From the Immunobiology Program, Department of Medicine, The University of Vermont College of
Medicine, Burlington, Vermont 05405
Little is understood of the anatomical fate of activated T lymphocytes and the consequences
they have on the tissues into which they migrate. Previous work has suggested that damaged
lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)-restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of
CD4
CD8
B220+ T cells occurs more frequently from a CD8+ precursor than from CD4+ T
cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8+ cells with only
the high affinity peptide, there is also a dramatically increased number of liver lymphocytes
with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC-restricted TCR. The findings
show that CD4
CD8
B220+ T cells preferentially derive from a CD8+ precursor after a high
intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte
damage, and thereby serve as a model of autoimmune hepatitis.
Key words:
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Introduction |
The activation of lymphocytes is accompanied by numerous changes in surface phenotype. Many of these
changes, such as increased surface LFA-1 and the hyaluronate receptor/memory marker CD44, as well as decreased
L-selectin (1), are critical to lymphocyte homing. Lymphocyte activation also results in clonal expansion, often followed by cell death through Fas-dependent apoptosis (2, 3). In some instances of T lymphocyte apoptosis, there may be an accompanying change in surface phenotype with
downmodulation of CD4 or CD8 and upregulation of the
B cell isoform of CD45 known as B220 (4, 5).
Collectively, the dynamic alterations in the trafficking of
lymphocytes allow binding to vascular endothelium and the
subsequent egress to extravascular tissues. 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 good model system in which to examine these events, as it contains a significant population of T
cells expressing a previously activated phenotype, including
expression of CD44, and in some instances a significant
proportion of cells manifesting the phenotype CD4
CD8
B220+ TCR-
/
intermediate (4, 6). The origin of the latter population is unresolved. Some studies have argued that this subset represents a proliferating T cell population whose lineage
is unique to the liver (7), whereas other investigations suggest
that cells of the same phenotype might migrate to the liver after activation elsewhere, and undergo apoptosis within the
liver (4). The lack of any significant expression of recombination-activating gene RAG-1 or RAG-2, proteins essential for
rearrangement of the TCR
and
chains, by murine liver
lymphocytes also argues against the adult liver being a site
of T lymphocyte development (8). A third, although not mutually exclusive, view has shown that a subset of CD4
CD8
and CD4+ T cells that express the natural killer marker
NK1.1 use a highly limited TCR repertoire (V
14, V
8.2,
V
7, or V
2) and are restricted in their response to the
class I-like molecule CD1 (9).
We have previously proposed a model whereby
CD4
CD8
B220+TCR-
/
+ cells result from a high intensity TCR signal that leads in most instances to apoptosis (10).
A large proportion of the precursors of this subset are probably previously expressed CD8, as reflected by demethylation
of the CD8
gene in CD4
CD8
TCR-
/
+ cells in the
thymus (11) and periphery (12). The further observation that
mice lacking class I MHC expression are nearly devoid of
CD4
CD8
TCR-
/
+ cells (13) suggests that these unusual cells require an active signal conferred by class I MHC.
In this study this model has been tested in two different TCR
transgenic mice that recognize chicken OVA restricted to
either class I MHC (OT-1 mice) or class II MHC (DO.11.10
mice). Our findings demonstrate that after equivalent doses of
the appropriate OVA peptide, only the CD8+ T cells from
OT-1 mice showed increased expression of surface B220,
with a portion becoming CD4
CD8
. The activated CD8+
OT-1 T cells also migrated to the liver in considerably higher numbers and produced more hepatocyte damage than did
activated CD4+ cells in DO.11.10 mice. These findings were
not observed in OT-1 mice that received lower affinity variants of the native OVA peptide. This suggests a model of
autoimmune hepatitis in which stimulation of the T lymphocytes can occur at distant lymphoid sites, with subsequent
migration to the liver where they provoke hepatocyte injury.
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Materials and Methods |
Mice.
