirely
to the destruction
479
of
For all of these reasons, we suggest that although the liver disease in viral hepatitis is certainly due to the destructive potential of the CTL response, viral clearance probably requires additional CTL functions in addition to their ability to kill infected cells.
CTLs can inhibit HBV gene expression and replication without killing the hepatocyte We have recently examined the cytopathic and curative antiviral properties of the CTL response in transgenic mice constructed to replicate HBV in the liver at high levels without any evidence of cytopathology [S]. Following adoptive transfer of CDB+, MHC class I restricted, hepatitis B surface antigen (HBsAg)-specific CTL lines and clones, however, the mice develop a necroinflammatory liver disease resembling acute viral hepatitis [9,10]. Interestingly, the CTL clones cannot induce a second episode of hepatitis if they are readministered to the mice within 3-4 weeks of the first CTL injection [ 111.
These results suggest that a strong intrahepatic immune response to HBV can suppress viral gene expression and replication and perhaps even ‘cure’ infected hepatocytes without killing them. Additionally, the results illustrate that the infected cells can become active participants in the antiviral response by responding to cytokine-induced signals and activating specific intracellular pathways that interrupt the viral life cycle. Indeed, we have identified two independent antiviral events that occur in the CTL-activated hepatocytes: one degrades the viral nucle-
480
Immunity to infection
ocapsids and their cargo of replicative and the other post-transcriptionally RNA. This is a significant which views the infected viral infection.
DNA intermediates, degrades the viral
departure from current dogma, cell merely as a victim of the
Additionally, the data suggest that a weak immune response or incomplete viral inactivation could contribute to viral persistence and chronic liver disease by reducing the expression of viral antigens just enough for some of the infected cells to escape immune recognition. If this scenario is accurate, the influence of CTL-derived cytokines on HBV gene expression and replication could be viewed either as a survival strategy by the virus, contributing to persistence, or as a tissue-sparing antiviral strategy by the host, contributing to viral elimination, depending on the strength and cytokine profile of the immune response.
HBV gene expression and replication are abolished during an unrelated infection of the liver, even in the complete absence of liver disease One might predict from the foregoing evidence that coinfection or superinfection of the HBV-infected liver by other pathogens could facilitate HBV clearance if they induce the local production of antiviral cytokines such as IFNy or TNFa to which HBV is susceptible. This prediction would be consistent with isolated case reports that chronic HBV infection resolved during an intermittent infection of the liver by hepatitis A or hepatitis C [17,18]. To investigate this hypothesis, we infected adult and newborn HBV-transgenic mice with lymphocytic choriomeningitis virus (LCMV) which, like HBV, is known to infect the liver and cause an inflammatory liver disease that is initiated by virus-specific CTLs [4*,19]. In experimentally infected immunocompetent adult mice LCMV is cleared by a MHC class I restricted, CD8+ CTL response [20,21] that is absent when newborn mice are infected [22,23]. In newborns, the lack of a CTL response results in a lifelong persistent noncytopathic infection of many organs, including the liver [24,25]. In these studies we demonstrated that within two days of LCMV infection of adult HBV-transgenic mice virtually all of the intrahepatic macrophages but less than 1% of the hepatocytes were infected by LCMV. At the same time TNFa and IFNc# were induced in the liver and hepatic HBV replication was noncytopathically abolished. It appears, therefore, that LCMV activates the production of TNFcl and IFNcx//P in the liver by the infected macrophages and that these cytokines activate the hepatocytes to reduce their burden of HBV While all these events occur before the appearance of an inflammatory infiltrate in the liver, this antiviral effect is amplified by the subsequent LCMV-induced cellular immune response in acutely infected animals due to the production of increased amounts of TNFcx and the late induction of
IFNy by the LCMV-specific inflammatory cells that they
T cells and the non-specific activate [26*].
