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Immunobiology Program, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and Fas ligand. The
liver is one of the main sites for infiltration by activated
CD8+ T cells, and this is followed by the death of
hepatocytes. The contribution of the various cytolytic components to
this process is unclear. Hepatocyte damage by CD8+ T cells
was studied using the MHC class I-restricted OVA-specific TCR
transgenic mouse (OT-1) to examine the contribution of Fas to
hepatocyte death. Activated CD8+ T cells from both OT-1 and
Fas-deficient OT-1lpr mice migrated to the liver in
similar numbers after OVA administration, but only in OT-1 mice was
there evidence of significant hepatocyte damage histologically and by
elevation of serum aspartate transaminase. These differences were not
the result of inefficient induction of cytolytic activity in
OT-1lpr liver T cells, since they were as cytolytic in
vitro as OT-1 liver T cells. This was supported by findings of similar
high levels of message for perforin, TNF-, and Fas ligand in liver
lymphocytes from both mice. These findings demonstrate that following
Ag activation, infiltrating liver CD8+ T lymphocytes induce
hepatocyte damage in a Fas-dependent manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fate of lymphocytes once they have extravasated into tissues is less well understood, as are the consequences to the tissues that are infiltrated. The liver is a useful model system in which to examine these events, as it contains a resident population of T cells that expresses an activated phenotype, including expression of CD44 (4). Following Ag stimulation, the number of lymphocytes infiltrating the liver increases enormously (1). We have previously observed that the administration of Ag in vivo to TCR transgenic mice causes liver infiltration of Ag-specific CD4+ and CD8+ T cells, but only the CD8+ T cell subpopulation caused significant liver damage (5). Less apparent was the mechanism responsible for the liver damage.
Cytolytic T cells kill their targets in vitro primarily through
perforin and Fas ligand
(FasL)4 (6, 7). In vivo this may also involve additional effector molecules
such as TNF- (8, 9). Hepatocytes are exquisitely
sensitive to Fas-induced death following in vivo administration of
anti-Fas Ab (10). However, studies of
lymphocyte-mediated liver injury have achieved various results
regarding which pathways were critical for hepatocyte death. These
differences depended somewhat on the lymphocyte activation method used.
Studies using in vivo administration of Con A observed that hepatocytes
were killed by either the perforin pathway (11) or
required IFN-
(12, 13), with little involvement of FasL
or TNF-. The findings in one study of a murine model of hepatitis B
found little involvement of perforin or FasL in hepatocyte injury
(14); however, conflicting data were reported by Kondo et
al. (15), supporting a role for FasL. Still a third model
used OVA-primed liposomes targeted to the liver and observed that
adoptive transfer of OVA-specific Th1, but not Th2
CD4+ T cells induced liver injury that was
mediated by IFN- and TNF-, but not FasL (16). In
none of these studies were liver lymphocytes actually purified and
analyzed. It is thus unclear what was the actual level of cytolytic
activity or expression of cytolytic mediators by the infiltrating T
cells.
In the current studies, we have extended our earlier findings of hepatocyte injury mediated primarily by Ag-specific CD8+ T cells to further examine the specific method of cytolysis used by the liver-infiltrating T cells. For this purpose, we used the OT-1 mouse whose T cells recognize the OVA peptide (OVAp) (3), SIINFEKL, in the context of the MHC class I molecule H-2 Kb (17). Fas-deficient OT-1lpr mice manifested markedly less hepatocyte damage despite similar CD8+ T cell activation, liver infiltration, and expression of cytolytic effector molecules. They also did not develop any accumulation of Ag-activated T cells in lymphoid tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strains of C57BL/6, C57BL/6-lpr, and transgenic OT-1
and OT-1lpr mice were bred at the animal facilities of the
University of Vermont College of Medicine. Original breeding pairs of
C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor,
ME). OT-1 mice bear a transgenic TCR that recognizes chicken OVAp
257–264 restricted to class I MHC, Kb, and were
provided by F. Carbone (Monash University Medical School, Victoria,
Australia) and M. Bevan (University of Washington, Seattle, WA). OT-1
mice were maintained by breeding TCR transgenic males to normal C57BL/6
females. Offspring were screened for the clonotype TCR using
anti-V2 mAb. Breeding OT-1 mice with C57BL/6lpr mice
created OT-1lpr mice. Offspring were screened for the
lpr mutation by PCR, as previously described
(18), and for the clonotype TCR using anti-V2
mAb.
