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Cutting Edge |
*
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037;
Fourth Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan; and
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-galactosylceramide inhibit hepatitis B virus
replication noncytopathically in the liver of transgenic mice. This
effect is mediated by antiviral cytokines directly produced by
activated NK T cells and/or by other cytokine-producing inflammatory
cells that are recruited into the liver. In this study, we demonstrated
that IFN-
produced by activated NK T cells induced parenchymal and
nonparenchymal cells of the liver to produce high levels of CXC
chemokine ligands 9 and 10, which mediated the intrahepatic
recruitment of lymphomononuclear inflammatory cells. Recruitment of
these cells was not necessary for the antiviral activity, indicating
that direct activation of the intrahepatic resident NK T cell is
sufficient to control viral replication in this
model. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-galactosylceramide
(-GalCer) (1), a glycolipid Ag presented to
V14+NK1.1+ T cells by
the nonclassical MHC class I-like molecule CD1d (2).
Within 24 h of -GalCer injection, antiviral cytokines such as
IFN- are detected in the liver of these animals and HBV replication
is abolished (1). This is associated with the recruitment
of activated NK cells and T cells into the organ and the rapid
disappearance of NK T cells from the liver, presumably reflecting their
activation (1). Based on these results, it is possible
that -GalCer inhibits HBV replication by directly activating NK T
cells to produce antiviral cytokines. Alternatively, antiviral
cytokines could be produced in the liver by recruited inflammatory
cells.
The recruitment of inflammatory cells into the liver following
injection of -GalCer is likely to be regulated by NK T
cell-derived cytokines (i.e., IFN-) that induce the intrahepatic
production of chemokines. At this time, the chemokines that are induced
in the liver following NK T cell activation have not been defined. CXC
chemokine ligand (CXCL)9 (monokine induced by IFN-) and CXCL10
(chemokine responsive to -2/IFN- inducible protein) are known to
be induced by IFN- (3, 4), to bind the same chemokine
receptor (CXCR3) (5), and chemoattract lymphocytes and
monocyte/macrophages (3, 4). Both chemokines are produced
by macrophages and recent studies have also shown that these same
chemokines can be produced by primary hepatocyte cultures
(6) or by hepatocytes in vivo (27). CXCL9 and
CXCL10 are induced in the mouse liver during vaccinia virus infection
(7) and they accelerate viral clearance and the associated
hepatitis (8). CXCL9 also contributes to protection
against hepatic mouse cytomegalovirus infection (9).
Collectively, the foregoing results suggest that CXCL9 and CXCL10 might
play a role in the recruitment of inflammatory cells into the liver
following NK T cell activation by -GalCer. It is also possible that
these chemokines could mediate the antiviral effects of activated NK T
cells. To test these hypotheses, we performed a series of experiments
aimed at monitoring the following: 1) the ability of -GalCer to
induce CXCL9 and CXCL10 in the liver; 2) the source, kinetics, and
regulation of CXCL9 and CXCL10 expression; 3) the ability of -GalCer
to recruit inflammatory cells into the liver; and 4) the ability of
chemokine-specific neutralizing Abs to block the proinflammatory and
antiviral effects of activated NK T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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HBV-transgenic mouse lineage 1.3.32 used in this study have been previously described (10). In all experiments, the mice were matched for age (8 wk), sex, and hepatitis B e Ag (HBeAg) levels in their serum before experimental manipulations.
Reagents
-GalCer was kindly provided by Dr. Y. Koezuka (Kirin Brewery,
Gumma, Japan) and prepared as previously described (1).
Hamster mAbs H22 specific for murine IFN- was generously provided by
Dr. R. Schreiber (Washington University, St. Louis, MO)
(11). The control hamster IgG was purchased from Jackson
ImmunoResearch Laboratories(West Grove, PA). Rabbit polyclonal Abs to
mouse CXCL9 or CXCL10 and preimmune normal rabbit serum (NRS) have been
previously described (12, 13).
Tissue DNA and RNA analysis
Total DNA and RNA were isolated from frozen livers and analyzed for HBV DNA by Southern blot, and for chemokine, chemokine receptor, cytokine, T cell and macrophage marker RNAs by RNase protection assay (RPA) exactly as previously described (10, 14, 15). The relative abundance of specific DNA and RNA molecules was determined by phosphor-imaging analysis, using Optiquant image analysis software (Packard, Meriden, CT).
