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* First Department of Oral and Maxillofacial Surgery,
Department of Oral Pathology, and
Clinical Laboratory, Kanagawa Dental College, Kanagawa, Japan;
Department of Medicine, Kitasato Institute Hospital, Tokyo, Japan; and
¶ Institute of Molecular Biotechnology of the Austrian Academy of Sciences and Department of Medical Biophysics and Immunology, Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1% of people infected with hepatitis
viruses develop acute fulminant hepatitis with no effective treatment
option (1). Moreover, the liver is a target for
autoreactive lymphocytes resulting in autoimmune hepatitis that very
often require transplants (2). In both viral and
autoimmune hepatitis, activation of macrophages and T cells are
critical initial steps in the pathogenesis of liver damage and
fulminant disease (3, 4).
In mice, fulminant T cell-dependent hepatitis and acute liver damage
can be modeled using in vivo administration of ConA. ConA is a T cell
mitogenic lectin that induces polyclonal T cell activation in vitro and
causes severe and acute liver injury in mice that is dependent on the
presence of T lymphocytes (5). Following ConA
administration, T cells and macrophages release high levels of
cytokines such as IL-2, IL-1, IL-6, IFN-
, IL-4, IL-12, or TNF-
as
well as nitric oxide that have been linked to the development of
hepatic lesions (6, 7, 8, 9, 10, 11, 12, 13). In addition, cytotoxicity
mediated via Fas ligand
(FasL)4 and perforin
have been implicated in ConA-induced hepatitis (14, 15, 16).
FasL- and perforin-mediated cytotoxicity is dependent on the attachment
to target cells by the effector cells (17, 18). Moreover,
IFN- and TNF- can induce the expression of ICAM-1 on rat
parenchymal hepatocytes and LFA-1-ICAM-1 interactions play an important
role in the interaction between hepatocytes and lymphocytes
(19). Moreover, it has been shown that Abs to LFA-1 or
ICAM-1 can inhibit T cell cytotoxicity to hepatocytes
(14, 15, 16), suggesting that cell adhesion between activated
T cells and liver cells might be an important initial step in liver
damage.
The leukocyte integrin LFA-1 was initially defined by mAbs that inhibit
CTL-mediated cytotoxicity in the absence of complement
(20). However, LFA-1-deficient mice display
normal CTL responses against systemic choriomeningitis virus and
vesicular stomatitis virus infections (21) and loss
of LFA-1 alone has only minor effects on the cytotoxicity of
NK1.1+TCR
+ T cells
(22), suggesting that the consequences of anti-LFA-1
mAbs may reflect nonspecific effects of Ab treatment, such as capping
of other surface molecules. Thus, the in vivo function of LFA-1 in the
generation of cytotoxic T cell responses was unclear. Moreover, no
adhesion receptor has been genetically identified yet that is indeed
critical for liver damage.
In this study, we investigated the role of the adhesion receptor LFA-1 in ConA-induced hepatitis, using LFA-1-/- gene-deficient mice (21). We show that LFA-1 has an essential role in the ConA-induced hepatitis. The cytotoxic machinery of mutant ConA-activated hepatic T cells appeared functional but the adhesion between activated hepatic T cells and liver cells was impaired in the absence of LFA-1 expression. Importantly, adoptive transfer of hepatic T cells from LFA-1+/+ mice, but not from LFA-1-/- mice, sensitized LFA-1-/- mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutant mice lacking LFA-1 (H-2b) generated by gene-targeting technology have been described previously (21). The mutant mice were backcrossed more than six times to a C57BL/6 (H-2b) background and maintained in specific pathogen-free conditions. As control, age-matched LFA-1+/+ littermate mice were used. All mice used were 8- to 10-wk-old and maintained at the Kanagawa Dental College (Kanagawa, Japan) in accordance with college guidelines. TLR2 and NMuLi cell lines were purchased from the Riken Cell Bank (Tsukuba, Japan).
ConA-triggered disease
ConA (Seikagaku Kogyo, Tokyo, Japan) was dissolved in PBS and i.v. injected to mice via the tail vein in a dose of 20 mg/kg. Serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by a standard photometric method using a Hitachi type 7070 automatic analyzer (Tokyo, Japan). To analyze the in vivo effects of ICAM-1 inhibition in Con A-induced hepatitis, LFA-1+/+ mice were i.v. injected with anti-ICAM-1 mAbs (clone 3E2) at 24 and 2 h before Con A injection.