Abs, cell preparations, and flow cytometry
Monoclonal anti-murine CD8 conjugated to Red613 was
purchased from Life Technologies (Gaithersburg, MD). Monoclonal
anti-murine CD4 conjugated to Tricolor or PE was purchased from
Caltag Laboratories (Burlingame, CA). Monoclonal anti-murine V2
conjugated to PE, monoclonal anti-murine CD69 conjugated to PE,
monoclonal anti-murine CD80 (B7.1) conjugated to FITC, monoclonal
anti-murine CD86 (B7.2) conjugated to PE, and monoclonal
anti-murine H-2 Kb conjugated to biotin were
purchased from BD PharMingen (San Diego, CA).
Single cell suspensions were made by homogenizing tissues in RPMI 1640 medium (Life Technologies) supplemented with 5% bovine calf serum (BCS; HyClone Laboratories, Logan, UT). For flow cytometry, 750,000 cells were incubated in 0.1 ml PBS containing 0.5% BSA Fraction V, 0.001% (w/v) sodium azide (PBS-azide) (Sigma, St. Louis, MO), and the Abs listed above (3 µg/ml) at 4°C for 30 min. After washing with PBS-azide, cells were fixed in 1% methanol-free formaldehyde (Ted Pella, Reading, CA) in PBS-azide. Samples were stored at 4°C until they were analyzed with a Coulter Elite flow cytometer calibrated using DNA check beads (Coulter, Hialeah, FL).
OVAp and treatment of TCR transgenic mice
Peptide to chicken OVA 257–264 (SIINFEKL) (OVAp) was produced at Macromolecular Resources (Colorado State University, Fort Collins, CO). Mice received two i.p. injections 24 h apart of 190 µl 100 µM peptide solution in PBS or PBS alone. Tissues were harvested 2, 3, 5, or 7 days after the first injection of peptide.
Isolation of liver lymphocytes
Mice were euthanized, and the peritoneal cavity was opened and the portal vein identified. This was cannulated with a 27-gauge needle and perfused with 5–10 ml PBS until all the lobes of the liver blanched. With the needle remaining in the portal vein, the inferior vena cava was cut above the liver. The liver was then excised with forceps, and the gall bladder was identified and removed. The liver was washed once in RPMI/5% BCS and then cut into small pieces and homogenized in a tissue grinder. Cells were then spun once at 1200 rpm for 10 min. Supernatant was removed and the cells were resuspended in 10 ml digestion mix consisting of serum-free RPMI containing 0.05% collagenase IV and 0.002% DNase I (Sigma), and incubated at 37°C for 40 min, mixing the tube frequently. A total of 30 ml serum-free RPMI was then added and spun at 300 rpm for 3 min. This sedimented the majority of hepatocytes, but left lymphocytes in the supernatant. The supernatant was transferred to another 50-ml tube and spun at 1200 rpm for 10 min. The supernatant was aspirated, and the cells were then resuspended in a total volume of 1.6 ml serum-free RPMI and transferred to a 15-ml tube. A total of 2.4 ml 40% (w/v) metrizamide (Sigma) in PBS was added to the cells and mixed well. This solution was overlaid with 1 ml serum-free RPMI and spun at 2500 rpm for 20 min. Liver lymphocytes were identified at the interface, carefully aspirated, and transferred to another 15-ml tube, then washed with RPMI/5% BCS and spun at 1600 rpm for 10 min. Cells were then resuspended in RPMI/5% BCS for analysis or placed in culture in complete medium (RPMI 1640, 5% FCS, 25 mM HEPES, 292.3 µg/ml glutamine, 2500 µg/ml glucose, 10 µg/ml folate, 110.4 µg/ml pyruvate, 5 x 10-5 M 2-ME, 100 U/ml penicillin, and 100 U/ml streptomycin).
