Fas is not essential for lamina propria T lymphocyte homeostasis
David L. Boone,*
Themistocles Dassopoulos,*
Sophia Chai,
Marcia Chien,
James Lodolce, and
Averil Ma
Department of Medicine, Inflammatory Bowel Disease Research Center and
Committee on Immunology, The University of Chicago, Chicago, Illinois
60637
Submitted 30 August 2002
; accepted in final form 9 April 2003
 |
ABSTRACT
|
---|
IL-2 receptor
-deficient
(IL2R
-/-) mice spontaneously
accumulate vast numbers of intestinal lamina propria (LP) T cells and develop
bowel inflammation. The accumulation of T cells in
IL2R
-/- mice is thought to result, in
part, from defective Fas-induced cell death. To understand the role of cell
proliferation and death in regulating LP T cells in
IL2R
-/- mice, we have directly
examined the proliferation and Fas sensitivity of wild-type, lpr/lpr, and
IL2R
-/- LP T cells. In wild-type mice,
5'-bromodeoxyuridine (BrdU) labeling and Fas susceptibility are greatest
in CD44Hi LP T cells. Fas-deficient lpr/lpr mice have normal total
numbers of LP T cells, despite an increased proportion of BrdU+ T
cells. By contrast, IL2R
-/- mice
possess increased total numbers of LP T cells, despite normal proportions of
BrdU+ LP T cells. Finally, wild-type and
IL2R
-/- LP T cells are equivalently
Fas sensitive. These results demonstrate that LP T cells proliferate and are
Fas-sensitive cells. IL2R
-/- mice
accumulate a large number of these Fas-sensitive LP T cells and clearly differ
from Fas-deficient lpr/lpr mice in this regard. Thus our studies reveal that
Fas is dispensable for LP T cell homeostasis and suggest that the intestinal
inflammation observed in IL2R
-/- mice
is independent of defective Fas-induced cell death.
intestinal inflammation; colitis
THE MAJORITY OF INTESTINAL lamina propria (LP) lymphocytes
(LPLs) display markers consistent with prior activation
(23,
34). In addition to their
phenotypic resemblance to activated lymphocytes, LPLs are functionally
competent to perform effector functions such as cytolysis and cytokine
elaboration (5,
12,
15,
16). In this way, LPLs
effectively monitor antigen presence in the intestine and regulate the
duration and intensity of mucosal immune responses. The importance of properly
regulating the number and activity of intestinal LP T cells is underscored by
the fact that disturbances in the regulation of these cells are central to the
pathogenesis of many animal models of spontaneous bowel inflammation
(9,
21,
30). These cells are likely to
be important in the pathogenesis of human inflammatory bowel disease (IBD) as
well. Thus understanding the signals that regulate LPL activation, cycling,
survival, and differentiation into effectors is paramount to understanding the
regulation of mucosal immunity.
One of the most potent regulators of LP T cells in vivo is the cytokine
IL-2. Spontaneous bowel inflammation occurs in both IL-2-deficient
(IL-2-/-) and IL-2 receptor
-deficient
(IL-2R
-/-) mice, indicating that IL-2
signals mediated through IL-2R
are critical for downregulating these
cells in vivo (26,
33). The inflammation in
IL-2-/- mice is a T cell-dependent process
(20) and depends on
IL-12-mediated Th1 type cytokines
(10). Moreover, several
studies (14,
19,
22) have suggested that
IL-2-/- T cells may not differentiate into
cells that are fully susceptible to Fas-mediated programmed cell death (PCD).
IL-2-induced T cell activation and proliferation may increase T cell
expression of Fas ligand (FasL) and suppress expression of c-FLIP, an
inhibitor of Fas receptor signals
(24). Together, these studies
suggest that the accumulation of activated T cells, which occurs in the
absence of IL-2R
signals, is associated with muted induction of FasL
expression and a relatively Fas-resistant state of activated T cells, at least
compared with activated peripheral lymph node T cells in normal mice.
Although IL-2 receptor signals are likely associated with Fas sensitivity
of activated T cells, it remains unclear whether IL-2 and Fas receptors play
the same roles in regulating PCD of LP T cells as they do in regulating
peripheral lymph node (PLN) T cells. Although Fas has been suggested to play
an important role in regulating the numbers of activated LPLs
(2), the progressive
accumulation of activated T cells (including stereotypical CD3+
B220+ T cells) seen in peripheral lymph nodes and spleens of
Fas-deficient lpr/lprmrl mice is not seen in intestines of these
mice (1). Moreover,
Fas-deficient lpr/lprmrl mice are not known to develop bowel
inflammation similar to IL-2-/- and
IL-2R
-/- mice. Hence, the Fas receptor
may play distinct roles from IL-2R in regulating the homeostasis of LP T
cells.
To better understand the roles of Fas and IL-2 receptors in regulating LP T
cells in vivo, we have used in vivo 5'-bromodeoxyuridine (BrdU) labeling
to identify LP T cells that have passed through the S phase of the cell cycle.