Importantly, HBV gene expression and replication were totally and persistently abolished, without any accompanying liver disease, when the HBV-transgenic mice were persistently infected by LCMV and their intrahepatic macrophages expressed high levels of TNFa and IFNo$ [26’]. This demonstrates, for the first time, that activated hepatic macrophages can persistently abolish HBV gene expression and replication from the hepatocytes in the absence of an inflammatory liver disease, strongly suggesting that this cell type, as well as HBV-specific CTLs, plays a key role in the control of HBV infection. Indeed, hepatic macrophages are likely to be activated during HBV infection because they are characteristically hypertrophic and hyperplastic during human viral hepatitis [27] as well as in mammalian [28] and avian [29] models of hepadnavirus infection. Rapid clearance of Woodchuck hepatitis virus (WHV) is also the rule after experimental infection of immunocompetent adult animals even when 100% of the hepatocytes are infected. Viral clearance occurs in the absence of massive destruction or regeneration of hepatocytes and in the presence of inflammatory cells and Kupffer cell hyperplasia [27], suggesting that cytokine-dependent pathways like those described in the transgenic mouse model may also play an important role in viral clearance during WHV infection. Collectively, these results suggest, that during acute HBV infection in man, viral clearance is probably achieved by the cooperation of at least two mechanisms, as illustrated in Fig. 1. First, HBV-specific CTLs recognize and kill a small fraction of infected hepatocytes. Second, they secrete inflammatory cytokines that directly or indirectly (via macrophage activation) potentially cure most of the infected hepatocytes by the intracellular inactivation pathways described above.
What about other viral infections? The current observations raise the possibility that cytokine-activated pathways may be operative in other viral infections. Indeed, Martz and Howell have previously introduced the concept of cytokine-dependent ‘intracellular inactivation’ for viruses such as the herpes simplex virus [30]. If cytokines do play an effector role in the antiviral immune response, each virus can be expected to display its own individual cytokine sensitivity profile, and some viruses (including different subtypes of the same virus) may simply be insensitive to cytokine-mediated control. For example, the ability of LCMV to establish a persistent infection in aiuo in the presence of concentrations of TNFa and IFNo@ that abolished HBV replication correlates with the relative resistance of this virus to control by these cytokines. Additionally, perforin-deficient mice are unable to clear certain LCMV strains [2,3], suggesting that the specific destruction of infected cells by CTLs is required for viral clearance, although these experiments
Host defense
Figure
against
viral infection
Guidotti and Chisari
481
1
Mechanisms
of viral clearance
from
the hepatocyte. (a) Upon antigen activation, CTLs deliver an apoptotic signal mediated by both Fas ligand (FasL) and perforin to their target hepatocytes (Hc), killing them. (b) They also secrete IFNy and TNFcq cytokines that have been shown to abolish HBV gene expression and viral replication in viva, infection. TNFa the intrahepatic
curing the
can also be produced by macrophages (Kupffer
cells, KC) following activation by IFNy. The curative effect of the CTL response is much more efficient than its destructive effect (1 O-l OO-fold more cells are cured
Antigen recognition
& Hc
-
Perforin
than destroyed). The outcome of an infection may depend on the relative balance of these two effects, with a predominantly curative response leading to viral clearance, and a predominantly destructive response leading to viral persistence and chronic liver disease. +, infected with virus; -, not infected with virus.