Detection of apoptosis by TUNEL
To analyze apoptotic cells by flow cytometry, the TUNEL assay
was used as described (19, 20). Lymphocyte populations
were initially incubated in complete medium at 37°C for 4 h.
Cells were initially stained for expression of CD4, CD8, and V2, and
then fixed for 15 min in 1% formaldehyde. Cell membranes were then
permeabilized for 15 min using 70% ethanol at 4°C. Samples were
incubated at 37°C for 1 h in 50 µl containing 10 U TdT and 0.5
nM d-UTP-biotin (Roche Diagnostics, Indianapolis, IN). Specimens were
washed twice with PBS/1% BSA and incubated with a 1/50 dilution of
streptavidin Tricolor (Caltag Laboratories) at 4°C for 30 min. Cells
were washed twice and analyzed by flow cytometry. Negative controls
consisted of staining of cells with the same protocol, but in the
absence of d-UTP-biotin.
A modification of Gavrieli et al. (21) was used to measure apoptosis in situ. Tissue samples were fixed in 10% neutral buffered Formalin for 24 h and embedded in paraffin, and 5-µm sections were cut and mounted on slides and heated at 60°C overnight. Slides were hydrated by washing twice in xylene for 8 min and then 5 min each in 96%, 90%, and 80% ethanol, and finally twice in double-distilled water (ddH20). Nuclear proteins were stripped by incubating with 25 µg/ml proteinase K (Sigma) in 10 mM Tris, 5 mM CaCl2, pH 7.4, at room temperature for 15 min, followed by washing in ddH20. Endogenous peroxidase was inactivated by submerging the slides in 0.6% H2O2 at room temperature for 15 min. Sections were rinsed twice in ddH20 and treated with 30 mM Tris-HCl, pH 7.2, 140 mM sodium cacodylate, 4 mM MgCl2, 0.1 mM DTT, after which, positive control slides were treated with 10 µg/ml DNase I (Sigma) at room temperature for 15 min. All slides were washed three times in ddH20 before the addition of TdT buffer (30 mM Tris-HCl, pH 7.2, 140 mM sodium cacodylate, 1 mM CoCl2). Nick end labeling of DNA was performed by incubating slides with 30 U TdT (Roche Diagnostics) in 100 µl TdT buffer/0.05% BSA at 37°C for 60 min. TdT was omitted from negative control slides. The reaction was terminated by submerging slides in TB buffer (300 mM NaCl, 30 mM sodium citrate) at room temperature for 20 min. Sections were rinsed with ddH20, covered with 2% BSA in TBS (20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, pH 7.4), and incubated with 100 µl streptavidin-peroxidase (Zymed Laboratories, San Francisco, CA) at room temperature for 20 min. Following three washes of TBS, 90 µl 1:20 aminoethylcarbazole (Zymed Laboratories) and 0.6% H2O2 were added at room temperature for 20 min. Sections were mounted using GVA mounting solution (Zymed Laboratories) and photographed with ASA 100 Kodak Gold 135 mm film.
Determination of serum aspartate transaminase levels
Blood from PBS- or OVAp-treated mice was collected from the orbital sinus and centrifuged using Microtainer serum separator tubes (BD Biosciences, Franklin Lakes, NJ). Serum was collected according to the manufacturer’s directions. Serum aspartate transaminase levels were determined by colormetric assay conducted by the chemistry laboratory at Fletcher Allen Medical Center (Burlington, VT).
Semiquantitative PCR
A total of 5 x 106 liver lymphocytes
was lysed in Ultraspec RNA reagent (Biotecx Laboratories, Houston, TX),
and RNA was prepared according to the manufacturer’s directions.