We have investigated the role of Fas-mediated PCD in regulating the
homeostasis of these cells by studying the expression and functional
competency of FasL/Fas signaling in normal LP T cells and by assessing cycling
of LP T cells in lpr/lpr mice. Moreover, we have compared the homeostasis of
LPL T cells in lpr/lpr vs. IL-2R
-/-
mice (all on an inbred C57Bl/6J background) and studied the ability of
IL-2R
-/- LPLs to respond to Fas
signals. Our studies reveal novel insights into the homeostatic regulation of
intestinal T cells, including critical distinctions between the roles of Fas
and IL-2R signals in regulating these cells.
 |
MATERIALS AND METHODS
|
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Animals. Wild-type,
IL-2R
-/-, and Fas-deficient lpr/lpr
mice, all on a C57BL/6J background, were purchased from the Jackson Laboratory
(Bar Harbor, ME) and were maintained in the University of Chicago animal
facility under specific pathogen-free conditions.
Histology. Intestinal specimens were excised, fixed in buffered
formalin, embedded in paraffin, and stained with hematoxylin and eosin.
Inflammation was scored as described
(7) in the proximate, mid-, and
distal colon as follows: 1) leukocytic infiltration of the colon
(03); 2) mucin depletion (02); 3) crypt
abscesses (02); 4) epithelial erosion (02); 5)
hyperemia (02); 6) mucosal thickness (13), and the
average of the three sections was obtained. The index of the severity of
disease ranged from 1 (no disease) to 15 (severe disease). All specimens were
denoted, coded, and examined by the same person on 1 day to prevent observer
biases.
Lymphocyte collection. LPL and PLN T cells were collected as
described (20). Briefly,
intestines were excised, cleared of adhering fat, and flushed with sodium
bicarbonate free RPMI [supplemented with 10% FCS,
-mercaptoethanol,
penicillin, and streptomycin (RPMI)]. Minced pieces of this intestine were
then shaken in serum-free RPMI containing 1 mM EDTA to remove epithelial
cells, washed, and then incubated (37°C, 30 min) in RPMI with 1 mg/ml
collagenase B (Boerhinger Mannheim) and 0.25 mg/ml deoxyribonuclease I (Sigma)
in a rotary shaker. Pooled LPL from two sequential enzymatic digestions were
collected, layered over a discontinuous gradient of 44 and 70% Percoll, and
centrifuged at 800 g for 20 min. Lymphocyte-enriched fractions were
collected at the interface between 44 and 70% Percoll. The enzymes used for
isolating LPL did not affect the expression of any lymphocyte surface markers
used in this study. Resting T lymphocytes were obtained by magnetic bead
depletion of activated lymphocytes using anti-CD44 MAb (PharMingen, San Diego,
CA) and sheep anti-mouse IgG-coated Dyna-beads M-450 (Dynal, Oslo, Norway).
Activated T lymphocytes were obtained by culturing PLN T cells for 24 h in the
presence of ConA (5 µg/ml), followed by 24 h in recombinant murine IL-2 (10
ng/ml; PharMingen).
Flow cytometric analysis of lymphocytes. Lymphocytes were
incubated with FITC, phycoerythrin (PE), Cy-Chrome (CYC), or biotin-conjugated
MAbs specific for murine CD3, CD4, CD8a, CD44, Fas (PharMingen) or FasL
(kindly provided by J. Tschopp). Biotinylated MAbs were developed with CYC or
allophycocyanin-conjugated streptavidin (PharMingen). Cells were analyzed with
a FACScalibur flow cytometer using Cell Quest Software (Becton Dickinson, San
Jose, CA).
FasL-mediated lymphocyte killing. Fas-induced killing of
lymphocytes was assayed as described
(8). Briefly, lymphocytes were
cultured on 3T3 fibroblasts expressing either murine FasL (FasL fibroblasts)
or an empty PSR
vector (control fibroblasts) for 18 h, incubated with
FITC-conjugated anti-Thy1.2 MAb with or without anti-CD44 MAb, resuspended in
PBS containing propidium iodide (1.0 mg/ml), and analyzed by flow
cytometry.
In vivo analysis of lymphocyte cell cycling. BrdU was introduced
into mice by either twice-daily intraperitoneal injections (0.4 mg/injection)
or by continuous feeding in drinking water (0.8 mg/ml) for 4 days. Lymphocyte
cycling in vivo was determined by flow cytometric analysis of BrdU
incorporation into T cells using modifications of previously described
techniques (6,
32). Briefly, freshly isolated
lymphocytes were incubated with MAbs specific for lymphocyte surface antigens,
fixed, treated with DNase I, incubated with FITC-conjugated anti-BrdU antibody
(20 µl/ml; Becton Dickenson), and analyzed by flow cytometry. For some
studies, mice were thymectomized and allowed to recover for 4 wk.
Thymectomized mice were fed BrdU water for 1012 days and analyzed as
above or switched to fresh water and "chased" for the indicated
number of days.