Histology and TUNEL assay
Paraffin-embedded liver samples were cut into 5-µm sections,
deparaffinized in xylene, and rehydrated through a series of decreasing
concentrations of ethanol. Sections were stained with H&E. For in situ
detection of apoptosis, paraffin-embedded sections were pretreated for
10 min at room temperature with 20 µg/ml proteinase K (DAKO,
Carpinteria, CA). The TUNEL apoptosis assay was an Apo Tag Plus
Peroxidase In Situ Apoptosis Detection kit (Intergen, Purchase, NY).
For detection of ConA-induced apoptosis of hepatic T cells, hepatic T
cells were purified at different time points following ConA injection
using nylon wool. Cell death was detected by TUNEL staining (APO-DIRECT
kit; BD PharMingen, San Diego, CA). For immunohistochemical studies on
liver T cells, 4% paraformaldehyde-fixed and paraffin-embedded
sections were stained using an anti-CD3-
polyclonal Ab (clone
M-20; Santa Cruz Biotechnology, Santa Cruz, CA). The primary Ab was
detected using the Histofine SAB-PO (G) kit (Nichirei, Tokyo,
Japan) according to the manufacturer’s instructions.
Isolation of hepatic NK1.1-TCR+
cells
The liver was pressed through a stainless steel mesh and cells
were suspended in PBS. Total liver cells were resuspended in a 40%
isotonic Percoll solution (Amersham Pharmacia Biotech, Piscataway, NJ)
and underlaid with 80% of isotonic Percoll. Following centrifugation
for 20 min at 2000 x g, mononuclear cells (MNC) were
harvested at the 40–80% interface. The pellet was resuspended in RBC
lysis solution (0.17 M NH4Cl, 0.01 mM EDTA, and
0.1 M Tris-HCl, pH 7.3), washed twice in PBS, and subjected to flow
cytometry and PCR analysis. For the enrichment of hepatic
NK1.1-TCR+ cells,
cells were purified by negative selection using T cell enrichment
columns (R&D Systems, Minneapolis, MN) according to the manufacturer’s
instructions. Purity of the recovered cells was verified by two-color
flow cytometry analysis. The recovered cells contained >95%
NK1.1-TCR+ and
>93% B220-TCR+
cells.
Adoptive transfer of hepatic
NK1.1-TCR+ cells
LFA-1-/- host mice (6 wk of age)
received hepatic
NK1.1-TCR+ cells
(1 x 107 cells) from
LFA-1+/+ or
LFA-1-/- donor mice via tail vein
transfer. One hour after the T cell transfer, the mice were injected
i.v. with ConA (20 mg/kg). Serum AST and ALT levels and histological
changes of liver were determined 12 h after the ConA
injection.
Flow cytometry
Surface phenotypes of cells were identified by using mAbs in
conjugation with two- or three-color immunofluorescence. MNCs were
resuspended in immunofluorescence staining buffer (PBS, 4% FCS, 0.1%
NaN3) and incubated with the appropriate mAbs for
30 min. To block unspecific binding via FcRs, samples were
preincubated with a nonconjugated CD32/16 mAb (clone 2.4G2) for 15 min.
The following anti-mouse mAbs were used: anti-NK1.1
(PE-conjugated, clone NKR-PC1), anti-TCR (FITC-conjugated,
clone H57-597), anti-CD69 (biotin-conjugated, clone H1.2F3),
anti-LFA-1/CD11a (biotin-conjugated, clone M17/4), anti-CD4
(PE-conjugated, clone GK1.5), anti-CD8 (FITC-conjugated, clone
53-6.7), anti-CD25 (biotin-conjugated, clone 7D4), anti-ICAM-1
(FITC-conjugated, clone 3E2), anti-ICAM-2 (FITC-conjugated, clone
3C4), and anti-Fas (PE-conjugated, clone Jo2) (all from BD
Biosciences, Mountain View, CA). Biotin-conjugated mAbs were visualized
using streptavidin CyChrome (BD Biosciences). Cells were analyzed using
a FACScan and CellQuest software (BD
Biosciences).
