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Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40). We showed that a single i.v. injection of
CD40 was sufficient to inhibit HBV replication noncytopathically by
a process associated with the recruitment of dendritic cells,
macrophages, T cells, and NK cells into the liver and the induction of
inflammatory cytokines. The antiviral effect depended on the production
of IL-12 and TNF- by activated APCs; however, it was mediated
primarily by IFN-
produced by NK cells, and possibly T cells, that
were activated by IL-12. Collectively, these results suggest that
activated APCs can directly produce antiviral cytokines (IL-12,
TNF-) and trigger the production of other cytokines (i.e., IFN-)
by other cells (e.g., NK cells and T cells) that do not express CD40.
These results provide insight into a hitherto unsuspected antiviral
function of intrahepatic APCs, and they suggest that therapeutic
activation of APCs may represent a new strategy for the treatment of
chronic HBV infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-. Using this same transgenic mouse model, it was
shown that IL-12 inhibits HBV replication via an IFN--dependent
pathway (3), and similar cytokine-dependent mechanisms are
responsible for the antiviral activity observed after activation of
intrahepatic NK cells and NKT cells (4), or after
infection with unrelated hepatotropic viruses (5, 6), in
which case the antiviral effect is also triggered by IFN-/
, the
antiviral activity of which was confirmed by administration of the
IFN-/ inducer, polyinosinic-polycytidylic acid complex (7, 8). Finally, we have shown that HBV replication is also
controlled noncytopathically in acutely HBV-infected chimpanzees, and
this is temporally associated with the induction of IFN- in the
liver of these animals (9). Like the case of HBV, other
viruses, including adenovirus (10, 11), mouse hepatitis
virus (12, 13), coxsackievirus (14, 15), and
measles virus (13), are susceptible to the antiviral
activity of cytokines produced by CTLs or other immune cells, and it
has long been known that IFN-/ inhibits noncytopathically the
replication of these and other viruses, including retroviruses,
influenza viruses, vesicular stomatitis virus, HSV, vaccinia virus, and
reovirus (16, 17). While it is widely accepted that cytokines produced by NK, NKT, and T cells play an indispensable role in the control of viral infections, very little is known about the ability of APC-derived cytokines to contribute to this process. This is germane because APCs can become activated and produce antiviral cytokines after appropriate stimulation (18, 19, 20), suggesting that activated APCs might also participate in the control of viral infections by similar mechanisms.
Very large numbers of APCs are present in the liver (21, 22), and they can be activated by the administration of an
agonistic anti-CD40 mAb (CD40) (23, 24). CD40 is a
member of the TNF receptor family, found on professional APCs (B cells,
dendritic cells (DCs), and activated macrophages) and other cell types
(fibroblasts, epithelial cells, and endothelial cells)
(25, 26, 27). CD40 activation is a critical step in the final
maturation of DCs into fully competent APCs and is required for the
generation of CTLs (28). CD40 ligation on monocytes and
DCs enhances their survival and their ability to secrete TNF-,
IL-1-, IL-1-, IL-6, IL-8, IL-10, IL-12, IL-18, and IFN- and to
produce NO (18, 20, 26, 29, 30).
In the current study we took advantage of our HBV transgenic mouse
model to determine whether systemic administration of CD40 could
activate intrahepatic APCs to produce antiviral cytokines and inhibit
HBV replication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HBV transgenic mouse lineages 1.3.32 and 1.3.46 used in this
study have been previously described (31). Both lineages
of mice replicate HBV at high levels in the liver and kidney without
any evidence of cytopathology. Lineage 1.3.32 was expanded by
repetitive backcrossing (>20 generations) against the C57BL/6 parental
strain and then bred one generation against BALB/c mice to produce the
F1 hybrids used in this study. Transgenic mice
from lineage 1.3.46 were backcrossed against mice genetically deficient
for the IFN-/R-/- (32)
exactly as previously described (7). The knockout mice
were provided by M. Aguet (IFN-/R-/-;
Genentech, South San Francisco, CA). In all experiments the mice were
matched for age (8 wk), sex (male), and serum hepatitis B e Ag (HBeAg)
levels before experimental manipulations. All animals were housed in
pathogen-free rooms under strict barrier conditions.