Destruction
Inactivation of virus
of cell
0 1996 Current Oprlon in lmmunolog~
do not rule out the possibility that a threshold of cell destruction is needed to induce an effective concentration of antiviral cytokines in an infected organ. The fact that some strains of LCMV replicate uncontrollably in mice that lack IFNy receptors [31], or that have been treated with IFNy-specific antibodies [32] might suggest that this cytokine either directly participates in the control of LCMV replication or contributes to viral clearance by inducing an effective cytolytic T cell response. In support of the former hypothesis, it has been shown previously that LCMV infection in mice can be cleared, without evidence of cytopathology [33], before the onset of detectable cytolytic activity and by extremely small numbers of adoptively transferred CTLs [34]. Thus, the hypothesis that T-cell-derived cytokines may control viral infections noncytopathically was already formulated by Lehmann-Grube and co-workers more than ten years ago [34]. In addition to this indirect evidence in the LChlV model, control of viral replication if/ cie,o by IFNa/P, IFNy and TNFcl has also been suggested for several other viral infections, including vaccinia virus [35,36,37’], measles virus [38*], herpes simplex virus (HSV) [39,40*], influenza virus [41*], Semliki Forest virus (SFV) [31,42’] and Theiler’s virus [43’]. Furthermore, a variety of cellular antiviral proteins are induced in response to IFN activation, some of which have been shown to impair influenza and vesicular stomatitis virus transcription i/l ah-o [44,45] while others lead to cleavage of single-stranded picornaviral RNA [46]. Additionally, TNFcx and/or TNFP have been reported to inhibit the replication of HSV, adenovirus and vesicular stomatitis virus ia oh-o [47,48], and HIV replication has
been shown to be inhibited in vitro by non-cytolytic factors produced by HIV-specific class I restricted CTLs upon antigen recognition [49]. Although the precise nature of the antiviral factors produced by the CDS+ T cells is a matter of some conjecture at present [50]. Finally, a number of viruses encode proteins that block the transcriptional activation of the IFN-activatable genes [51,52], downregulate TNFa production in vitro [53], or represent receptor analogues for IFNc@, IFNy and TNFa [54-57,58*], apparently as a strategy to blunt the antiviral activity of these cytokines. However, these viral functions may be more important for protecting the virus against cytokines produced by the infected cells rather than cytokines produced by antiviral CTLs.
Conclusion Viral clearance might depend heavily on noncytopathic curative mechanisms, especially in massive infections of vital organs. If this is correct, the dual destructive/curative process could explain the co-evolution of cytopathic and noncytopathic antiviral functions in the effector limb of the immune response and could influence the mutational threshold required for cytokine-sensitive viruses to escape immunological control. It could also explain the spectrum of outcomes observed during certain noncytopathic viral infections in different individuals, with dominance of the curative process leading to viral clearance and recovery, and dominance of the destructive pathway leading either to low-grade chronic inflammation and viral persistence, or massive tissue destruction and the death of the host. hluch more attention to the curative potential of antiviral cytokines produced by antigen-specific and antigen-non-specific responses to viral infections, as well
482
Immunity
to infection
as the relative susceptibility of individual viruses to control by these cytokines, is certainly warranted.
Acknowledgements We thank Bonnie W&r for help with manuscript preparation. This work was supported by grants ROIAI20001 and R37CA40489 from the National Institutes of Health. This is manuscript number 10086-MEM from the Scripps Research Institute.
References
and recommended
1 7.
Davis GL, Hoofnagle JH, Waggoner JG: Acute type A hepatitis during chronic hepatitis B virus infection: association of depressed hepatitis B virus replication with appearance of endogenous alpha interferon. J Med Viral 1984, 14:141-l 47.
18.
Sheen I-S, Liaw Y-F, Lin D-Y, Chu C-M: Role of hepatitis C and delta viruses in the termination of chronic hepatitis B surface antigen carrier state: a multivariate analysis in a longitudinal follow-up study. J Infect Dis 1994, 170:1358-l 361.
19.
Lohler J, Gossman J, Kratzberg T, Lehmann-Grube F: Murine hepatitis caused by lymphocytic choriomeningitis virus. I. The hepatic lesions. Lab Invest 1994, 70~263-277.
reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
. ..
Tsui, LV, Guidotti LG, lshikawa T, Chisari FV: Post-transcriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyteactivated hepetocytes. Proc Nat/ Acad Sci USA 1995, 92:12396-l 2402. HBV-specific CTLs noncyiopathically downregulate most HBV RNAs in the hepatocyte nucleus by a cytokine-dependent post-transcriptional mechanism that involves recognition of virus-specific sequences contained in the longest viral transcripts. These results suggest that noncytopathic cytokine-dependent clearance of a virus may occur only if the virus genome (or its products) carries appropriate responsive elements. 16. .
of special interest of outstanding interest
1.
Lukacher AE, Braciale VL, Braciale TJ: In viva effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific. I Exp Med 1984, 160:814-826.
2.