Oligo(dT) priming and reverse transcriptase were used to prepare cDNA
from RNA samples. PCR amplifications were performed as follows:
94°C x 1 min, 55°C x 1 min, 72°C x 1 min (35
cycles). Primers used for amplification of perforin (22),
FasL (23), TNF-, and hypoxanthine
phosphoribosyltransferase (HPRT) (24) have been described
previously. PCR reactions were separated on 1.5% agarose gels, and
products were visualized by ethidium bromide staining. Densitometry was
quantitated using a digital camera and Imager software.
Cytotoxicity assays
Adherent Hepa 1.6 cells (American Type Culture Collection, Manassas, VA) were grown in flat 96-well plates. A total of 3 x 104-5 x 104 cells/well was labeled with 30 µl/ml 5 mCi Na251CrSO4 (New England Nuclear, Boston, MA) for 1 h at 37°C directly in the wells. Wells were washed five times with 200 µl RPMI + 5% BCS. Liver lymphocytes were serially diluted in a separate 96-well plate, followed by a 2-h incubation at 37°C in the presence of 50 nM or 150 nM (final concentrations) concanamycin A (CMA), similarly diluted DMSO, or media alone. Following the incubation, liver lymphocytes were added to wells containing Hepa 1.6 targets. Additionally, 20 µg/ml Fas-Fc (Alexis, San Diego, CA) was added to wells to block FasL-mediated apoptosis. A total of 50–100 µl supernatant was removed at 6 h and 16–18 h, and radioactivity was determined using a gamma counter. HCl (3 N) was diluted 1/1 into select wells to determine maximal 51Cr release. Percent specific lysis was calculated as follows: ((experimental cpm - spontaneous release cpm)/(maximum release cpm - spontaneous release cpm)) x 100.
Cell surface expression of FasL
A total of 0.5 x 106 lymph node or liver lymphocytes was stained for cell surface expression of FasL using the Enzymatic Amplification Staining Kit (EAS Kit; Flow-Amp Systems, Cleveland, OH). Cells were washed twice with staining buffer (PBS, pH 7.4, 1% BSA, 1% FBS), then incubated at 4°C for 20 min with 6 pg/ml of either isotype control hamster IgG-biotin or hamster anti-murine FasL-biotin (BD PharMingen). After two washes with staining buffer, all samples were incubated with a 1/50 dilution of streptavidin-HRP secondary reagent (EAS kit) at 4°C for 20 min. Cells were subsequently washed twice with staining buffer, then once with PBS, pH 7.4. Cells were reacted with a 1/20 dilution of amplifier solution (EAS kit) at room temperature for 20 min, followed by two washes with staining buffer. Cells were then stained with directly conjugated Va2-FITC (BD PharMingen) simultaneously with streptavidin-PE (Caltag Laboratories) and incubated at 4°C for 20 min. Following two washes with staining buffer, cells were fixed in methanol-free 1% formaldehyde/PBS and stored at 4°C until analyzed by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymph node and spleen composition and cell numbers were
very similar between the OT-1 and OT-1lpr mice used in these
studies, as was the proliferative capacity to OVAp (data not shown).
Following administration of OVAp, the livers of OT-1 mice typically
appeared very pale and friable by day 2, which was never observed in
the livers of OT-1lpr mice. Furthermore, one of the three
OT-1 mice for each time point died on day 2 or 3 in each of the three
experiments. By contrast, no OT-1lpr mice died. Paralleling
these findings, OVAp administration induced extensive infiltration of
V2+ CD8+ lymphocytes
into the livers of both OT-1 and OT-1lpr mice by day 2 (Fig. 1
). Importantly, no difference was
observed in either the kinetics of the OVAp response or the number of
V2+ CD8+-infiltrating
lymphocytes in the livers of the two types of mice. In addition, the
rates of decline of liver lymphocytes after day 2 were essentially
identical (Fig. 2).