Anti-CD40 and anti-cytokine Abs
The FGK45 hybridoma producing rat IgG2a mAb against mouse CD40
(CD40) was provided by Dr. A. Rolink (Basel Institute for
Immunology, Basel, Switzerland) (33). CD40 was purified
from FGK45 culture supernatants by affinity chromatography using a
protein G column and was tested for LPS levels with the
Limulus amebocyte lysate test (<0.16 endotoxin
units/ml) using a commercially available kit (Sigma-Aldrich, St.
Louis, MO). The mice were injected i.v. either with 100 µg of CD40
or 100 µg of purified rat IgG2a (BD PharMingen, San Diego, CA)
control Ab. The mice were sacrificed at different time points after
injection, their livers were perfused via the inferior vena cava with
10 ml of PBS (Invitrogen, Carlsbad, CA), and they were harvested for
histological, histochemical, and FACS analyses or were snap-frozen in
liquid nitrogen and stored at -80°C for subsequent molecular
analyses (see below).
Twenty-four hours before CD40 injection, mice were injected i.p.
(250 µg/mouse) with 1) hamster mAb H22 specific for murine IFN-
(34), 2) hamster mAb TN3 19.12 specific for murine TNF-
(35) (both of which were provided by Dr. R. Schreiber
(Washington University, St. Louis, MO), 3) control hamster IgG (Jackson
ImmunoResearch, West Grove, PA), 4) goat polyclonal Ab specific for
murine IL-12 (36) (provided by Dr. M. Gately, Hoffmann-La
Roche, Nutley, NJ), or 5) control goat IgG (Sigma-Aldrich).
In vivo depletion of macrophages, DCs, CD4+ T cells, CD8+ T cells, and NK cells
To deplete macrophages and DCs in the liver, mice were injected
i.v.y (100 µl/mouse) with liposome-encapsulated dichloromethylene
diphosphonate (37, 38) (L-MDP; provided by Dr. M. Naito,
Niigata University School of Medicine, Niigata, Japan) 24 h before
CD40 injection. Liposome-encapsulated PBS was used as a negative
control (37, 38). To deplete CD4+
and CD8+ T cells, mice were injected i.v. (2
mg/mouse) with rat anti-mouse CD4 (YTS191.1) and rat anti-mouse
CD8 (YTS169.4) mAb (39) (provided by Dr. R. Zinkernagel,
University of Zurich, Zurich, Switzerland). YTS191.1 or YTS169.4 was
injected twice, 3 and 1 days before CD40 injection. Purified rat
IgG2b (BD PharMingen) was used as a negative control. To deplete NK
cells in the liver, mice were injected i.v. with either rabbit
anti-mouse asialoGM1 Ab (50 µg/mouse; Wako Pure Chemical, Osaka,
Japan) or anti-mouse NK1.1 Ab (200 µg/mouse; BD
PharMingen) 24 h before CD40 injection. Purified rabbit IgG
(Sigma-Aldrich) was used as a negative control.
Tissue DNA and RNA analyses
Frozen liver was mechanically pulverized under liquid nitrogen, and total genomic DNA and RNA were isolated for Southern and Northern blot analyses for HBV DNA and 2',5'-oligoadenylate synthetase (2',5'-OAS), respectively, exactly as previously described (31). Quantification of various cytokine and chemokine RNAs was performed by RNase protection assay (RPA) exactly as previously described (1, 40, 41).
Biochemical and histological analyses
The extent of hepatocellular injury was monitored histologically and biochemically by measuring serum alanine aminotransferase (sALT) activity at multiple time points after infection. Serum ALT activity was measured in a Paramax chemical analyzer (Baxter Diagnostics, McGaw Park, IL) exactly as previously described. For histological analysis, liver tissue was fixed in 10% zinc-buffered formalin, embedded in paraffin, sectioned (3 µm), and stained with H&E.
Intrahepatic leukocyte preparation
Single-cell suspensions were prepared from liver that was perfused with PBS via the inferior vena cava and pressed through a 70-µm cell strainer (BD Biosciences, Mountain View, CA). Total liver cells were digested with 10 ml of RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 0.02% (w/v) collagenase IV (Sigma-Aldrich) and 0.002% (w/v) DNase I (Sigma-Aldrich) for 40 min at 37°C. Cells were washed with RPMI 1640 and then underlaid with 24% (w/v) metrizamide (Sigma-Aldrich) in PBS. After centrifugation for 20 min at 1500 x g, intrahepatic leukocytes (IHLs) were isolated at the interface.