Normal strains of C57BL/6 and BALB/c mice and
transgenic DO.11.10 or OT-1 mice were bred at the animal facilities of The University of Vermont College of Medicine. Original breeding pairs of normal mice were obtained from The Jackson Laboratory (Bar Harbor, ME). DO.11.10 TCR transgenic
mice recognize chicken OVA peptide 323-339 in the context of
class II MHC, I-Ad, and were the gift of Dr. Dennis Loh (Washington University, St. Louis, MO; reference 16). DO.11.10 mice
were maintained by breeding transgenic male mice to normal
BALB/c females. Offspring bearing the TCR transgene were
identified by expression of the clonotype TCR identified by mAb
KJ1-26. OT-1 mice bear a transgenic TCR that recognizes
chicken OVA peptide 257-264 restricted to class I MHC, Kb,
and were provided by Drs. Francis Carbone (Monash University Medical School, Victoria, Australia) and Michael Bevan (University of Washington, Seattle, WA; reference 17). OT-1 mice were
maintained by breeding TCR transgenic male mice to normal
C57BL/6 females. Offspring were screened for the clonotype
TCR using anti-V
2 mAb.
Antibodies, Cell Preparations, and Flow Cytometry.
Monoclonal anti-murine CD8
conjugated to Red613 and PE-conjugated B220 were purchased from Caltag Labs. (Burlingame, CA). Monoclonal anti-murine CD4 conjugated to Red613 was purchased from GIBCO BRL (Gaithersburg, MD). Monoclonal
anti-murine V
2 conjugated to FITC or PE was purchased from
PharMingen (San Diego, CA). The hybridoma KJ1-26, which
reacts to the clonotype TCR of DO.11.10 mice, was the gift of
Dr. Philippa Marrack (National Jewish Center for Immunology
and Respiratory Diseases, Denver, CO). KJ1-26 was purified
from mouse ascites on HiTRAP Protein G columns (Amersham
Pharmacia Biotech, Inc., Piscataway, NJ), and then conjugated to
fluorescein (Sigma Chemical Co., St. Louis., MO) using established methods (18). Fluorescein-conjugated antibody was purified from reaction components by chromatography on PD-10 columns (Amersham Pharmacia Biotech., Inc.).
Single cell suspensions were made by homogenizing tissues in
RPMI 1640 medium (GIBCO BRL) supplemented with 5%
(vol/vol) bovine calf serum (BCS)1 (Hyclone Laboratories, Logan, UT). Cells excluding trypan blue were counted. For flow
cytometry, 106 cells were incubated in 0.1 ml PBS containing
0.5% BSA Fraction V, 0.001% (wt/vol) sodium azide (Sigma
Chemical Co.), and the antibodies listed above (3 µg/ml) at 4°C
for 30 min (PBS azide). After washing with PBS-azide, cells were
fixed in 1% (vol/vol) methanol-free formaldehyde (Ted Pella
Inc., 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, Inc., Hialeah, FL).
Apoptosis was quantified using staining with FITC-conjugated
Annexin V (Nexins Research B.V., Hoeven, The Netherlands), which binds to phosphatidylserine residues that are found on the inner leaflet of cytoplasmic membranes of living cells but translocate to the outer leaflet upon initiation of apoptosis (19). Data were gated using Elite software by forward and side light scatter. Negative controls were set by using isotype-matched Ig directly conjugated to fluorochromes (Caltag Labs.).
OVA Peptides and Treatment of TCR Transgenic Mice.
Peptides to
chicken OVA 323-339 (ISQAVHAAHAEINEAGR) (OVAII),
257-264 (SIINFEKL) (OVAI), or OVAI variants E1(EIINFEKL)
and R4 (SIIRFEKL) were produced at Macromolecular Resources (Colorado State University, Fort Collins, CO). OVAI/Kb
binds to the OT-1 TCR with high affinity (Kd = 6.5 µM),
whereas E1 and R4 bind with lesser affinities of 22.6 and 57.1 µM, respectively (20). OVAII/I-Ad binds to the DO.11.10 TCR
with a Kd of 31 µM (21). Mice received one, two, or three daily
intraperitoneal injections of 250 µl of 100-µM peptide solutions
in PBS or PBS alone. Tissues were harvested 1, 2, 3, 5, or 7 d
after the last injection of peptide.
Isolation of Liver Lymphocytes.