In situ hybridization
This procedure was conducted exactly as described (10). For CXCL10, a 726 bp cDNA fragment was synthesized and cloned in pGEM4 (Promega, Madison, WI) as described previously (14). For CXCL9, a 302-bp cDNA fragment spanning nucleotides 101–402 of the murine CXCL9 gene (GenBank accession no. M34815) was synthesized by RT-PCR and after sequence verification cloned in pGEM4. The 33P-labeled RNA probes used for in situ hybridization were generated by T7-driven transcription of 1 µg of linearized plasmids containing CXCL9- or CXCL10-specific sequences.
Histological analysis
For histological analysis, liver was fixed in 10% zinc-buffered formalin (Anatech, Battle Creek, MI), embedded in paraffin, sectioned (3 µm), and stained with H&E as described (1).
Isolation and analysis of intrahepatic leukocytes (IHLs)
IHLs were isolated from two liver lobes and analyzed by flow cytometry. Single-cell suspensions were prepared and analysis of the IHL population was performed by flow cytometry, exactly as described (1). The cells were surface stained with FITC- or PE-labeled anti-CD3, CD4, CD8, DX5, NK1.1, CD19, Gr-1, CD11b, or CD11c Abs (BD PharMingen, San Diego, CA). Samples were acquired on a FACSCalibur flow cytometer, and the data were analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-GalCer injection
We have previously reported that hepatic NK T cell activation by
-GalCer inhibits HBV replication in the liver of transgenic mice
(1). This occurs within 24 h and is associated with
the intrahepatic induction of antiviral cytokines such as IFN-
(1). To determine whether the IFN- inducible chemokines
CXCL9 and CXCL10 along with their common receptor CXCR3 were induced in
the liver of transgenic mice injected with -GalCer, we monitored the
expression of CXCL9, CXCL10, and CXCR3 by RPA. HBV-transgenic mice
(three mice per group) were i.v. injected with 10 ng of -GalCer and
sacrificed at the indicated time points (Fig. 1
). Total hepatic RNA was extracted and
analyzed and the results were compared with those observed in livers
from transgenic littermates injected with saline (NaCl) that were
sacrificed on day 1 after NaCl injection (Fig. 1).
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for two representative mice per group, the
intrahepatic RNAs for CXCL9 and CXCL10 were strongly induced over 1000-
and 200-fold, respectively, when compared by phosphor imaging analysis
with preinjected levels by day 1 after -GalCer injection. CXCL9 and
CXCL10 transcripts decreased thereafter returning to baseline levels by
days 14 and 5, respectively (Fig. 1). The kinetics of CXCL9 and
CXCL10 induction coincided with the induction of IFN- and the
inhibition of HBV replication (Fig. 1) as also previously reported
(1). CXCR3 RNA increased in these livers as well, albeit
with delayed kinetics, reaching its peak by day 3 and returning toward
baseline levels by day 7–14. This is consistent with our previous
observation that by day 3 after -GalCer injection, lymphomononuclear
cells are maximally recruited into the liver and small, widely
scattered necroinflammatory foci are detectable in the organ
(1). In keeping with this, the signal for T cell (CD8,
CD4, and CD3) and macrophage (F4-80) RNAs were maximal by day 3 (Fig. 1). Altogether, these results demonstrate that the IFN- inducible
chemokines CXCL9 and CXCL10 are induced in the liver after NK T cell
activation due to -GalCer injection and they also suggest that these
chemokines may be involved in the recruitment of
CXCR3+ inflammatory cells into the liver.
To determine which cells produce CXCL9 and CXCL10, the hepatic content
of both chemokine RNAs was analyzed by in situ hybridization analysis
of livers derived from HBV-transgenic animals sacrificed at day 1 after
-GalCer injection. CXCL9 and CXCL10 were detected not only in
nonparenchymal cells (Kupffer cells and infiltrating inflammatory
cells) but, even more abundantly, in parenchymal (hepatocytes) of the
liver (not shown). This indicates that the hepatocytes represent a
major source of CXCL9 and CXCL10 RNAs in the -GalCer-injected livers
and it is consistent with previous experiments that showed expression
of CXCL9 and CXCL10 by the hepatocyte following the transfer of
HBV-CTLs into HBV-transgenic mice (27) and by primary
hepatocyte cultures derived from mice treated with IL-2/IL-12
(6).