Flow cytometry
Single-cell suspensions of IHLs were washed in PBS (containing 1% BSA and 0.02% sodium azide), and incubated for 20 min on ice with culture supernatant from the hybridoma cell line 2.4G2 (American Type Culture Collection, Manassas, VA) to block FcR. The cells were surface-stained with fluorochrome-conjugated mAb for 20 min on ice. The following Abs were used: anti-CD3, anti-CD4, anti-CD8, anti-NK1.1, anti-DX-5, anti-CD11b, anti-CD11c, anti-B220, anti-Gr-1, anti-CD40, and anti-CD86 (all from BD PharMingen). Samples were acquired on a FACSCalibur flow cytometer, and the data were analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA).
Detection of intracellular cytokines
IHLs were harvested from HBV transgenic mice at the indicated
times after injection of CD40, and they were cultured ex vivo for
4 h in brefeldin A (BD PharMingen) to allow the intracellular
cytokines to be sequestered in the Golgi apparatus. Cells were then
surface-stained with anti-CD3-FITC, anti-NK1.1-PE,
anti-CD11b-FITC, and anti-CD11c-PE mAb; washed in FACS buffer
(PBS with 1% FCS); and fixed in 2% paraformaldehyde for 30 min at
room temperature. After fixation, cells were permeabilized for 30 min
in 25 µl of PBS plus 0.5% saponin. Anti-mouse IFN-- and
TNF--allophycocyanin or isotype control-allophycocyanin mAb were
added at a final dilution of 1/100, and cells were incubated for 30 min
at room temperature. Cells were washed and resuspended in 1 ml of FACS
buffer for analysis on a FACScan flow cytometer as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40 inhibits HBV replication
To determine whether CD40 injection can inhibit HBV replication
in the liver of transgenic mice and to relate this effect to the
kinetics of cytokine and chemokine expression and liver disease, eight
groups (three mice per group) of age-, sex-, and serum HBeAg-matched
transgenic mice from lineage 1.3.32 received a single i.v. injection of
CD40 (100 µg/mouse). Mice were bled and sacrificed, and livers
were harvested at 4, 8, and 12 h and 1, 3, 5, 7, and 14 days after
injection. The results were compared with those observed in livers from
age-, sex-, and serum HBeAg-matched transgenic littermates (three mice)
injected with control rat IgG2a (rat IgG) and sacrificed 1 day after
injection.
As shown in the Southern blot at the top of Fig. 1
for two representative mice per group,
HBV replicative intermediates disappeared from the liver between 12 and
24 h after injection of CD40, remained virtually undetectable
for at least 7 days, and returned to baseline by day 14 (Fig. 1). As
shown by RPA in the lower portion of Fig. 1, CD40
injection induced sequential activation of various cytokines and
chemokines in an orderly, hierarchical, and biphasic manner. The first
wave of genes to be induced included TNF-, IL-12p40, and the
chemokine CXCL10 (chemokine responsive to -2/IFN--inducible
protein), whose mRNA levels were induced in the liver as early as
4 h after CD40 injection, reached peak levels by 12 h, and
slowly subsided thereafter until a second peak was observed on day 5,
which rapidly decreased to the baseline level on day 7 (Fig. 1). The
second wave of responsive genes included 2',5'-OAS (a well-known
IFN-/-inducible gene) and IFN-, whose mRNA were first
detectable by 8 h, peaked by 12 h, and followed the same
biphasic time course as that described above (Fig. 1). Interestingly,
the IL-12 p40 and p35 mRNAs were not coordinated, since p40 gene
expression was maximal by 12 h, and p35 gene expression was
maximal on day 5 (Fig. 1). CXCL9 (monokine induced by IFN-), whose
mRNA was also first induced at 8 h, peaked by 12 h, declined,
and reached a transient second peak on day 5. The chemoattractant for
monocytes/macrophages CCL5 (RANTES) was induced by 8–12 h and remained
constantly elevated for 
5–7 days (Fig. 1).