After death, the peritoneal cavity
was opened and the portal vein was identified. This was cannulated
with a 27-gauge needle and perfused with 5 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 1,200 rpm for 10 min. Supernatant was removed and the cells were resuspended in 10 ml of digestion mix
consisting of serum-free RPMI containing 0.05% collagenase IV
and 0.002% DNAse I (both from Sigma Chemical Co.), and then
they were incubated at 37°C for 40 min, mixing the tube frequently. 30 ml of 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 1,200 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. 2.4 ml of 40% (wt/vol) metrizamide (Sigma Chemical
Co.) in PBS was added to the cells and mixed well. This solution
was underlaid with 1 ml serum-free RPMI and spun at 2,500 rpm
for 20 min. Liver lymphocytes were identified at the interface,
carefully aspirated with a pasteur pipette, and transferred to another
15-ml tube, then washed with RPMI/5% BCS and spun at 1,600 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, 2,500 µg/ml
glucose, 10 µg/ml folate, 110.4 µg/ml pyruvate, 5 × 10
5 M
2-ME, 100 U/ml penicillin, and 100 U/ml streptomycin).
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Results |
Trafficking of CD8+ T Cells to the Liver Is Induced only by
High Affinity Peptide Antigen.
To assess the relative dynamics and consequences of CD8+ versus CD4+ T cell migration to the liver after antigen stimulation, two TCR transgenic mouse models were used, both of which recognize chicken OVA. T cells from OT-1 mice recognize OVA
peptide 257-264 (OVAI) restricted to class I MHC, Kb (17),
whereas those from DO.11.10 mice recognize OVA peptide 323-339 (OVAII) in the context of class II MHC, I-Ad (16).
A concentration range of OVA peptide was initially examined to determine what dose in vivo would produce a
moderate increase of lymphocyte number in peripheral
lymphoid tissues. In the case of OT-1 mice, 250 µl of 10-µM
OVAI produced only a slight increase in lymphoid number
(data not shown), whereas 100-µM OVAI produced a
more substantial and reproducible increase and was consequently used in subsequent studies. A single administration
of 250 µl of 100-µM OVAI produced an abrupt decrease
in thymocyte numbers by day 2, concomitant with a moderate twofold increase in lymph node cell numbers and little change in the number of splenocytes (Table 1, Exp. no.
1). During this period, the number of liver lymphocytes
rose from a mean of 0.8 to 5.6 × 106. Less pronounced effects were observed with the lower affinity OVA peptide
variants E1 and R4. The affinities of the OT-1 TCR for
the E1/Kb complex (Kd = 22.6 µM) and R4-Kb complex
(Kd = 57.1 µM) are reduced 3.5- and 8.8-fold, respectively, compared with native OVAI (Kd = 6.5 µM) (20).
Although a single dose of E1 and R4 produced a slight decrease in thymocyte numbers on day 2, there was otherwise only a modest increase in the size of peripheral lymphoid cell numbers by day 2, but no increase in the
number of liver lymphocytes (Table 1, Exp. no. 1). This was also true even when E1 and R4 were administered
three times at 24-h intervals (Table 1, Exp. no. 3).
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Table 1
Kinetics of Lymphocyte Cell Numbers after Administration of OVAI Peptide Variants to Class I-restricted (OT-1) OVA-specific
TCR Transgenic Mice
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Two doses of OVAI resulted in a pronounced decrease of
thymus size by day 2 and an increase in the number of lymph
node cells and splenocytes, which peaked on day 3 (Table 1,
Exp. no. 2). At the same time, there was a particular increase
in the number of liver lymphocytes, which peaked on day 2. When administered at three 24-h intervals, OVAI produced
a profound increase (to 27 × 106) in the number of liver
lymphocytes (Table 1, Exp. no. 3), but this dose also resulted
in severe hepatic damage (see below). Thus, two 250-µl administrations of 100-µM OVAI were optimal for producing
moderate expansion of peripheral lymphoid cell numbers and an increase in liver infiltration by lymphocytes.
Compared with the effects of OVAI in OT-1 mice, the
same doses of OVAII administered to DO.11.10 mice
yielded a similar loss of thymocytes and initial expansion of
peripheral lymphoid tissues, but there was a somewhat less
profound loss of lymphocytes at later time points (Tables 1
and 2 and Fig. 1). The DO.11.10 TCR binds OVAII/I-Ad
with an affinity of 31 µM (21). After OVA administration
in both OT-1 and DO.11.10 mice, there was an initial expansion of lymph node cell number on days 2 and 3. After
this, the rate and extent of decline in cell numbers was
more pronounced in OT-1 mice. Thus, by day 5, lymph
node and splenocyte cell numbers in OT-1 mice receiving
OVAI were the same as or considerably below those of
control OT-1 mice, whereas peripheral lymphocyte cell numbers in DO.11.10 mice were often still above those of
control mice. Similar differences were observed with three
doses of the respective native OVA peptides (Tables 1 and 2).