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2+
CD8+ expansion and contraction was observed in
the lymph nodes of both types of mice, except for an isolated increase
in OT-1 mice on day 3 that was not observed at other time points, nor
in the spleen cells. These findings were consistent over three
experiments and highlight that the kinetics of loss of OT-1 T cells was
no different on the lpr background. We (25) and
others (1, 26) have observed the appearance of
CD4-8- T cells following
activation of CD8+ T cells. Although these cells
increased in number following OVAp, they did not accumulate in
OT-1lpr mice (Fig. 1
, lower panels).
Fig. 2 shows H&E staining of liver sections before and after OVAp
treatment. Livers from OT-1 (Fig. 2A) or OT-1lpr
(Fig. 2D) mice that received PBS showed a normal morphology
of hepatocytes characterized by distinct membranes and vacuolar
appearance of the cytoplasm. There were also few lymphocytes within the
sinusoids or periportal regions of the liver. However, by day 2 after
OVAp, large numbers of lymphocytes infiltrated the livers of both OT-1
and OT-1lpr mice. Infiltrating lymphocytes in OT-1 livers
were seen clustered initially in the periportal regions (Fig. 2B), followed shortly by migration into the parenchyma by
day 3 (Fig. 2C). A similar degree of periportal infiltrates
(Fig. 2E) and parenchymal invasion (Fig. 2F) was
visible in OVAp-treated OT-1lpr mice. Concomitant with
lymphocyte infiltration, hepatocyte damage was discernable in livers
from primarily OT-1 mice given OVAp. Damaged hepatocytes stained more
darkly and displayed a hazy ground-glass morphology with few
intracellular vacuoles and indistinct cellular membranes (Fig. 2C, left half). Additionally, areas of more
extensive liver damage were frequently observed by pink staining of the
cytoplasm with nearly complete loss of cellular detail (Fig. 2C, right half). Markedly less damage was noted
in livers from OT-1lpr mice given OVAp. In this case,
hepatocytes clearly displayed vacuolar cytoplasm and distinct
membranes, despite similar degrees of lymphocytic infiltration (Fig. 2E).
Differences in hepatocyte damage were even more evident when liver
sections are stained for nicked DNA using the TUNEL method. Three and
five days after the initial OVAp injection, extensive hepatocyte death
was visible in OT-1 mice (Fig. 3A–C). However, livers from
OT-1lpr mice given OVAp displayed only minimal damage (Fig. 3D–F). Serum levels of the hepatocyte enzyme aspartate
transaminase (AST) also confirmed the differences in liver injury. Mice
treated with PBS manifested normal basal AST values (88–200 U/L).
Following OVAp administration, however, serum AST values rose
dramatically at day 2 in OT-1 mice, but only slightly in
OT-1lpr mice (Fig. 4). The AST
values for OT-1 mice at day 2 were actually an underestimate, as the
serum tested gave a reading beyond the 750 U/L upper limit of the
linear range of the assay. Despite this, the difference in AST values
between the OT-1 and OT-1lpr mice on day 2 was highly
significant (p < 0.0007, paired Student’s
t test). Additionally, whereas the difference in AST values
on day 2 from OT-1 mice given OVAp vs PBS was significant
(p < 0.0006), the same comparison in
OT-1lpr mice did not reach statistical significance.
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, perforin, and FasL. Liver lymphocytes manifest similar phenotypes and rates of cell death in OT-1 and OT-1lpr mice
Activated T cells undergo apoptosis by a process known as activation-induced cell death. A phenotypic change that often occurs concomitant with the entry of T cells in apoptosis is the surface expression of B220 (1). As lymphocytes entering the liver have been reported to die (27), differences in hepatocyte damage might merely reflect variability of lymphocyte death and the release of proteases. Thus, the TUNEL assay and B220 expression were used to assess the kinetics and degree of lymphocyte death following OVAp.