Hepatic NK1.1-TCR+ cell activation
Hepatic NK1.1-
TCR+ cells (2 x
105) from LFA-1+/+
and LFA-1-/- mice were cultured in the
presence of 4 µg of ConA in 96-well round-bottom plates
(Costar, Cambridge, MA) at 200 µl/well. After incubation for
36 h, 100 µl of culture supernatants were harvested to detect
IL-2 production using the Biotrac mouse IL-2 ELISA system (Amersham
Pharmacia Biotech) and [3H]thymidine at 1
µCi/50 µl medium was added per well and incubated for another
12 h. Proliferation was measured by
[3H]thymidine incorporation using an Aloka type
LSC-5100 liquid scintillation counter (Tokyo, Japan). Serum levels of
IL-4, IFN-, and TNF- were determined in individual mice using
mouse IL-4, IFN-, and TNF- ELISAs (Amersham Pharmacia
Biotech).
Cytotoxicity assays
Cytotoxic activity of hepatic NK1.1-
TCR+ cells was tested against murine NMuLi
liver cells (23) using 51Cr-release
assays. Briefly, target cells were loaded with
51Cr for 1 h, washed, and
51Cr-loaded target cells (1 x
104/well) were plated in triplicate into 96-well
V-bottom plates (Costar). Target cells were incubated with hepatic
NK1.1-TCR+ cells at
the indicated E:T ratios in a total volume of 200 µl of 10% FCS in
RPMI 1640 medium for 4 h. After incubation, the supernatant was
harvested and counted. Specific cytotoxicity was calculated as
described (22). In some experiments, cytotoxicity assays
were performed in the presence of anti-LFA-1 (clone M17/4),
anti-ICAM-1 (clone 3E2), or anti-ICAM-2 (clone
3C4)-blocking mAbs.
Western blotting and RT-PCR
Hepatic
NK1.1-TCR+ cells
(2 x 106 cells) were lysed in 10 mM EDTA,
1% SDS, 60% (v/v) glycerol, and 0.5% bromophenol blue. Proteins were
separated by 10% SDS-PAGE, transferred to polyvinylidene
difluoride membranes (Millipore, Bedford, MA), and membranes
incubated in blocking solution (5% skim milk; 0.1% Tween 20 in PBS)
at room temperature for 1 h to block nonspecific Ab binding.
Membranes were then incubated with a polyclonal anti-FasL Ab (clone
C-178; Santa Cruz Biotechnology) at a dilution of 1/1000 or
anti-perforin mAb (clone CB5.4; Wako Pure Chemical) at a dilution
of 1/1000 at room temperature for 1 h and washed three times in
PBS/0.1% Tween 20. As a control, membranes were probed with a
polyclonal anti-actin Ab (clone I-19; Santa Cruz Biotechnology).
Membranes were then incubated with HRP-conjugated secondary Abs
(Amersham Pharmacia Biotech) and washed three times in PBS/0.1% Tween
20. Immune complexes were detected by ECL (Amersham Pharmacia Biotech)
according to the manufacturer’s protocol. For RT-PCR, total cellular
RNA was extracted using TRIzol (Life Technologies, Grand Island, NY).
Single-strand cDNA was synthesized with reverse transcriptase from 2
µg of RNA and used for PCR. Primer sequences were as follows: IL-4,
5' primer-ATGGGTCTCAACCCCCAGCTAGT and 3'
primer-GCTCTTTAGGCTTTCCAGGAAGTC; IFN-, 5'
primer-GCTCTGAGACAATGAACGCT and 3'
primer-AAAGAGATAATCTGGCTCTGC; TNF-, 5'
primer-GATCTCAAAGACAACCAACTAGTG and 3' primer-CTCCAGCTGGAAGACTCCCAG;
-actin, 5' primer-CGTGACATCAAAGAGAAGCTGGTCC and 3'
primer-GCTCAGGAGGAGCAATGATCTTGAT. Various amounts of cDNA were
amplified in PCR under the following conditions: 94°C for 1 min,
62°C for 1 min, and 72°C for 1 min with 25 cycles. After
amplification, PCR products were separated by electrophoresis on 1.5%
agarose gels.