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Table 2
Kinetics of Lymphocyte Cell Numbers after Administration of OVAII Peptide to Class II-restricted (DO.11.10) OVA-specific TCR
Transgenic Mice
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Fig. 1.
OVA antigen induces greater increase of liver lymphocytes
in OT-1 than in DO.11.10 mice. OT-1 or DO.11.10 mice from experiment no. 2 received two doses of PBS or, respectively, OVAI and OVAII
at 24-h intervals. Total lymphocyte numbers were determined for each
organ on the day indicated after the last injection.
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Paralleling the temporal differences in lymphocyte loss between OT-1 and DO.11.10 mice after administration of their
respective OVA peptides, both the kinetics and magnitude of
lymphocyte accumulation in the liver were greater in the
OT-1 mice. As mentioned above and shown in Table 1, after
two doses of OVAI, OT-1 liver lymphocyte numbers increased from a control of 1.4 × 106 to 11.2 × 106 on day 2, and after three doses of OVAI this increased further to 27 × 106 on days 2 and 3. Thereafter, the degree of lymphocyte infiltration rapidly declined. In contrast, equivalent doses of
OVAII in DO.11.10 mice produced only modest increases in
liver lymphocytes on day 2 and these continued to increase
throughout days 5-7, although they never reached the magnitude seen in OT-1 mice (Table 2). The greater liver lymphocyte infiltrate in OT-1 mice was paralleled by an increased
mortality in this group. At two doses, one out of nine OVAI-treated OT-1 mice died on day 5, whereas at three doses, two
out of nine mice died, also on day 5. No DO.11.10 mice died
from OVAII administration at any of the doses studied.
Cell Size Changes and Induction of CD4
CD8
B220+
Phenotype in OT-1 T Lymphocytes after OVAI Administration.
In addition to the changes in the number of lymphocytes after administration of OVAI, there were also substantial changes in their morphology and phenotype. We
(10) and others (22) have considered that the CD4
CD8
phenotype for T cells reflects those that have received a
high intensity TCR signal and are prone toward apoptosis.
A useful additional marker for dying T cells is B220, the
high molecular weight B cell isoform of CD45 (4). Thus,
expression of B220 and loss of CD4 and CD8 were used
along with cell size and Annexin V staining of apoptosis to
determine the phenotype and fate of lymphocytes after
treatment of OT-1 and DO.11.10 mice with OVA.
The shifts in cell size and phenotype after administration
of OVA peptides were also more dramatic in the OT-1
mice than in DO.11.10 mice. Although most of the TCR
transgenic V
2+ cells in lymphoid tissues of control OT-1
mice were CD8+, after OVAI there was a large but transient increase in the proportion of peripheral CD4
CD8
V
2+ cells. A striking shift from CD8+ to CD4
CD8
could be seen as soon as 1 d after administration of only the high affinity OVAI peptide. As shown in Fig. 2, the low
and moderate affinity OVAI variants, R4 and E1, respectively, produced little or no substantial change from PBS-injected control mice in the phenotypic composition of the
lymph nodes, which were predominantly CD8+ and manifested only 8-9% surface B220. However, within 24 h of a
single dose of OVAI there was a decrease in the proportion
of CD8+ cells, from 60% with PBS to 28% with OVAI.
Accompanying this was a reciprocal increase in the proportion of CD4
CD8
cells, from 35% with PBS to 69% with
OVAI, 45% of which were V
2+ (Fig. 2). These phenotypic shifts were confirmed by absolute numbers. The
number of CD8+V
2+ cells decreased only after treatment
with OVAI. The number of CD4
CD8
V
2+ lymph
node cells did not change with R4 and increased nearly twofold with E1, whereas with OVAI this subset increased
fourfold. These changes in phenotype and cell number
were very consistent in three separate experiments. In addition to the induction of CD4
CD8
V
2+ cells with
OVAI, the residual CD8+ cells after OVAI treatment now
expressed increased amounts of B220 (24%). B220 expression by the CD4
CD8
V
2+ subset was already significant before antigen exposure (62-78%) and did not increase any further after OVAI administration (Fig. 2). This
may reflect the fact that the CD4
CD8
T cells in normal
mice have also arisen by a high intensity TCR signal from
endogenous or environmental antigens.

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Fig. 2.