CD8+ liver lymphocytes for control PBS-treated
mice contained high proportions of TUNEL+ as well
as B220+ cells compared with splenocytes from the
same mice (Fig. 5 and Table I). There was little difference between
OT-1 and OT-1lpr mice for these parameters (Table I). This
is consistent with the view that many of the lymphocytes entering the
liver eventually die. However, the initial wave of
CD8+ cells entering the liver on days 2 and 3
after OVAp administration manifested few TUNEL+
or B220+ cells, whereas the
CD8+ cells in spleens at the same time showed
marked expansions of both TUNEL+ and
B220+ cells (Fig. 5). Thus, the initial wave of
hepatic lymphocytes was not dying in either group of mice. However, by
day 7, as liver lymphocyte numbers diminished, there was a return to
initial levels of both TUNEL+ and
B220+ cells (Table I). Throughout this 7-day
period following OVAp administration, there was little difference
between OT-1 and OT-1lpr liver lymphocytes in either the
kinetics or magnitude of TUNEL+ and
B220+ cells (Table I). These findings are in
agreement with the similar kinetics of liver lymphocyte infiltration
and decline between OT-1 and OT-1lpr mice. Furthermore,
following OVAp in OT-1lpr mice, there was no sustained
accumulation of B220+ cells in either lymph node,
spleen, or liver (Table I, and data not shown). This raises questions
regarding to what extent the accumulating B220+
cells in lpr mice result from an Ag-driven process.
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Cytotoxic T cells kill targets primarily through a process
dependent on perforin, TNF-, or FasL (28). It was thus
possible that differences in hepatocyte damage between OT-1 and
OT-1lpr mice might result from differences in their ability
to produce these effector molecules. This was examined in two ways,
either by measuring direct cytolytic potential of liver lymphocytes in
vitro, or by their expression of mRNA for perforin, TNF-, and FasL.
The hepatoma cell line Hepa 1.6 (H-2b) was used
as a syngeneic target to determine which cytolytic pathways were
activated in liver lymphocytes from mice given OVAp. Freshly isolated
liver lymphocytes from day 2 OVAp-treated OT-1 or OT-1lpr
mice killed Hepa 1.6 targets even in the absence of exogenously added
OVAp (Fig. 6A). This
contrasted with minimal cytolytic activity by spleen or lymph node T
cells from the same mice (data not shown). In addition, there was very
little cytolysis by liver lymphocytes from PBS control mice (Fig. 6A). Liver lymphocytes from OT-1lpr mice were
consistently slightly more cytolytic than those from OT-1 mice in three
separate experiments. The lack of a requirement of exogenous OVAp Ag
for cytolysis of the Hepa 1.6 targets was supported by the ability of
liver lymphocytes to also efficiently lyse allogeneic P815
(H-2d) targets (Fig. 6B). OT-1 T cells
do not manifest natural cross-reactivity to H-2d.
This suggested that liver lymphocytes expressed lytic molecules when
freshly isolated and did not require further Ag stimulation.
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C). Fas-Fc also partially blocked target killing to similar
degrees in both mice (Fig. 6C). This is consistent with the
view that both FasL and perforin were partly responsible for the lysis
of the hepatocytes. Positive control for Fas-Fc blocking of FasL
killing was defined using FasL-expressing 293 T cells (Fig. 6D). To confirm that the perforin pathway was activated in T
cells from mice given OVAp, further cytolysis assays were performed
using the thymoma target EL4 that is not sensitive to FasL-mediated
apoptosis (N. J. Kennedy, unpublished observations). Liver
lymphocytes isolated from OT-1 or OT-1lpr mice given OVAp
killed EL4 targets, and this was completely blocked by CMA, but
unaffected by Fas-Fc (Fig. 6E). By contrast,
anti-TNF- did not block cytolytic activity from liver
lymphs of either OT-1 or OT-1lpr mice (data not
shown). These observations show that liver lymphocytes from mice
administered OVAp kill targets in vitro using both the perforin- and
FasL-mediated pathways, and support the view that these same
pathways are involved with liver damage in vivo in OVAp-treated
OT-1 mice.