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CD40
inhibits HBV replication in the liver of transgenic mice, and this
effect is preceded by the intrahepatic induction of cytokines (i.e.,
IFN-, TNF-, IFN-/, and IL-12) known to have antiviral
activity against HBV (1, 3, 7) and by the intrahepatic
induction of chemokines (i.e., CXCL9 and CXCL10) that have no direct
antiviral activity against HBV, but mediate the recruitment of
lymphomononuclear cells into the liver (42).
The severity of liver disease after CD40 injection was measured
biochemically and histologically. Serum ALT (an enzyme that is released
into the circulation by injured hepatocytes) activity was only mildly
elevated at 12 and 24 h and on day 5, similar to the biphasic
pattern of cytokine activation in these animals (Fig. 1
). In keeping
with the early induction of chemokines (Fig. 1), histological analysis
revealed widely scattered inflammatory foci in the liver parenchyma
containing mostly lymphomononuclear cells and a few apoptotic
hepatocytes at 12 and 24 h after CD40 injection (Fig. 2, A and B). Given
that HBV replication was completely abolished within 24 h and the
fact that the liver disease was very mild at this time point, these
results indicate that CD40 inhibits HBV replication
noncytopathically. On day 5 the inflammatory infiltrate (predominantly
lymphomononuclear cells) increased considerably in both portal tracts
as well as parenchyma (Fig. 2, C and D), and
there was increased cytological evidence of hepatocellular injury,
reflecting the higher levels of sALT activity detected at this time
point (Fig. 1).
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CD40 recruits inflammatory cells into the liverTo determine the characteristics of the intrahepatic inflammatory cell infiltrate in the same livers, we quantified the absolute number of IHLs recovered, and we determined the phenotype of the recruited inflammatory cell subsets by FACS analysis.
The IHLs detected in control transgenic mice (three mice) that had been
injected with rat IgG and sacrificed 24 h later served as a
baseline for this experiment. Those results are represented as the day
0 point (Fig. 3).
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CD40
injection and gradually rose thereafter until it had increased 6-fold
above baseline by day 5, after which it rapidly decreased (Fig. 3, top panel), corresponding well with the histological
analysis shown in Fig. 2. Based on these results, the absolute number
of each IHL subset at each time point was calculated by multiplying the
total number of IHLs by the frequency of the subset determined by FACS
analysis (Fig. 3). The
CD11b+/CD11c- macrophage
population was the first to increase, rising as early as 4 h after
CD40 injection and reaching a peak at 20-fold induction by day 1,
where it remained until day 5. The next populations to change included
the NKT cells
(CD3+/NK1.1+), which
decreased by 12 h, and the NK cells
(CD3-/NK1.1+), which
increased by 12 h, with the latter remaining elevated for 5 days,
and the former remaining depressed for the duration of the experiment.
CD3+/NK1.1- cells (T
cells) also increased by 12 h after injection, reached peak levels
on day 5, and returned to preinjection levels by day 7 (Fig. 3). While
CD11b-/CD11c+ DCs (mostly
lymphoid dendritic cells) showed a modest increase by days 3–5,
CD11b+/CD11c+ DCs (mostly
myeloid dendritic cells) were more strongly induced by 24 h,
peaked by day 5, and returned to baseline levels by day 7 (Fig. 3). The
proportion of B cells
(B220+/CD3-) in the liver
decreased slightly by 12 h and returned to the baseline by day 7
(data not shown). These results demonstrated that macrophages expand
first in the liver following CD40 injection, followed by increase in
the NK and T cell populations, the disappearance of NKT cells, and,
finally, a slower, but progressive, accumulation of both myeloid and
lymphoid DCs.
The antiviral activity of CD40 requires APCs and is independent
of T cells
To evaluate the role of APCs in the CD40-induced inhibition of
HBV replication, macrophages and DCs were depleted from the liver of
age- (8–10 wk), sex- (male), and serum HBeAg-matched transgenic mice
by L-MDP, which induces apoptosis of macrophages and DCs in vivo and in
vitro (37, 43). One day later the mice were injected i.v.
with CD40 or rat IgG, and they were sacrificed 24 h later. The
results were compared with those observed in the liver of transgenic
littermates injected with liposome-encapsulated PBS (L-PBS) prior to
the injection of either CD40 or rat IgG.