Induction of CD4
CD8 V 2+ and CD8+B220+
phenotype only by high affinity
OVAI peptide. OT-1 mice,
three per group, received a single
injection of the indicated peptide
or PBS. OVAI confers a high affinity (Kd = 6.5 µM) interaction
with the OT-1 TCR when
complexed with Kb. R4 and E1
are OVAI variants that manifest
affinities of 57.1 and 22.6 µM,
respectively. On day 1 after injections, lymph node cells from
each group were pooled and analyzed for expression of CD4,
CD8, V 2, and B220. Numbers
in quadrants indicate the percentage of positive cells. The
forward and side scatter histograms at the left show that the
gates set for this analysis were
uniform and did not include
small dying cells. Numbers in parentheses represent the percentage of V 2+ cells that express
B220. The right hand column
displays absolute numbers of
CD8+V 2+ and CD4 CD8
V 2+ cells. The findings are representative of three experiments.
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Various sized lymphoid populations appeared in rapid
succession after two doses of OVAI to OT-1 mice, as
shown for lymph node cells in Fig. 3. On day 2 a blast population was observed that was enriched in the proportion of
CD4
CD8
V
2+ cells compared with control OT-1 mice.
This increase occurred at the expense of the CD8+ subset,
which on day 2 diminished from 63 to 43%, but at the same time manifested increased B220 expression (34%)
compared with control mice (17%) (Fig. 3 A). Concomitantly, a subpopulation of small cells gradually increased
during the 5 d after administration of OVAI. As shown in
Fig. 3 B, these small cells were considerably enriched in the
proportion of CD4
CD8
B220+V
2+ cells, even in PBS-treated control mice. Furthermore, the minor CD8+ subset
also expressed more B220 than was seen in the blast cells. These morphologic and phenotypic changes corresponded
to an initial expansion in peripheral lymphoid cell numbers
by day 2 followed by a rapid decline by day 5 in OT-1
mice that had received OVAI (Fig. 4 A). Throughout this
period there was an increase in the expression of B220
by the CD8+ subset in thymus, lymph node, and spleen
(Fig. 4 B). In contrast to OT-1 mice, the appearance of
CD4
CD8
B220+ T cells did not occur in DO.11.10
mice at the same dose of OVAII (Fig. 5). In fact, the absolute number of CD4
CD8
KJ1-26+ cells often decreased
with OVAII, whereas the number of CD4+ lymph node
cells increased. Even at a 100-fold higher dose of OVAII
there was still no significant induction of B220 expression by T cells, nor of CD4
CD8
T cells (data not shown).

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Fig. 3.
OVAI induces blast formation of OT-1 T cells followed by
the appearance of small T cells that express B220 and are enriched for a
CD4 CD8 V 2+ phenotype. OT-1 mice received two injections of
OVAI or PBS in experiment no. 2. Lymph node cells were analyzed on
the indicated days after the last injection. (A) An initial blast population
appears on day 2 following OVAI which then becomes a smaller transition cell population over the next 3 d. Surface phenotype is based on the
large and moderate sized cells as shown by the enclosed gate. (B) The small sized population of lymphocytes is enriched for CD4 CD8 V 2+B220+
cells. OVAI administration also increased the proportion of CD8+ cells expressing B220.
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Fig. 4.
Cell counts and percentage of B220 expression by the CD8+V 2+ and CD4 CD8 V 2+ subsets of T cells from OT-1 mice after two doses
of OVAI. Analysis is based on the same mice as used in experiment no. 2 in Fig. 1 and phenotypes were determined on the living cells based on flow cytometric size gates. Tissues were pooled from three OT-1 mice per group. (A) Actual lymphocyte counts of subsets in various organs. (B) Percentage of
cells in the indicated subset that expressed B220.
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Fig. 5.
OVAII does not
induce B220 expression or
CD4 CD8 phenotype by responding CD4+ cells from
DO.11.10 mice. DO.11.10 mice
received OVAII injections and
lymph node cells (top) or liver
lymphocytes (bottom) were examined for expression of CD4, CD8,
B220, and clonotype TCR using
antibody KJ1-26. The right column indicates the absolute numbers of KJ1-26+ cells that were
CD4+ or CD4 CD8 . Phenotypes were based on gates set for
living cells as in Fig. 2.
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Liver Lymphocyte Phenotype.