To further assess the cytolytic capacity of liver lymphocytes from OT-1
vs OT-1lpr mice, semiquantitative PCR was performed to
assess the levels of expression of perforin, TNF-, and FasL, using
the levels of expression of the hprt gene as a comparison.
An example of the PCR results of cDNA titrations from liver lymphocytes
is illustrated in Fig. 7A and
summarized in Fig. 7B for all three experiments analyzed. A
striking finding was the significantly higher levels of message for
perforin, TNF-, and FasL, and in liver lymphocytes compared with
lymph node or spleen cells from the same animals. As T cells in lymph
nodes, spleens, and livers in OT-1 mice are nearly all
CD8+, these differences did not reflect different
composition of CD4+ vs CD8+
cells. This underscores the notion that liver
CD8+ lymphocytes are enriched for activated T
cells following Ag stimulation. A less striking feature was the
slightly increased expression in lymph nodes of perforin, TNF-, and
FasL by day 2 after OVAp. A somewhat opposite trend was apparent in the
spleen. These trends paralleled the initial increase of
V2+ CD8+ T cells in
lymph nodes after OVAp, but their rapid decline in number in the
spleen. Conceivably, the activated splenic T cells may have rapidly
migrated to the liver. There were only minimal nonstatistical
differences between both strains of mice in message levels of these
molecules within liver lymphocytes. This further supports the view that
the difference in hepatocyte cell death in vivo was not the result of
diminished cytolytic capacity of OT-1lpr
CD8+ cells. Finally, in lymph node and spleen
cells, there was a moderate increase in message for FasL from
OT-1lpr mice compared with OT-1 mice. This is in agreement
with earlier findings (31) and might reflect a
compensation by lymphocytes for the lack of Fas (32, 33).
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C). These levels were also higher than from lymph nodes
from the same day 2 animals, especially OT-1 mice. Surface FasL was
also much higher on OT-1lpr lymph node T cells than from
OT-1 mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although hepatocytes are clearly sensitive to death following in vivo
injection of anti-Fas Ab (10), it has been less
certain to what extent Fas contributes to hepatocyte damage by
liver-infiltrating lymphocytes. In three studies using in vivo Con A,
liver damage induced by T cells was mediated by perforin or IFN-,
but not by FasL (11, 12, 34). Other studies using
viral-specific T cells from mice bearing transgenically expressed
hepatitis B virus yielded conflicting results, either supporting a role
for Fas in liver damage (15), or arguing against any
involvement of Fas (14, 35). These differences may have
resulted in part from the heterogeneity of infiltrating T cells.
However, in none of these studies were liver lymphocytes isolated and
studied directly for their cytolytic activity or expression of
cytolytic effector molecules. The current studies represent the first
analysis of the cytolytic activity and mechanisms used by the actual
infiltrating lymphocytes in the liver. Hepatocyte damage in OT-1 mice
given OVAp results from both perforin- and FasL-mediated killing
pathways, as both the perforin inhibitor CMA as well as Fas-Fc block in
vitro killing of hepatocyte targets by OT-1 liver lymphocytes. The
partial involvement of perforin would be consistent with our findings
in vivo that OT-1lpr mice were not completely devoid of
histological evidence of damage of hepatocytes or elevation of serum
AST, although it was dramatically less than in Fas-bearing OT-1
mice.