Conjugate formation assays
Hepatic NK1.1-
TCR+ cells were isolated and prepared as
effector cells. Murine liver NMuLi cells were labeled with PKH2
fluorescence (Zynaxis Cell Science, Malvern, PA) for 10 min at
37°C and washed five times in 10% FCS in RPMI 1640 medium to remove
unbound fluorochromes. Labeled NMuLi cells (1 x
105 cells) and effector cells (2 x
105 cells) were then mixed together in a total
volume of 1 ml of 10% FCS in RPMI 1640 medium and centrifuged for 5
min at 1000 x g. Pellets were incubated for 10 min at
37°C and resuspended in ice-cold PBS to stop conjugate formation.
Binding between NMuLi target and effector cells was determined using
FACScan. Hepatic NK1.1-
TCR+ cells were gated and analyzed for PKH2
expression representing NMuLi cells and effector cells conjugate
formation (22). In some experiments, conjugate formation
assays were performed in the presence of anti-LFA-1,
anti-ICAM-1, or anti-ICAM-2 blocking mAbs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of the LFA-1 adhesion receptor in
ConA-induced hepatitis, we injected ConA into
LFA-1-/- mice, and the induction of
hepatitis and liver damage was evaluated by measuring the activities of
the hepatic enzymes ALT and AST in the serum 3, 6, 12, and 24 h
after ConA injection. Following ConA administration, the activities of
both ALT and AST were rapidly increased in
LFA-1+/+ mice, reaching their peak values
at 12 h (Fig. 1
). In contrast, no
increase in the levels of either ALT or AST was detected in
LFA-1-/- mice (Fig. 1). We then
conducted histological examinations of the liver 3, 6, 18, and 24
h after ConA injection (Fig. 2). In
LFA-1+/+ mice, massive hepatic lesions
were observed starting at 6 h after ConA challenge and the onset
of these lesions correlated with the levels of ALT and AST in the
serum. By contrast and in line with the normal ALT and AST levels,
livers from LFA-1-/- mice appeared
normal in histology even at late time points after ConA injection.
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). In contrast, TUNEL-positive cells
were not detectable in LFA-1-/- mice.
LFA-1-/- mice are healthy even days
after ConA injection, that is, there is not a delayed response. In
ConA-injected LFA-1+/- mice, the extents
of ALT and AST levels and histological liver damage and apoptosis were
comparable to that of LFA-1+/+ littermates
(not shown). As expected, LFA-1+/-
mice also showed fulminant hepatitis as observed in
LFA-1+/+ mice (not shown). It should be
noted that the livers of
LFA-1-/- mice were histologically
identical to that of LFA-1+/- and
LFA-1+/+ mice at baseline. These results
show that loss of LFA-1 expression results in complete protection from
ConA-induced hepatitis and liver damage.
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It has been shown that LFA-1-ICAM-1 interactions play an important
role in the interaction between hepatocytes and lymphocytes and that
Abs to LFA-1 or ICAM-1 can inhibit T cell cytotoxicity to hepatocytes
in vitro (14, 15, 16). Therefore, we analyzed whether
administration of an anti-ICAM-1 blocking mAb in vivo inhibits the
liver injury induced by Con A. However, inhibition of ICAM-1 in
LFA-1+/+ mice did not alter the course of
ConA-induced hepatitis and massive liver damage was still evident as
detected by increased serum ALT and AST levels (Fig. 4A). Moreover, the
histological lesions in the livers were comparable between nontreated
and anti-ICAM-1-treated LFA-1+/+ mice
following ConA challenge (Fig. 4B). These results indicate
that treatment with anti-ICAM-1 mAb does not inhibit Con A-induced
hepatitis.
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+ cells
in LFA-1-/- mice
ConA is a T cell mitogen that induces polyclonal T cell activation
and liver injury (5). The total numbers and proportions of
T cells and B cells in the liver before and after ConA injection were
comparable among LFA-1+/+ and
LFA-1-/- mice (Fig. 5A). Following ConA injection,
the proportion of activated hepatic
NK1.1-TCR+ as
detected by expression of the CD69 activation marker increased with a
similar kinetic in the livers of both
LFA-1+/+ and
LFA-1-/- mice (Fig. 5B).
Similar to CD69 expression, the kinetics and activation-dependent
expression levels of CD25 (IL2R chain) were comparable between
ConA-injected LFA-1+/+ and
LFA-1-/- mice (Fig. 6A). Surface levels and the
kinetics of induction of CD69 and CD25 expression on both
CD4+ (not shown) and CD8+
(Fig. 6B) T cells were also comparable among
LFA-1+/+ and
LFA-1-/- mice.