By contrast with other
lymphoid tissues, after OVAI treatment of OT-1 mice the
liver lymphocytes contained an initial dramatic increase in
the proportion and absolute number of CD8+V
2+ and
CD4
CD8
V
2+ cells (Fig. 4 A). This caused the ratio of
CD4
CD8
V
2+ to CD8+V
2+ cells to decrease rather
than increase as observed in the lymph nodes (compare
Figs. 2 and 6). However, by day 5 the proportion of CD8+
cells had decreased to nearly their initial levels, with a
somewhat slower decline in the proportion of CD4
CD8
V
2+ cells. In addition, after OVAI administration the
liver lymphocytes did not manifest significant B220
expression in either the CD8+ or CD4
CD8
subsets over
the 5-d course of the experiment. These findings suggested
that migration of lymphocytes to the liver was a selective process in OT-1 mice, initially of activated CD8+ cells;
however, they may have changed their phenotype to
CD4
CD8
within the liver.

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Fig. 6.
Some resident liver T lymphocytes in OT-1 mice express
B220, but after OVAI administration the initial influx is of CD8+ cells that
lack B220. OT-1 mice received two injections of OVAI in experiment no.
2 and liver lymphocytes were isolated on the indicated days after the last injection. The forward and side scatter histograms at the left show that the
gates set for this analysis were uniform and did not include small dying cells.
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Annexin V staining confirmed that lymphocytes in the
liver were undergoing increased apoptosis. Fig. 7 A displays
Annexin V staining from experiment no. 3 and illustrates
that the resident CD8+ liver lymphocytes in OT-1 mice
contained a higher proportion of Annexin V+ cells (57.1%)
than either lymph node (13.8%) or spleen (11.9%, data not
shown). After OVAI administration, there was a significant increase on day 2 of Annexin V+ CD8+ cells in lymph node
(42.8%) and spleen (34.4%, data not shown), which decreased only partially by day 5. In contrast, the liver lymphocytes maintained an already high proportion of Annexin V+
CD8+ cells (58.8%). By day 5, the Annexin V+ subset of
CD8+ liver lymphocytes had decreased to levels seen in the
periphery, and were clearly less than was observed in liver
lymphocytes from control mice. As before, DO.11.10 mice
demonstrated less evidence of lymphocyte apoptosis after
OVAII (Fig. 7 B). Although DO.11.10 liver lymphocytes revealed more apoptosis than lymph node cells from the same
mice, these levels did not approach those seen in OT-1
mice. The findings were similar in two other experiments.

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Fig. 7.
Liver lymphocytes and lymph node cells are undergoing increased apoptosis in OT-1 mice compared with DO.11.10 mice. Mice
are from experiment no. 3 and received three doses of PBS, OVAI (OT-1
mice), or OVAII (DO.11.10 mice) at 24-h intervals. Lymphoid cells were
isolated at the indicated time points and stained with Annexin V and either anti-CD8 (for OT-1 mice) or anti-CD4 (for DO.11.10 mice). Analysis is gated on CD8+ cells from OT-1 mice (A) or on CD4+ cells from
DO.11.10 mice (B).
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Infiltrating CD8+ Liver Lymphocytes Induce Hepatocyte
Death.
Similar to the more profound influx of liver lymphocytes in OT-1 mice versus DO.11.10 mice after OVA
treatment, serum levels of the hepatocyte enzyme aspartate
transaminase (AST) rose dramatically in OT-1 mice, but
not at all in DO.11.10 mice. As shown in Table 3, AST elevation was maximal and statistically significant on day 2 in
OT-1 mice (P = 0.030), which corresponded to the time of greatest numbers of liver lymphocytes.
Because serum AST elevation parallels hepatocyte injury,
liver histology was examined before and after OVA administration to assess the degree of hepatocyte damage. Fig. 8
shows hematoxylin and eosin-stained liver sections from
OT-1 mice (left) and DO.11.10 mice (right). Livers from
nontransgenic C57BL/6 mice that received OVAI peptide
(Fig. 8 A) and from BALB/c mice that received OVAII (Fig.