Liver lymphocytes were directly cytolytic when freshly isolated and did not require in vitro restimulation with OVAp, unlike spleen or lymph node T cells from the same mice. This is consistent with the view that CD8+ cells may become activated in peripheral lymphoid tissues, but then migrate to the liver, where cytolytic activity would become concentrated. This model would suggest that OVAp-induced liver injury in mice represents an "innocent bystander" phenomenon. In this system, Ag presentation by the target organ may not be necessary for either the trafficking of lymphocytes to the organ or the subsequent tissue injury induced by infiltrating lymphocytes. We (J. Q. Russell, unpublished observations) and others (2) have observed that Ag-activated T cells also traffic to the lung and kidneys, and we are examining the degree of tissue injury that results at these sites. This model might also serve to explain the liver dysfunction that is often observed after situations in which the immune system has been strongly activated largely outside the liver, such as by a superantigen in toxic shock syndrome (36, 37). It also suggests that the normal number of resident liver lymphocytes in wild-type mice might induce tonic low levels of hepatocyte apoptosis in a Fas-dependent manner. This would be consistent with the observation that Fas-deficient mice have larger livers than wild-type mice (38).
The current findings also raise questions regarding the origin of the
CD4-CD8-
B220+ T cells that accumulate in lpr
mice. Prevailing evidence suggests that these unusual lpr T
cells derive from a CD8+ precursor. This is
supported by persistent demethylation of the CD8 gene in
lpr CD4-CD8- T
cells (39), and the absence of these cells in
lpr mice deficient in
2-microglobulin (18, 40).
Although we have previously observed that administration of OVAp to
OT-1 mice results in the transient appearance of
V2+
CD4-CD8- T cells
(25), the current studies did not observe an accumulation
of these cells in OT-1lpr mice. Conceivably more persistent
administration of OVAp to OT-1lpr mice might result in
accumulation of V2+
CD4-CD8- T cells.
However, even with the limited administration of OVAp in this protocol,
V2+ cells, either CD8+
or CD4-CD8-, were largely
eliminated from lymphoid tissues of OT-1lpr mice by day 7
with kinetics that was nearly identical to OT-1 mice. These findings
suggest that the derivation of
CD4-CD8- T cells from
CD8+ precursors in lpr mice may not
result from chronic antigenic activation, but by a different
process.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biochemistry, Howard Hughes Medical Institute, University of Massachusetts Medical Center, 373 Plantation Street, Worcester, MA 01605.
3 Address correspondence and reprint requests to Dr. Ralph C. Budd, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Medical Building, D-305, Burlington, VT 05405-0068. E-mail address: rbudd{at}zoo.uvm.edu
4 Abbreviations used in this paper: FasL, Fas ligand; AST, aspartate transaminase; BCS, bovine calf serum; CMA, concanamycin A; HPRT, hypoxanthine phosphoribosyltransferase; OVAp, OVA peptide.
Received for publication June 5, 2001. Accepted for publication September 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ cells in lpr/lpr mice lacking 2-microglobulin. J. Immunol. 154:2063.[Abstract]
+ CD4-CD8- B220+ cells based on high affinity TCR signals. J. Immunol. 162:5747. and IFN- at the effector phase of liver injury mediated by Th1 cells specific to hepatitis B virus surface antigen. J. Immunol. 165:956.This article has been cited by other articles:
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) and
OT-1lpr mice (
) received two 190-µl doses of either
PBS or 100 µM OVAp administered i.p. 24 h apart. On the day
indicated following the first injection, lymphocytes were isolated from
liver, lymph node, and spleen and assessed for the number of
V
). No cytolytic activity was observed in
lymph node or spleen cells from the same mice (data not shown).
B, Cytolysis of allogeneic P815 (H-2d)
target cells by liver lymphocytes. C, Inhibition of
cytolytic activity by Fas-Fc (20 µg/ml) to block FasL, or by CMA (150
µM) to block perforin. Cytotoxicity is expressed as the percentage of
maximal killing observed by lymphocytes in the presence of control Ig
(20 µg/ml). D, Lysis of Hepa 1.6 cells by
FasL-transfected 3T3 cells in the presence of control Ig (20 µg/ml),
Fas-Fc (20 µg/ml), or CMA (150 µM). E, Lysis of
Fas-resistant EL-4 cells by liver lymphocytes in the presence of
control Ig, Fas-Fc, or CMA.