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+ activated T
cells increased in percentages and remained activated in
LFA-1+/+ mice, the increase in activated
CD69+CD25+TCR+
T cells was only transient in LFA-1-/-
mice and activated
NK1.1-TCR+ T cells
of both CD4 and CD8 lineages were rapidly depleted from the livers of
these mice (Fig. 5
B). In contrast to the activation of
NK1.1-TCR+ cells,
expression of CD69 and CD25 on hepatic NK1.1+
TCR- NK cells and
NK1.1+TCR+ cells was
not elevated 4–8 h after ConA injection in both
LFA-1+/+ and
LFA-1-/- mice (not shown) indicating
that ConA administration primarily activates hepatic
NK1.1-TCR+ cells T
cells in vivo. In addition, ConA injection markedly induces the
expression of LFA-1 on the hepatic
NK1.1-TCR+ cells
(Fig. 6C). Because T cells on activation undergo
activation-induced cell death, we measured the apoptosis of
ConA-activated liver T cells by flow cytometry. Apoptosis induction in
T cells peaked at 12 h and declined by 24 h. However, the
percentages of apoptotic hepatic T cells in the livers were comparable
between ConA-injected LFA-1+/+ and
LFA-1-/- mice at all time points
analyzed (Fig. 6D). These results show that the activation
and cell death of hepatic TCR+ T cells are
similar in LFA-1+/+ and
LFA-1-/- mice; however, in the absence
of LFA-1 expression these activated T cells fail to induce liver damage
and hepatocyte apoptosis.
Normal in vitro ConA responsiveness of hepatic
NK1.1-TCR+T cells from
LFA-1-/- mice
To extend the apparently normal activation of
NK1.1-TCR+ cells in vivo (Figs. 5 and 6), we isolated "naive" NK1.1-TCR+ T
cells from nontreated LFA-1+/+ and
LFA-1-/- mice and assayed their response
to ConA stimulation in vitro. The ConA-induced proliferative response
(Fig. 7A) of hepatic
NK1.1-TCR+ cells as
well as production of the cytokine IL-2 (Fig. 7B) were
comparable among hepatic
NK1.1-TCR+ cells
from LFA-1-/- and
LFA-1+/+ littermates. Moreover,
"naive" hepatic
NK1.1-TCR+ cells
from both LFA-1-/- and
LFA-1+/+ littermates increased the
expression of the activation markers CD69 and CD25 to similar levels
(Fig. 7C) and at similar kinetics (not shown). Similar to in
vivo activated liver
NK1.1-TCR+ T cells
(Fig. 6C), the expression of LFA-1 on
NK1.1-TCR+ cells
from LFA-1+/+ mice significantly increased
after in vitro ConA stimulation (Fig. 7C). Thus, loss of
LFA-1 expression has no apparent effect on ConA-induced proliferation
and activation of hepatic
NK1.1-TCR+ cells in
vitro and in vivo. However, the potential role of LFA-1 in cell
survival needs to be tested.
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+ cells
Our results indicated that the absence of LFA-1 does not impair
the activation of hepatic
NK1.1-TCR+ cells. We
next examined the cytotoxic activity of ConA-activated hepatic
NK1.1-TCR+ cells
from LFA-1+/+ and
LFA-1-/- mice against the mouse liver
cells NMuLi, that express ICAM-1, ICAM-2, and Fas on the cell surface
(not shown). Whereas ConA-activated liver
NK1.1-TCR+ cells
from LFA-1+/+ mice rapidly lysed NMuLi
cells, the cytotoxic activity of ConA-activated liver
NK1.1-TCR+ cells
from LFA-1-/- mice was significantly
impaired (Fig. 8A). Similar
results were obtained when we used the hepatocyte cell line TLR2 (data
not shown). Thus, loss of LFA-1 expression results in impaired
cytotoxicity toward liver cells.