8 F) showed a normal morphology of hepatocytes with few lymphocytes in the liver sinusoids. Similarly, PBS administration to OT-1 mice (Fig. 8 B) and DO.11.10 mice (Fig. 8
G) had similar normal appearances. However, there was an
intense infiltration of liver lymphocytes over the 5 d after
OVAI administration to OT-1 mice. On day 2 there was a
prominent infiltrate that was confined to the periportal region (Fig. 8 C). By day 3 this had progressed to an intense
pansinusoidal infiltration of lymphocytes accompanied by
extensive hepatocyte damage (Fig. 8 D). Thus, the peak of
observed hepatocyte damage histologically occurred 24 h after the peak serum AST elevations. By day 5 the lymphocyte infiltrate was largely resolved (Fig. 8 E). Accompanying this recovery was the appearance of massive levels of hepatocyte
mitosis (Fig. 8, E and J). Although DO.11.10 mice also manifested periportal lymphocytic infiltrates on day 2 after
OVAII (Fig. 8 H), these quickly dissipated as liver lymphocytes became more loosely scattered throughout the liver on
days 3-5 (Fig. 8 I). Throughout this process there was no evidence of hepatocyte injury in DO.11.10 mice.

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Fig. 8.
Infiltration of lymphocytes into the liver and resulting hepatocyte damage after OVA administration are both more pronounced in OT-1
mice than DO.11.10 mice. Livers were taken from mice of experiment no. 2 that had received two doses of OVA and were stained with hematoxylin
and eosin. Original magnification is ×160 except for J, which is ×1,000. Administration of OVAI to nontransgenic C57BL/6 mice (A) and OVAII to
nontransgenic BALB/c mice (F) yielded no influx of liver lymphocytes and no hepatocyte damage. Similarly, PBS administration to OT-1 mice (B) or
DO.11.10 mice (G) also produced no infiltrates. Beginning on day 2 after administration of OVAI to OT-1 mice or OVAII to DO.11.10 mice, periportal
lymphocytic infiltrates were observed in both mice but were more intense in OT-1 (C) than DO.11.10 mice (H). By day 3 a massive lymphocytic infiltrate is observed throughout the livers of only OT-1 mice with extensive hepatocyte damage (D). By day 5 the lymphocytic infiltrate in OT-1 livers is resolved (E) and in its wake is observed a dramatic burst of hepatocyte mitotic activity, shown magnified in J. By contrast, DO.11.10 mice never manifested
extensive liver sinusoidal lymphocytes as infiltrates were completely gone by day 5 without any evidence of hepatocyte damage or mitotic rebound (I).
|
|
 |
Discussion |
Our findings support a model in which the liver is a destination for lymphocytes that have been activated by a high
intensity TCR signal, many of which are destined to undergo apoptosis. Although lymphocytes may not be actively undergoing apoptosis as they enter the liver, given
the generally higher levels of Annexin V staining of intrahepatic lymphocytes compared with peripheral lymphoid tissues, it is most likely that a considerable portion are targeted for apoptosis after entry. In this study, this process
appeared to be more dynamic for CD8+ than CD4+ T
cells. Furthermore, the entry of activated CD8+ lymphocytes into intrahepatic sinusoids was responsible for extensive hepatocyte death.
The notion that "damaged" lymphocytes might not traffic
normally to peripheral lymphoid tissues but might rather
migrate to the liver has been appreciated for some time.
Treatment of lymphocytes with trypsin (23) or glycosidases
(24) before intravenous infusion prevented the cells from
circulating normally. After neuraminidase treatment of lymphocytes to remove terminal sialic acid residues, the cells
selectively migrated to the liver (25). Conceivably, recognition of such altered lymphocytes might occur via the
asialoglycoprotein receptor expressed by hepatocytes (26). One of the early events in apoptosis is the expression of surface phosphatidylserine residues on the outer surface of the
plasma membrane, which are normally confined to the inner
leaflet (19). Receptors for phosphatidylserine are expressed
by macrophages such as the Kupffer cells in the liver (27).
This might further enhance the tendency of T cells that are
targeted for apoptosis to migrate to the liver. It is conceivable
that the abrupt increase in OT-1 liver lymphocytes following administration of OVAI might represent clonal expansion of resident liver lymphocytes rather than migration to
the liver. However, this is unlikely as the fold increase of liver
lymphocytes vastly surpassed that of peripheral lymphocytes.
Furthermore, liver lymphocytes were not observed to be in
the G2/S phase of the cell cycle by propidium iodide analysis
(Russell, J.Q., unpublished observations). Thus, it is more
likely that the activated lymphocytes migrated to the liver.