As shown in Fig. 4, CD40 inhibited HBV
replication in the liver of L-PBS-treated mice, but not in
L-MDP-treated mice, indicating that intrahepatic APCs are required for
the CD40-induced antiviral effect. Compared with that in
L-PBS-treated mice, the induction of TNF-, IFN-/, and, to a
lesser extent, IFN- was diminished in L-MDP-treated mice (Fig. 4),
again suggesting that these cytokines are likely to play a role in the
CD40-dependent inhibition of HBV replication. Furthermore, no
elevation of sALT was observed in the L-MDP-treated mice (Fig. 4),
indicating that APCs are necessary for the initial cytopathic effect of
CD40. The induction of CCL5 and CXCL10 was also diminished in the
APC-depleted animals (Fig. 4), suggesting that less recruitment of
inflammatory cells should occur in these livers. Consistent with this,
CD40-induced expansion of all the intrahepatic inflammatory cell
subsets was blocked in L-MDP-treated mice (Fig. 4, bottom).
The lack of CCL5 expression in these mice also confirms the efficacy of
the APC depletion, since it has been previously shown that the hepatic
expression of this chemokine is dramatically reduced in L-MDP-treated
mice following LPS injection (44). Surprisingly, the
expression of CXCL9 was not particularly affected by APC depletion
(Fig. 4), thus suggesting that this chemokine may not contribute
substantially to the recruitment of inflammatory cells in this
system.
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CD40 requires the
activation of conventional T cells, age- (8–10 wk), sex- (male), and
serum HBeAg-matched transgenic mice (three mice per group) from lineage
1.3.32 were treated with rat anti-mouse CD4 and CD8 mAb, injected
i.v. with CD40 or control rat IgG, and sacrificed 24 h later.
Importantly, the total number of CD4 and CD8 cells that were
recoverable from the liver of the mAb-treated animals decreased 44- and
61-fold, respectively, compared with controls (not shown). Nonetheless,
CD40 treatment inhibited HBV replication in the liver on day 1
regardless of the presence or the absence of T cells (Fig. 5), suggesting that intrahepatic
CD4+ and CD8+ T cell
populations did not contribute to the antiviral activity of CD40 at
this time point. Importantly, TNF-, IFN-, and IFN-/ were
induced to a similar degree in the liver of both groups of animals
(Fig. 5). This suggests that either the remaining undepleted
intrahepatic T cells, representing 1.5–2.5% of the original T cell
population, are responsible for the production of these antiviral
cytokines on day 1 after CD40 injection or that other cells, e.g.,
NK cells, were responsible for this effect. The same is true for the
mild elevation of sALT activity, since similar values were detected in
the blood of both groups of animals (Fig. 5). Consistent with the idea
that CD40 directly activates APCs, and the activation products of
the activated APCs trigger the recruitment and activation of other
cells in the liver, T cell depletion did not affect the recruitment of
NK cells, macrophages, and DCs into the liver by CD40 (Fig. 5, bottom). This indicates that T cells are not necessary for
the recruitment of other inflammatory cells after CD40 injection.
This interpretation is supported by the fact that the chemokines CCL5,
CXCL10, and CXCL9 were similarly induced in animals in which T cells
were either depleted or not (Fig. 5).
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CD40, because treatment of the mice with
anti-mouse asialoGM1 or anti-mouse NK1.1 Ab that reduced the
intrahepatic NK cell population 10-fold also triggered the
production of IFN- and TNF-, which inhibited HBV replication
(data not shown).
Intrahepatic APCs are activated and produce high levels of TNF-
after CD40 injection
To determine which cells produced IFN- and TNF- after
CD40 injection, we stained the intrahepatic macrophage
(CD11b+/CD11c-), DC
(CD11b+/CD11c+,
CD11b-/CD11c+), NK cell
(CD3-/NK1.1+), and T cell
(CD3+/NK1.1-) subsets with
Abs to IFN- and TNF- 12 h after CD40 injection. As shown
in Fig. 6, macrophage and DC subsets
expressed high levels of TNF- (Fig. 6), but little or no IFN-
(data not shown). In contrast, IFN- was found to be produced at high
levels by NK cells and at lower levels by T cells (Fig. 6) at this time
point, even though these cells do not express CD40 (25).