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, and TNF- play pathogenic
roles in ConA-induced hepatitis (6, 7, 8, 9, 11, 12, 13). Moreover,
it has been shown that FasL and perforin expression in effector cells
is critical for their cytotoxic activities (15, 16, 17). To
investigate whether loss of LFA-1 resulted in decreased expression of
cytokines and/or cytotoxic effector molecules, we first examined the
serum levels of IL-4, IFN-, and TNF- by ELISA and the expression
levels of these cytokines in hepatic
NK1.1-TCR+ by
RT-PCR. Serum IL-4 and IFN- levels were increased after 6 h of
ConA injection but almost normalized after 24 h in both
LFA-1+/+ and
LFA-1-/- mice. Serum TNF- levels
could not be detected at these time points (not shown). In purified
liver NK1.1-TCR+ T
cells, ConA-induced expression of IL-4 mRNA was detectable within
3 h, and expression of IFN- and TNF- mRNA was detectable
within 6 h (Fig. 8B). Expression of all cytokines
returned to baseline levels at 18 h after ConA injection. Thus,
kinetics and extends of IL-4, IFN-, and TNF- expression levels
were comparable among liver MNC isolated from
LFA-1+/+ and
LFA-1-/- mice. Moreover, there was no
apparent difference in the expression levels of FasL and perforin
protein among hepatic
NK1.1-TCR+ cells
purified from LFA-1+/+ and
LFA-1-/- mice (Fig. 8C).
These results show that the lack of LFA-1 expression has no apparent
affect on the induction of the proinflammatory cytokines IL-4, IFN-,
and TNF- and expression of the death effector molecules perforin and
FasL in hepatic
NK1.1-TCR+ T cells
following ConA administration in vivo.
Activation of hepatic
NK1.1-TCR+ cells in
response to ConA was accompanied by an increase in the expression of
the cell adhesion receptor LFA-1 (Fig. 6C). To test whether
the reduction of cytotoxic activity of hepatic
NK1.1-TCR+ cells
against NMuLi liver cells after ConA injection was due to impaired
binding to target cells, we analyzed the formation of effector-target
cell conjugates by flow cytometry. As shown in Fig. 8D,
naive (0 h) hepatic
NK1.1-TCR+ cells
bound to target cells; however, there was no significant difference in
binding between LFA-1+/+ and
LFA-1-/- mice. Following ConA injection,
the adhesion of hepatic
NK1.1-TCR+ cells to
NMuLi target cells was markedly increased in
LFA-1+/+ mice. By contrast, following ConA
injection of LFA-1-/- mice, adhesion
between hepatic
NK1.1-TCR+ cells and
NMuLi target cells did not change (Fig. 8D). Similar results
were obtained when we used the hepatocyte cell line TLR2 (data not
shown).
We next examined whether anti-ICAM-1, anti-ICAM-2, or
anti-LFA-1 blocking mAbs inhibit the in vitro cytotoxic activity
and adhesion of Con A-activated LFA-1+/+
hepatic NK1.1-TCR+
cells to target cells. Cytotoxic activity (Fig. 8E) and cell
adhesion of Con A-activated hepatic
NK1.1-TCR+ cells to
NMuLi liver cells (Fig. 8F) or TLR2 hepatocytes (not shown)
were markedly inhibited by blocking mAbs to LFA-1. By contrast,
inhibition of ICAM-1 or ICAM-2 had only minor effects on the
cytotoxicity and cell adhesion of hepatic
NK1.1-TCR+ cells
(Fig. 8, E and F). The cytotoxicity assays (Fig. 8, A and E) and cell adhesion assays (Fig. 8, D and F) shown were performed with hepatic
NK1.1-TCR+ cells at
24 h after ConA injection and it should be noted that similar
results, i.e., defective cytotoxicity and impaired ConA-induced cell
adhesion of LFA-1-/- hepatic
NK1.1-TCR+ T cells,
were also observed at earlier time points (not shown). These results
indicate that LFA-1 expression is necessary for the ConA-inducible
adhesion between hepatic
NK1.1-TCR+ cytotoxic
effector cells and liver cell targets.
Restoration of ConA-induced hepatitis by adoptive transfer of
hepatic NK1.1-TCR+ cell from
LFA-1+/+ mice
To directly test whether LFA-1 positive hepatic
NK1.1-TCR+ cells are required and
sufficient for the development of ConA-induced hepatitis, we adoptively
transferred "naive" LFA-1+/+ and
LFA-1-/- hepatic
NK1.1-TCR+ donor
cells into LFA-1-/- mice followed by
ConA administration. Migration of donor
NK1.1-TCR+ cells
into the host livers was followed by flow cytometry. Interestingly,
migration of hepatic
NK1.1-TCR+ cells
into the livers was increased as compared with
LFA-1+/+ splenic
NK1.1-TCR+ cells
(Fig. 9A) suggesting the
existence of liver-specific homing mechanisms.