IFN- mediates the antiviral activity of -GalCer and the
induction of CXCL9 and CXCL10 in the liver
To define the role of IFN- in the -GalCer-dependent
antiviral activity and in the intrahepatic induction of CXCL9 and
CXCL10, we monitored the ability of hamster neutralizing Abs specific
for IFN- (IFN-) to modulate these processes. Two groups (three
mice per group) of HBV-transgenic mice were injected once i.p. with 250
µg of either IFN- or control hamster Ig 16 h before
-GalCer. Mice were sacrificed 48 h later and their livers were
harvested for HBV DNA analysis by Southern blot and IFN-, CXCL9, and
CXCL10 RNA analysis by RPA. The results were compared with those
observed in livers pooled from 10 age-, sex-, and serum HBeAg-matched
transgenic mice littermates injected with saline (NaCl).
As expected, HBV replication was suppressed in the mice that received
-GalCer and control Ig and this coincided with the induction of
IFN-, CXCL9, and CXCL10 RNAs in the liver (Fig. 2, left). In contrast, HBV
replication was still strongly detected in the livers of transgenic
mice that were treated with -GalCer and IFN- and the induction
of CXCL9 and CXCL10 was almost completely blocked (Fig. 2, left). These results confirm previous experiments that
showed that IFN- mediates most of the antiviral activity of
-GalCer (1) and they also indicate that IFN-
mediates the -GalCer-dependent induction of CXCL9 and CXCL10 in the
liver. The fact that the intrahepatic levels of HBV replication in
IFN--treated mice were slightly lower than those observed in
saline-injected controls (Fig. 2, left) suggests the
existence of a marginal antiviral activity of -GalCer which is
independent of IFN-. This is consistent with the notion that
IFN-
can also be induced by -GalCer (1) and
trigger IFN--independent antiviral pathways in this model (15, 16).
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-GalCer
To define the role of CXCL9 and CXCL10 in the -GalCer-induced
antiviral activity, we monitored the ability of rabbit neutralizing Abs
specific for CXCL9 and CXCL10 to modulate this process. Two groups
(three mice per group) of HBV-transgenic mice were injected i.p. with 1
ml of either a mixture of CXCL9 and CXCL10 neutralizing rabbit Ig
(CXCL9 and CXCL10) or NRS twice, first 16 h before and then
simultaneously with -GalCer injection (10 ng/mouse). Mice were
sacrificed and livers were harvested 48 h after -GalCer
injection and total hepatic DNA and RNA were analyzed as described
above.
HBV replication was abolished in mice that received -GalCer plus NRS
as well as in mice that received -GalCer plus Abs specific for CXCL9
and CXCL10, and this coincided with similar inductions of IFN-,
CXCL9, and CXCL10 RNAs in the liver in both groups of animals (Fig. 2, right). These results indicate that CXCL9 and CXCL10 do not
directly inhibit HBV replication themselves nor do they mediate the
antiviral potential of -GalCer in our system.
IFN-, CXCL9, and CXCL10 contribute to the recruitment of
inflammatory cells into the liver
To determine the characteristics of intrahepatic inflammatory
infiltrate in the same livers described in Fig. 2, the absolute number
of IHLs recovered was quantitated and the phenotype of the recruited
inflammatory cell subsets was determined by FACS analysis.
When compared with NaCl-injected controls, the total number of IHLs
significantly increased in the liver of -GalCer-treated mice that
received control Abs (Fig. 3A). The total number of IHLs
increased 7- to 8-fold at 48 h after -GalCer injection.
IFN- or anti-chemokine treatments reduced the number of total
IHLs recruited by 
2-fold (Fig. 3A). The most striking
decrease was observed for
NK1.1+CD3- cells (Fig. 3B), which increased 20-fold in control mice and
IFN- or anti-chemokine treatments reduced their number by
5-fold (Fig. 3B). CD8+ cells
(mostly CTLs; Fig. 3C) and CD4+ cells (mostly Th
cells) increased 4- to 7- and 3-fold in control mice, and IFN-
or anti-chemokine treatments reduced their numbers by 2-fold
(Fig. 3, C and D).