Collectively, these results suggest that cross-linking of CD40 directly
triggers the production of TNF- by APCs and, indirectly, IFN- by
NK cells and T cells. Thus, activated APCs may directly and/or
indirectly contribute to the inhibition of HBV replication via the
production of these antiviral cytokines.
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CD40 is mediated by IFN-, TNF-,
and IL-12
To determine whether the CD40-dependent inhibition of HBV
replication is mediated by IFN- or TNF-, we monitored the ability
of hamster mAb specific for either cytokine to modulate this process.
Groups (three mice per group) of age-, sex-, and serum HBeAg-matched
transgenic mice (lineage 1.3.32) were injected with either IFN-
or TNF- mAb or irrelevant hamster IgG prior the injection of
CD40 and were sacrificed 24 h later. The results were compared
with those observed in livers from age-, sex-, and serum HBeAg-matched
transgenic littermates (three mice) injected with an irrelevant
hamster IgG.
As shown in Fig. 7, administration of
either IFN- or TNF- mAb completely blocked the antiviral
effect of CD40 injection, suggesting that both these cytokines must
be induced for CD40 to inhibit HBV replication. Neutralization of
these cytokines reduced their own intrahepatic induction as well as the
induction of 2',5'-OAS mRNA, which is primarily induced by IFN-/
(Fig. 7). In addition, both treatments blocked the cytopathic effect of
CD40 (see the sALT activity values; Fig. 7), suggesting that they
function cooperatively in this regard as well. Consistent with the
observation, both Abs effectively diminished the induction of CCL5,
CXCL10, and CXCL9 (Fig. 7) as well as the recruitment of NK cells,
macrophages, and DCs into the liver (data not shown).
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CD40 injection (Fig. 1), experiments were performed to determine the
relative contribution of this cytokine to the antiviral effect CD40.
Groups (three mice per group) of age-, sex-, and serum HBeAg-matched
transgenic mice (lineage 1.3.32) were injected with IL-12 polyclonal
Ab (36) and sacrificed 24 h later. The results were
compared with those observed in livers from age-, sex-, and serum
HBeAg-matched transgenic littermates (three mice) injected with
polyclonal goat IgG control Ab.
As shown in Fig. 8, the administration of
IL-12 polyclonal Ab completely blocked the antiviral effect of
CD40, indicating that the IL-12 pathway plays an important role in
this model. The CD40-induced liver injury along with the induction
of 2',5'-OAS, IFN-, TNF-, CCL5, CXCL10, and CXCL9 were diminished
in these animals, indicating that these effects require IL-12 as well.
In keeping with the diminished cytokine and chemokine expression, the
administration of IL-12 also blocked cell recruitment of
inflammatory cells into the liver (data not shown).
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/ plays a role in this system, either
CD40 or rat IgG was injected into groups (three mice per group) of
age- (8–10 wk), sex- (male), and serum HBeAg-matched animals from
lineage 1.3.46 that were heterozygous
(IFN-/R+/-) or homozygous
(IFN-/R-/-) for the IFN-/R null
mutation (7). The animals were sacrificed 24 h
later.
HBV replication was inhibited in IFN-/R+/-
and IFN-/R-/- mice that were injected
with CD40. Indeed, the content of HBV replicative forms was
similarly reduced in both groups of mice (as measured by phosphor
imaging analysis using the transgene band for normalization; not shown)
compared with the respective rat IgG-injected controls (note that
IFN-/R-/- mice replicate HBV at higher
levels than IFN-/R+/- mice)
(7). As expected, no CD40-dependent induction of
2',5'-OAS was observed in the IFN-/R-/-
animals, although IFN- and TNF- were induced in these mice at
control levels (data not shown). These results indicate that
IFN-/-mediated signaling is not necessary for CD40 to exert
its antiviral effect. Given that blocking IFN-, TNF-, and IL-12
strongly suppressed 2',5'-OAS induction (Figs. 7 and 8), the results do
not rule out the possibility that IFN-/ may contribute to the
antiviral activity of CD40. Indeed, there is very strong evidence
from other experiments that HBV replication is strongly inhibited by
IFN-/ (7, 8 .