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+ cells
into LFA-1-/- mice was sufficient to
induce ConA liver damage as determined by the liver enzymes AST and ALT
(Fig. 9B). Histologically, adoptive transfer of hepatic
NK1.1-TCR+ cells
from LFA-1+/+ mice into
LFA-1-/- hosts resulted in massive
accumulation of RBCs in the sinusoidal area and tissue damage (Fig. 9C, right panel). In contrast, no significant
histological changes were observed in
LFA-1-/- mice following transfer of
hepatic LFA-1-/-
NK1.1-TCR+ T cells
(Fig. 9C, left panel). Moreover, adoptively
transferred hepatic
NK1.1-TCR+ cells
from LFA-1+/+ donors into
LFA-1-/- hosts are activated in vivo
after ConA injection as determined by CD69 expression (Fig. 9D). Taken together, our results show that LFA-1-positive
hepatic NK1.1-TCR+
cells are sufficient and necessary to trigger ConA-induced hepatitis
and liver damage. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following ConA administration, T cells and macrophages release
cytokines such as IL-2, IL-1, IL-6, IFN-, IL-4, IL-12, or TNF- as
well as nitric oxide that have been linked to the pathogenesis of
hepatic lesions (6, 7, 8, 9, 10, 11, 12, 13). Moreover, several cytotoxic
effector molecules are considered to be involved in the development of
ConA-induced hepatitis. For example,
perforin-/- mice fail to develop
ConA-induced hepatitis (16). The role of Fas/FasL system
in the development of ConA-induced hepatitis is controversial, because
FasL-defective gld/gld mice, but not Fas mutant
lpr/lpr mice, fail to develop ConA-induced hepatitis
(15). Our data show that the absence of LFA-1 has no
apparent effect on the serum cytokine production, activation of hepatic
T cells, and expression of the cytotoxic effector molecules FasL and
perforin. However, ConA-activated hepatic T cells express high levels
of the adhesion molecule LFA-1 and loss of LFA-1 results in reduced
cytotoxicity and reduced conjugate formation between activated T cells
and liver cells. Importantly, these data indicate that attachment of
activated T cells to hepatic cells is a critical step in the
pathogenesis of T cell-dependent hepatitis.
Recently, it has also been shown that
NK1.1+TCR+ cells may
play a role in ConA-induced hepatitis (13, 24). Moreover,
we (22) and other groups (25, 26) have
demonstrated that LFA-1-/- mice have
reduced numbers of NK T cells in the liver. However, the kinetics and
extents of ConA-induced CD69 and CD25 expression were comparable among
LFA-1+/+ and
LFA-1-/- NK T cells implying that
ConA-induced activation of NK T cells is normal in
LFA-1-/- mice. Although we cannot
exclude that the low numbers of NK T cells in the livers of
LFA-1-/- mice may contribute to the lack
of hepatic injury, the preserved IL-4 production and normal expression
of CD69 and CD25 suggests that LFA-1-deficient
NK1.1+TCR+ cells are
normally activated in vivo and hence may play a minor role in
ConA-mediated liver damage. Furthermore, adoptive transfer of hepatic
NK1.1-TCR+ T cells
from LFA-1+/+ mice, but not from
LFA-1-/- mice, sensitized
LFA-1-/- mice to ConA-induced hepatitis.
These results show that hepatic
NK1.1-TCR+ T cells
are sufficient and necessary to mediate liver damage in our model of
experimental hepatitis. Thus,
NK1.1-TCR+ T cells,
but not NK cells, appear to be involved in the development of
ConA-induced hepatitis.