In this study, CD8+ T cells from the class I MHC-
restricted TCR transgenic OT-1 mouse manifested a considerably more dramatic influx into the liver with resulting
hepatocyte damage after receiving OVAI than did CD4+ T
cells from DO.11.10 mice after an equivalent dose of OVAII. This was paralleled by a greater tendency for OT-1 CD8+
T cells in lymph node and spleen to acquire B220 expression
and to manifest a CD4
CD8
phenotype than was demonstrated by DO.11.10 CD4+ cells. These findings are in partial
agreement with those of Huang et al. (4), which showed
that after exposure to a class I MHC-restricted antigen, a portion of the responding peripheral CD8+ T lymphocytes became CD4
CD8
B220+. However, that study also observed
that the T cells that migrated to the liver after antigen exposure were predominantly of the CD4
CD8
B220+ phenotype. In contrast, we observed an influx of primarily
CD8+ cells with a smaller proportion of CD4
CD8
T cells,
although the absolute numbers of the latter population in
the liver did increase considerably. The difference in these two systems may reflect that in OT-1 mice the large proportion of CD4
CD8
B220+ T cells generated in the
lymph nodes and spleen with OVA never reached the liver
and died in situ or en route to the liver.
We (10) and others (22) have previously suggested that
the CD4
CD8
T cell phenotype reflects high intensity
TCR signaling and a tendency to undergo apoptosis. This
would be consistent with the findings that in OT-1 mice
only the high affinity OVAI peptide, and not the lower affinity E1 and R4 peptide variants, induced CD4
CD8
V
2+
cells and concomitant liver damage. It is possible that the
higher intensity signaling by OT-1 T cells was unique to this
system and not applicable to CD8+ versus CD4+ T cells in
general. In this regard, the OT-1 TCR has an affinity of 6.5 µM for OVAI/Kb (20), whereas the affinity of the DO.11.10
TCR for OVAII/I-Ad is 31 µM (21). However, in other
studies using class I- and class II-restricted TCR transgenic
mice different from those used in this study, the findings also
showed that the class I-restricted CD8+ T cells manifested a
much greater influx into the liver as well as a higher proportion of CD4
CD8
B220+ T cells (Crispe, I.N., personal
communication, and reference 4). Furthermore, as normal
mice age the liver accumulates a greater proportion of CD8+
and CD4
CD8
T cells than of CD4+ T cells (6). This may
reflect merely an increased tendency of CD8+ T cells to traffic to the liver after activation. Alternatively, the collective
findings also agree with other studies of thymocyte development, suggesting that on average CD8+ T cells may receive
higher intensity signals than do CD4+ cells (22).
It has been previously appreciated that infiltration of the
liver by Con A-activated lymphocytes produces collateral
liver damage (28). As such, that study and this may represent
models of autoimmune hepatitis in which T lymphocytes
become antigen activated in lymphoid organs and then
migrate to the liver and cause hepatocyte apoptosis. Hepatocytes may be merely innocent bystanders in this system, having little if anything to do with actual antigen presentation.
Hepatocytes express high levels of Fas and are highly susceptible to apoptosis induced by in vivo administration of anti-Fas antibody (29). However, in the Con A-induced liver
injury model, it appears that perforin plays a more prominent
role than Fas-mediated cell death (30). Studies are in progress
to examine this issue in OT-1/lpr mice. This mechanism of
lymphocyte-mediated tissue injury might also apply to other target organs and manifest itself as idiopathic autoimmune
damage. In this system, antigen 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 (Russell, J.Q., unpublished observations) and others (31) have observed that
antigen-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 where the
immune system has been strongly activated, such as by a
superantigen in Kawasaki disease (32, 33).
Beyond the parallels with autoimmune hepatitis, our
findings may also have implications for normal liver homeostasis. The resident liver lymphocytes that are observed before intentional antigen challenge may have also migrated to
the liver after activation by endogenous or environmental
antigens. This constant low level influx of activated lymphocytes may result in a continual minimal degree of hepatocyte apoptosis. In this regard, it is interesting to note that
liver enlargement due to actual increase in hepatocyte mass
has been observed in Fas-deficient mice (34).
Address correspondence to Ralph C. Budd, The University of Vermont College of Medicine, Given Medical Bldg., Burlington, VT 05405-0068. Phone: 802-656-2286; Fax: 802-656-3854; E-mail: rbudd{at}zoo.uvm.edu
We wish to thank Allison Stout for technical assistance and Colette Charland for assistance with flow cytometry.
This work was supported by grant AI-36333 from the National Institutes of Health.
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