Rather, the current results suggest that the induction of IFN-/
by CD40 is dependent on IFN-, TNF-, and IL-12, and these
cytokines can inhibit HBV replication in the absence of
IFN-/-mediated signaling. The CD40-induced liver injury was
not inhibited in IFN-/R-/- mice, and the
transcripts of IFN-, TNF-, CCL5, CXCL10, and CXCL9 were well
induced (data not shown), indicating that these effects do not require
IFN-/-mediated signaling. Consistent with these results, there
was no difference between IFN-/R+/- and
IFN-/R-/- mice with regard to the
CD40-induced cell recruitment of NK cells, macrophages, and DCs in
liver (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40 into
transgenic mice that replicate HBV at high levels in their hepatocytes
rapidly and profoundly inhibited viral replication. This effect was
associated with the recruitment of APCs, T cells, and NK cells into the
liver, the intrahepatic induction of various cytokines and chemokines,
and mild inflammatory liver disease. Follow-up experiments were
performed to determine the relative contributions of these events to
the antiviral activity of CD40.
The results indicate that the antiviral effect of CD40 is dependent
on APCs, since depletion of these cells abrogated the antiviral
activity of CD40. Given that CD40 has the potential to activate
other CD40-bearing cells (fibroblasts, epithelial cells, and
endothelial cells) (25, 26, 27), the results suggest that
those cells did not contribute to the antiviral effect of CD40.
Depletion of APCs also abrogated the intrahepatic recruitment of
inflammatory cells, most of the cytokine and chemokine induction, and
the liver disease, suggesting that all these events are downstream of
APC activation. It remains to be determined whether the CD40-induced
antiviral activity depends on activation of the resident intrahepatic
APC population or recruitment and activation of additional APCs into
the liver. It is noteworthy that macrophages were recruited in high
numbers into the liver as early as 4 h following CD40
injection, while intrahepatic DCs increased with delayed kinetics. This
suggests that if there is an antiviral role for the recruited APCs, it
would most likely involve the macrophages. Future experiments will
attempt to monitor the antiviral effect of CD40 in animals in which
the recruitment of APCs into the liver will be inhibited by the
administration of anti-CXCL9 and anti-CXCL10 Abs, a treatment
that we have previously shown to inhibit the intrahepatic recruitment
of APCs after the transfer of virus-specific CTLs (42) or
the activation of intrahepatic NKT cells in these mice
(46). Resident and/or recruited activated macrophages are
also likely to be involved in the antiviral activity against HBV that
is observed in these animals during the blood stage of malaria
infection (47). Under these conditions, the resident
macrophages of the liver are initially activated by phagocytosis of
infected erythrocytes, and this is associated with the intrahepatic
recruitment of macrophages, NK cells, NKT cells, and T cells, all of
which are likely to produce the inflammatory cytokines that eliminate
HBV from the hepatocyte (47).
Importantly, APCs isolated from the liver of CD40-injected animals
were found to be activated and produce high levels of TNF-, but
little or no IFN-, suggesting that it was produced by other cells.
The importance of TNF-, IFN-, and IL-12 in the antiviral activity
of CD40 was underscored by experiments showing that the inhibition
of HBV replication was blocked by Ab neutralization of these cytokines.
These results are reminiscent of several studies that previously
defined the antiviral activity of TNF-, IFN-, and IL-12 in this
model (1, 3, 7). Furthermore, the results obtained in
IFN-/R-/- mice do not exclude the
possibility that IFN-/ may contribute to the antiviral activity
of CD40, although its induction was found to be dependent on
IFN-, TNF-, and IL-12 (Figs. 7 and 8). This idea is supported by
previous studies showing that HBV replication can be abolished in
transgenic mice in response to IFN-/ that is produced in the
liver during lymphocytic choriomeningitis virus (5, 7) and
adenovirus (6, 7) infections or after
polyinosinic-polycytidylic acid complex injection (7, 8)
as long as they express the IFN-/ receptor.