Kagi D, Ledermann B, Burki K, Seiler P, Odermatt B, Olsen J, Podack ER, Zinkemagel R, Hengartner H: Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 1994, 369:31-37.
20.
Oldstone MBA, Blount P, Southern PJ, Lampert PW: Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system. Nature 1986, 3211239-243.
3.
Walsh CM, Matloubian M, Liu C, Ueda R, Kurahara CG, Christensen JL, Huang MTF, Young JD-E, Ahmed R, Clark WR: Immune function in mice lacking the perforin gene. Proc Nat/ Acad Sci USA 1994, 91 :I 0854-I 0858.
21.
Ahmed R, Jamieson BD, Porter DD: Immune therapy of a persistent and disseminated viral infection. J Viral 1987, 61:3920-3929.
22.
Fung-Leung W-P, Kunding TM, Zinkernagel RM, Mak TW: Immune response against lymphocytic choriomeningitis virus infection in mice without CD6 expression. J fxp Med 1991, 174:1425-1429.
23.
Moskophidis D, Cobbold SP, Waldmann H, Lehmann-Grube F: Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response. J Viral 1987, 61 :1867-l 874.
4. Chisari FV, Ferrari C: Hepatitis 6 virus immunopathogenesis. . Annu Rev lmmunoll995, 13:29-60. A comprehensive review of HBV immunobiology and pathogenesis in humans and transgenic mice. The immunological basis for viral clearance and viral persistence is critically discussed. 5.
Razvi ES, Jiang Z, Woda BA, Welsh RM: Lymphocyte apoptosis during the silencing of the immune response to acute viral infections in normal, Ipr and Bcl-2 mice. Am J Pathol 1995, 147:79-91.
6.
Yoo JY, Howard R, Waggoner JG, Hoofnagle JH: Peroxidase-anti-peroxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis. J Med Viral 1987, 23:273-281.
24.
Pircher H, Bijrki K, Lang R, Hengartner H, Zinkemagel RM: Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 1969, 342:559-561.
7.
Rehermann B, Lau D, Hoofnagle JH, Chisari FV: Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection. J C/in invest 1996, 97:1655-l 665.
25.
King CC, Jamieson BD, Reddy K, Bali N, Conception RJ, Ahmed R: Viral infection of the thymus. J Viral 1992, 66:3155-3160.
0.
Guidotti LG, Matzke B, Schaller H, Chisari FV: High level hepatitis B virus replication in transgenic mice. J Viral 1995, 69:6158-6169.
9.
Ando K, Guidotti LG, Wirth S, lshikawa T, Missale G, Moriyama T, Schreiber RD, Schlicht HJ, Huang S, Chisari FV: Class I restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in viva. J lmmunol 1994, 152:3245-3253.
10.
Moriyama T, Guilhot S, Klopchin K, Moss 8, Pinked CA, Palmiter RD, Brinster RL, Kanagawa 0, Chisari FV: lmmunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 1990, 248:361-364.
11.
Wirth S, Guidotti LG, Ando K, Schlicht HJ, Chisari FV: Breaking tolerance leads to autoantibody production but not autoimmune liver disease in HBV envelope transgenic mice. J lmmunol 1995, 154:2504-2515.
12. ..
Guidotti LG, lshikawa 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. The first direct evidence that physiologic concentrations of antiviral cytokines induced by antigen-specific CTLs can noncytopathically abolish viral gene expression and replication. 13.
14.
15.
Gilles PN, Fey G, Chisari FV: Tumor necrosis factor-alpha negatively regulates hepatitis B virus gene expression in transgenic mice. J Viral 1992, 66:3955-3960. Guidotti LG, Guilhot S, Chisari FV: Interleukin-2 and interferon alpha/beta downregulate hepatitis B virus gene expression in viva by tumor necrosis factor dependent and independent pathways. J Viral 1994, 68:1265-l 270. Guilhot S, Guidotti LG, Chisari RI: Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mica by a post-transcriptional mechanism. J Viral 1993, 67:7444-7449.
26. .