The leukocyte integrin LFA1 was initially defined by mAbs that inhibit
CTL-mediated cytotoxicity in the absence of complement
(20). However, LFA1-deficient mice displayed
normal CTL responses against systemic choriomeningitis virus and
vesicular stomatitis virus infections (21) and loss of
LFA-1 alone has only minor effects on the cytotoxicity of
NK1.1+TCR+ T cells
(22) suggesting that the consequences of anti-LFA1
mAbs may reflect nonspecific effects of Ab treatment, such as capping
of other surface molecules. Therefore, the in vivo function of LFA-1 in
the generation of cytotoxic T cell responses was unclear and it has
been speculated that other adhesion receptors such as CD44 can
functionally substitute for the loss of LFA-1 in vivo
(22). Therefore, it was very surprising to find that
LFA-1-/- mice are completely protected
from ConA-induced liver damage and that adoptive transfer of hepatic
LFA-1+/+ T cells conferred susceptibility
to ConA-induced hepatitis in LFA-1-/-
mice. Moreover, although LFA-1 and CD44 cooperate in cell adhesion of
lymphokine-activated killer cells to their targets and together control
NK cytotoxicity (22),
CD44-/- mice show massive ConA-induced
hepatitis (our own data (not shown) and Ref. 27). Thus, it
appears that the requirement for LFA-1 in T cell-mediated
cytotoxicity and tissue damage might depend on the disease model
and/or the expression of specific adhesion receptors on the target
organ.
Based on our results and previous experiments by other groups the
following scenario of ConA-mediated acute liver damage emerges: in vivo
administration of ConA triggers polyclonal T cell activation
(5) and the differentiation of T lymphocytes into cells
that produce cytokines such as TNF-, IFN-, or IL-4
(6, 7, 8, 9, 10, 11, 12, 13) and cytotoxic effector cells that express perforin
and FasL (14, 15, 16). ConA stimulation also triggers
up-regulation of the LFA-1 adhesion receptor (and possibly other
adhesion receptors) on T cells. Cytokines such as IFN- and TNF-
can induce the expression of ICAM-1 on hepatocytes and nonparenchymal
stromal hepatic cells, e.g., sinusoidal endothelial cells
(28). LFA-1-dependent attachment of the activated T cells
to liver cells is then required to initiate liver cell apoptosis and
acute hepatitis. Intriguingly, our genetic data indicate that cell
adhesion is a critical and essential step involved in liver cell damage
and the pathogenesis of acute hepatitis. Whether LFA-1-dependent T cell
adhesion to liver cells is also essential for the development of
fulminant disease in other hepatitis models such as endotoxin-induced
hepatitis (29) remains to be
examined.
More than 2 billion people have been infected with hepatitis viruses
and 1% of people infected with hepatitis viruses develop acute
fulminant hepatitis with no effective treatment option
(1). Moreover, the liver is a target for autoreactive
lymphocytes resulting in autoimmune hepatitis that very often requires
transplants (2). In both viral and autoimmune hepatitis,
activation of macrophages and T cells are critical initial steps in the
pathogenesis of liver damage and fulminant disease (3, 4).
Our data in a mouse model of acute T cell-dependent liver damage show
that the adhesion receptor LFA-1 is important for the development
ConA-induced acute hepatitis. Loss of LFA-1 expression in mutant mice
results in a complete protection from disease. Whether LFA-1 would be a
potential target for the treatment of T cell-mediated hepatitis and the
prevention of liver damage in acute hepatitis needs to be
determined.
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Acknowledgments |
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Footnotes |
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2 G.M. and S.T. contributed equally to this work and are listed in alphabetical order.
3 Address correspondence and reprint requests to Dr. Josef M. Penninger, Institute of Molecular Biotechnology of the Austrian Academy of Sciences and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, M5G 2C1, Canada. E-mail address: Jpenning{at}uhnres.utoronto.ca
4 Abbreviations used in this paper: FasL, Fas ligand; ALT, alanine aminotransferase; AST, aspartate aminotransferase; MNC, mononuclear cell.
Received for publication April 29, 2002. Accepted for publication September 25, 2002.
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plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology 111:462.[Medline]
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14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J. Immunol. 163:93.14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J. Exp. Med. 191:105.+ cell development: evidence that liver NK1.1+TCR+ cells originate from multiple pathways. J. Immunol. 162:3753.This article has been cited by other articles:
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). Serum AST and
ALT activity levels were assessed at different times following ConA
injection. Five mice were used in each group. Mean transaminase
activities of AST and ALT ± SD are shown.
) or 4 µg/ml ConA (
). Results
are presented as mean [3H]thymidine uptake (± SD) from
one of two similar experiments. Similar results were obtained using
different concentrations of ConA (not shown). B, IL-2
production of hepatic NK1.1-TCR