CD11b+/CD11c+ cells (mostly myeloid dendritic
cells) and CD11b+/CD11c- cells (mostly
macrophages) were also induced in control animals (30- and 7-fold,
respectively; Fig. 3, E and F) and IFN- or
anti-chemokine treatments reduced their number by 3- and 2-fold,
respectively (Fig. 3, E and F).
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induced by NK T cell
activation recruits different subsets of lymphomononuclear inflammatory
cells into the liver and that most of this activity is mediated by the
hepatic induction of CXCL9 and CXCL10. The recruitment of cell subsets
(NK1.1+CD3- cells,
CD8+ cells, and
CD11b+/CD11c+ cells) that
are known to express CXCR3 (3, 17, 18) was inhibited by
both IFN- and anti-chemokine treatments. The recruitment of
CD4+ T cells was also reduced by IFN- but
to a much lesser extent by the anti-chemokine treatment. Because
CD4+ T cells are known to express CXCR3
(19, 20), these results may suggest that other
chemoattractant factors inducible by IFN- play a more important role
in the recruitment of these cell subsets into the liver. Similar
reasons may explain the partially reduced recruitment of cells such as
macrophages, which are not known to express CXCR3 (Fig. 3F).
Despite a much higher number of recruited inflammatory cells, the
intrahepatic levels of IFN- RNA in control mice were similar to
those observed in mice treated with anti-chemokine Abs (Fig. 2, right). This indicates that the recruited inflammatory cells
do not contribute to the intrahepatic production of IFN- and, thus,
it is likely that most of the IFN- produced in the liver at this
time point is the result of the direct activation of NK T cells by
-GalCer. Because IFN- mediates the
-GalCer-dependent inhibition of HBV replication (Fig. 2), these results suggest that the recruitment of inflammatory cells is
not necessary to control HBV replication after NK T cell
activation.
In conclusion, we showed in this study that IFN-
secreted by -GalCer-activated NK T cells rapidly and strongly
induces nonparenchymal and parenchymal cells of the liver to produce
CXCL9 and CXCL10. These chemokines recruit different cell subsets into
the organ, particularly those that express CXCR3. Nonetheless, neither
these chemokines nor the cells that they recruit mediate the
IFN--dependent antiviral activity of NK T cells, suggesting that the
NK T cells themselves inhibit HBV replication in this system by
secreting IFN-. It is known that activated NK T cells can recruit
and activate NK in an-IFN--dependent manner (21, 22, 23),
raising the possibility that NK cells are actually responsible for most
of the antiviral activity of NK T cells (24). Our results
suggest that NK T cells themselves can play a direct antiviral role if
they are sufficiently activated. Because it is apparent that the
percentage of resident intrahepatic NK T cells is much higher in mice
(30%) (25) than humans (4%) (26), the
extent to which naturally or artificially activated NK T cells could
contribute to the control of viral replication in humans remains to be
determined.
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Acknowledgments |
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-GalCer; Robert
Schreiber for providing anti-IFN- Abs; Ian Campbell, Valerie
Asensio, and Monte Hobbs for providing the chemokine and cytokine gene
probes used in the RPA and in situ hybridization experiments; and Heike
Mendez, Rick Koch, and Margie Chadwell for excellent technical
assistance. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Luca G. Guidotti, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: guidotti{at}scripps.edu
3 Abbreviations used in this paper: HBV, hepatitis B virus; -GalCer, -galactosylceramide; HBeAg, hepatitis B e Ag; NRS, preimmune normal rabbit serum; RPA, RNase protection assay; IHL, intrahepatic leukocyte; IFN-, hamster neutralizing Abs specific for IFN-; CXCL, CXC chemokine ligand.
Received for publication September 18, 2001. Accepted for publication October 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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14 NKT cells by glycosylceramides. Science 278:1626. (Mig) and chemokine responsive to -2 (Crg-2). J. Immunol. 166:3763. with patterns of tissue expression that suggest nonredundant roles in vivo. J. Immunol. 157:4511.[Abstract]
-interferon which differentially modulate macrophage activation and antiviral activity. J. Immunol. 134:1609.[Abstract]
) is important in T lymphocyte recruitment and host defense following viral infection of the central nervous system. J. Immunol. 166:1790. and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of -galactosylceramide. Eur. J. Immunol. 31:1720.[Medline]
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