Guidotti LG, Borrow P, Hobbs MV, Matzke B, Gresser I, Oldstone MBA, Chisari FV: Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Nat/ Acad Sci USA 1996, 93:4569-4594. HBV replication in the hepatocytes of HBV-transgenic mice is noncytopathically abolished by cytokines (particularly TNFa and IFNaIP) produced by LCMV-infected macrophages in the absence of an LCMV-specific T-cell response. Evidence is provided of an important role for cytokines produced by macrophages in viral clearance, and that LCMV itself is resistant to the antiviral cytokines it induces even though they readily control HBV replication. 27.
Uchida T, Kronborg I, Peters RL: Acute viral hepatitis: morphological and functional correlations in human livers. Hum Pathol 1984, 15:267-277.
28.
Kajino K, Jilbert AR, Saputelli J, Aldrich C, Cullen J, Mason WS: Woodchuck heDatitis virus infections: verv raoid recoverv after a prolonged viiemia and infection of virt&lly’every hepetocyte. J Viral 1994, 68:5792-5803.
29.
Jilbert AR, Wu T-T, England JM, De La Hall PM, Carp NZ, O’Connell AP, Mason WS: Rapid resolution of duck hepetitis B virus infections occurs after massive hepatocellular involvement. J Viral 1992, 66:1377-l 388.
30.
Martz E, Howell DM: CTL: virus control cells first and cytolytic cells second? lmmunol Today 1989, 10:79-86.
31.
Mijller U, Steinhoff U, Reis LFL, Hemmi S, Pavlovic J, Zinkernagel RM, Aguet M: Functional role of type I and type II interferons in antiviral defense. Science 1994, 264:1916-l 921.
32.
Moskophidis D, Battegay M, Bruendler M-A, Laine E, Gresser I, Zinkemagel R: Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and gamma interferon. J Viral 1994, 68:1951-i 955.
33.
Gegin C, Lehmann-Grube F: Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present
Host defense
an immunodominant viral cytotoxic T lymphocyte lmmunol1992,149:3331-3338.
epitope. I
34.
Lehmann-Grube F, Assmann U, Loliger C, Moskophidis D, Lohler J: Mechanism of recovery from acute virus infection. I. Role of T lymphocytes in the clearance of lymphocytic choriomeningitis virus from spleens of mice. J lmmunol 1985, 134:608-615.
35.
Sambhi SK, Kohonen-Corish MRJ, Ramshaw IA: Local production of tumor necrosis factor encoded by recombinant vaccinia virus is effective in controlling viral replication in ho. Proc Nat/ Acad Sci USA 1991, 88:4025-4029.
36.
Kohonen-Corish MRJ, King NJC, Woodhams CE, Ramshaw IA: lmmunodeficient mice recover from infection with vaccinia virus expressing interferon-y. Eur J lmmunol 1990, 20:157-l 61.
Van Den Broek MF, Mijller U, Huang S, Aguet M, Zinkernagel RM: Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors. J Viral 1995, 69:4792-4796. LCMV and vaccinia virus replicate much faster in mice that lack both IFN u/p and y receptors. Finke D, Brinckmann UG, Ter Meulen V, Liebert UG: Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis. J Viral 1995, 695469-5474. Measles virus is not cleared from the central nervous system in mice depleted of IFNy. 39.
Rossol-Voth R, Ross01 S, Schijtt KH, Corridori S, De Cian W, Falke D: In viva protective effect of tumor necrosis factor a against experimental infection with herpes simplex virus type I. J Gen Viral 1991, 72:143-147.
Bouley DM, Kanangat S, Wire W, Rouse BT: Characterization of herpes simplex virus type-l infection and herpetic stromal keratitis development in IFN-y knockout mice. J lmmunol 1995, 165:3964-3971. IFNy is required to clear herpes simplex virus from the eye.