While APCs may represent the major source of TNF- in this system,
IFN- was found to be mostly produced by NK cells and to a much
lesser extent by T cells 12 h after CD40 injection. Lack of an
important role for T cells either in the early cytokine-dependent
antiviral activity of CD40 or in the accompanying inflammatory
processes was indicated by the experiment in which T cells were
depleted before CD40 injection. In this setting HBV replication was
abolished, and both IFN- and several IFN--dependent chemokines
(CXCL9 and CXCL10) were fully induced, again suggesting that non-T
cells, especially NK cells, were the most abundant source of this
cytokine. It is unfortunate that we could not directly prove that NK
cells contribute to the antiviral effect of CD40, but our attempts
to deplete NK cells with anti-mouse asialo-GM1 or anti-mouse
NK1.1 resulted in the inhibition of HBV replication, probably because
the Ab treatment not only killed NK cells, but also activated them to
produce IFN- in the liver. Future experiments using HBV transgenic
mice genetically deficient for NK cells will be required to prove that
NK cells play a central role in this system. Finally, the high levels
of IL-12 mRNA detected in the liver of CD40-injected animals
together with the results of the IL-12 neutralization experiments
demonstrated a critical role for this cytokine as a mediator of the
CD40-induced antiviral effect. Future studies will be necessary to
identify the cell(s) that produces this cytokine in our system, but we
expect that these studies will confirm published reports that
macrophages and DCs produce IL-12 following activation by CD40
(20, 48). Collectively, these results suggest that
activated APCs can directly produce antiviral cytokines (IL-12,
TNF-) and trigger the production of other cytokines (i.e., IFN-)
by other cells (e.g., NK cells and T cells) that do not express CD40
(25).
Injection of CD40 was accompanied by a very mild liver disease,
particularly at the time points that just preceded (12 h) or were
concomitant with (24 h) the disappearance of HBV replicative
intermediates from the liver and the first peak of intrahepatic
cytokine and chemokine expression. These results indicate that the
antiviral activity of CD40 relies primarily on noncytopathic
mechanisms. A more severe liver disease was observed in these animals
on day 5, when the influx of T cells, NK cells, and myeloid DCs into
the liver was maximal and when we observed a rebound in cytokine and
chemokine expression. It must be noted, however, that even at this time
point the overall severity of liver disease as well as the elevation of
sALT activity were quite modest compared with those in previously
published models of liver cell injury (1, 49). We should
note that we do not understand the reason for the transient and delayed
second peak of liver inflammation, but the inflammatory rebound may
have contributed to the prolonged duration of the antiviral effect of a
single injection of CD40.
In conclusion, these results demonstrate that activation of
intrahepatic APCs, especially macrophages, can initiate a cascade of
events that begins with the production of IL-12 and TNF-, followed
by activation of intrahepatic NK cells and probably NKT cells and T
cells to produce IFN-, and subsequent induction of IFN-/, all
of which contribute to the inhibition of HBV replication in the
hepatocyte and the recruitment of additional inflammatory cells into
the liver. The cost of this process is a mild inflammatory liver
disease that is probably induced by the same mediators. Nonetheless,
the disease is very modest, in contrast to the major impact of these
events on viral replication. Thus, pharmacological activation of the
resident intrahepatic macrophage population may represent a novel
therapeutic approach for the treatment of chronic HBV infection.
|
Acknowledgments |
|---|
and TNF- mAb; Dr.
Maurice Gately (Hoffmann-La Roche) for providing anti-mouse IL-12
Ab; Drs. Monte Hobbs and Iain Campbell (The Scripps Research Institute)
for providing the probe sets used in the RPAs; and Alana Altage, Rick
Koch, Amber Morris, Heike Mendez, and Margie Chadwell for excellent
technical assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Francis V. Chisari, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: fchisari{at}scripps.edu
3 Abbreviations used in this paper: HBV, hepatitis B virus; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; DC, dendritic cell; HBeAg, hepatitis B e Ag; IHL, intrahepatic leukocyte; L-MDP, liposome-encapsulated dichloromethylene diphosphonate; L-PBS, liposome-encapsulated PBS; 2',5'-OAS, 2',5'-oligoadenylate synthetase; RPA, RNase protection assay; sALT, serum alanine aminotransferase.
Received for publication May 20, 2002. Accepted for publication September 11, 2002.
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