Topham DJ, Tripp RA, Sarawar SR, Sangster MY, Doherty PC: Immune CD4+ T cells oromote the clearance of influenza virus from major histocompatibility complex class II -I- respiratory epithelium. J Viral 1996, 70:1288-i 291. CD4+ effector T cells can eliminate influenza virus in respiratory epithelial cells that MHC lack class II expression. The involvement of cytokines and/or antibody is discussed. Hwang SY, Hertzog PJ, Holland KA, Sumarsono SH, Tymms MJ, Hamilton JA, Whiny G, Bertoncello I, Kola I: A null mutation in the gene encoding a type I interferon receptor component eliminates anti-proliferative responses to interferons a and p and alters macrophage responses. Proc Nat/ Acad Sci USA 1995, 92:11284-l 1288. Semliki Forest virus replicates uncontrollably in several organs of mice that lack a component of the IFN a@ receptor.
42. .
Fiette L, Aubert C, Miller U, Huang S, Aguet M, Brahic M, Bureau J-F: Theiler’s virus infection of 129Sv mice that lack the interferon a/p or interferon y receptors. J Exp Med 1995, 181:2069-2076. IFN a@ and y play a non-redundant role in the control of Theiler’s virus infection. 43. .
Guidotti and Chisari
483
Horisberger M, Staeheli P, Hailer 0: Interferon induces unique protein in mouse cells bearing a gene for resistance to influenza virus. Proc Nat/ Acad Sci USA 1983, 60:191 O-l 914.
45.
Staeheli P, Pavlovic J: Inhibition of vesicular stomatiiis virus mRNA synthesis by human MxA protein. J Viral 1991, 66:4498-4501.
46.
Kumar R, Choubey D, Lengyel P, Sen GC: Studies on the role of 2’-6’-oligoadenylate synthetase-RNase L pathway in beta interferon-mediated inhibition of encephalomyocarditis virus replication. J Viral 1988, 62:3175-3181.
47.
Mestan J, Digel W, Mittnacht S, Hillen H, Blohm D, Moller A, Jacobsen H, Kirchner H: Antiviral effect of recombinant tumor necrosis factor in vitro. Nature 1986, 323:816-819.
48.
Wong GHW, Goeddel DV: Tumor necrosis factor a and p inhibit virus replication and synergize with interferons. Nafure 1986, 323:819-822.
49.
Walker CM, Erickson AL, Hsueh FC, Levy JA: Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD6+ cells involves a non-cytotoxic mechanism. J Virol 1991, 65:5921-5927.
50.
Baiter M: Elusive HIV-suppressor 270:1560-1561.
51.
Foster GR, Ackrill AM, Goldin RD, Kerr IM, Thomas HC, Stark GR: Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA. Proc Nat/ Acad Sci USA 1991,88:2888-2892.
52.
Kalvakolanu DVR, Bandyopadhyay SK, Harter ML, Sen GC: Inhibition of interferon-inducible gene expression by adenovirus EIA proteins; block in transcriptional complex formation. froc Nat/ Acad Sci USA 1991, 88:7459-7463.
53.
Adler H, Jungi TW, Pfister H, Strasser M, Sileghem M, Peterhans E: Cytokine regulation by virus infection: bovine viral diarrhea virus, a flavivirus, downregulates production of tumor necrosis factor alpha in macrophages in vitro. J Viral 1996, 70:2650-2653.
54.
Symons JA, Alcami A, Smith GL: Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 1995, 81:551-560.
55.
Upton C, Mossman K, McFadden G: Encoding a homolog of the interferon-y receptor by myxoma virus. Science 1992, 268:1369-i 372.
56.
Alcami A, Smith GL: Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity. J Viral 1995, 69:4633-4639.
57.
Schreiber M, McFadden G: The myxoma virus TNF-receptor homologue (T2) inhibits tumor necrosis factor-a in a speciesspecific fashion. Virology 1994, 204:692-705.
40. .
41. .
viral infection
44.
37. .
38. .
against
factors found. Science
1995,
McFadden G, Graham K, Ellison K, Barry M, Macen J, Schreiber M, Mossman K, Nash P, Lalani A, Everett H: Interruption of cytokine networks by poxviruses: lessons from myxoma virus. J Leukoc Biol 1995, 571731-738. A very detailed review describing the virokines and viroreceptors produced during myxoma virus infection and their interactions with the cytokine network. 58. .