Statistics. All data are derived from at least three independent
experiments and are shown as the means ± SD. Where indicated, paired
data sets were analyzed by Student's t-test. Groups of data were
analyzed by ANOVA followed by post hoc Tukey's. In all cases, significance was
inferred at P < 0.05.
 |
RESULTS
|
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Cycling of PLN and LP T lymphocytes. LPLs are thought to
proliferate poorly in vitro
(15,
31). However, in vitro studies
may neglect factors that normally support the proliferation of cells in vivo
(15). Moreover, many LP T
cells express high levels of the activation/memory marker CD44, which has been
associated with a higher rate of proliferation in PLN T cells in vivo
(32). We therefore determined
the extent of T cell cycling in the lamina propria relative to the periphery.
To detect cycling cells in vivo, we treated mice with BrdU for 4 days and then
isolated and immunostained LP and PLN T cells. Flow cytometric analyses of
these cells showed that the percentage of T lymphocytes that were
BrdU+ was consistently higher in the LP T cell population than in
the PLN population (10.1 ± 3.2 vs. 3.7 ± 1.1%; P <
0.001; Fig. 1A). This
was observed in mice fed BrdU water for a range of times from 2 to 5 days and
was independent of the route of BrdU administration (feeding vs. twice-daily
injections; data not shown). Consistent with prior work, the majority
(7080%) of LP T cells were CD44Hi memory phenotype cells
(Fig. 1B). Tough and
Sprent (32) demonstrated a
correlation between activation state (CD44 surface expression) and cycling
(BrdU incorporation) of PLN T cells. We therefore examined the relationship of
CD44 expression to BrdU incorporation in LP T cells. The majority of the
BrdU+ T cells in both the PLN and LPL compartments were
CD44Hi (Fig.
1B). Further analysis of these populations revealed that
a lower proportion of CD44Hi LP T cells incorporated BrdU than
CD44Hi PLN T cells (10.5 ± 2.0 vs. 16.3 ± 5.1%;
P < 0.01), although this amounted to many fewer cells in the PLN
population because of a fewer number of CD44Hi cells.

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Fig. 1. A: Lamina propria (LP) lymphocyte (LPL) T cells incorporate
5'-bromodeoxyuridine (BrdU) in vivo. Mice were fed BrdU in water for 4
days, and peripheral lymph node (PLN) or LP T cells were analyzed for BrdU
incorporation. Bars indicate the percentage of BrdU+ cells
(±SD) within the T lymphocyte population. PLN = 3.7 ± 1.1, and
LPL = 10.1 ± 3.2, n = 10. *P < 0.0001
by Student's t-test. B: CD44Hi PLN T cells
include a higher proportion of BrdU+ cells than CD44Hi
LP T cells. Representative FACSplot of T lymphocytes from mice fed BrdU water
for 4 days and analyzed for CD44 expression and BrdU incorporation is shown.
*P < 0.01, n = 8 by Student's
t-test.
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Thymocytes incorporate BrdU at a high rate in vivo and, after several days
of administration, begin to contribute significantly to the number of
peripheral BrdU+ T cells
(32). We therefore studied T
cell BrdU incorporation in thymectomized mice. After the recent thymic emigres
were removed from the analysis, we administered BrdU for longer periods
(1012 days) to obtain higher proportions of BrdU+ cells. The
percentage of BrdU+ T cells was again higher in LP T cells than in
PLN T cells (32 ± 5.4 vs. 16.6 ± 4.8%; P < 0.05;
Fig. 2A). On cessation
of BrdU administration to thymectomized mice, the percentage of
BrdU+ T cells decreased in both the LPL and PLN compartments but at
a faster rate in the LPL compartment (Fig.
2B), consistent with a more rapid turnover of T cells in
the LPL population. The rate at which the percentage of BrdU+ cells
increases is the sum of their proliferation rate minus the death rate, whereas
the rate of decay following cessation of BrdU treatment is sum of the death
rate minus the proliferation rate of BrdU-labeled cells
(4). Because substantial
proliferation is required to dilute BrdU to undetectable levels in a given
cell (4), it is unlikely that
proliferation of BrdU+ cells contributes to reduced percentages of
BrdU+ cells in a population. Instead, the loss of BrdU+
reflects the loss of labeled cells from a population. Thus the LP T population
contains significant numbers of cycling cells and is subject to negative
homeostatic regulation to prevent T cell accumulation within this
compartment.
Fas-mediated cell death in LP T lymphocytes. Because significant
numbers of LP T cells are cycling in vivo, it is apparent that this population
must be subject to negative homeostatic regulation. Because Fas-deficient mice
accumulate T cells in the periphery but not in the lamina propria, we sought
to determine whether LP T cells are insensitive to Fas-induced death or
whether Fas-deficient LP T cells display reduced cycling in vivo. The latter
possibility was considered in light of the observation that T cells deficient
for the critical Fas death-signaling molecule FADD have defective
proliferative responses to some stimuli
(35). The cell surface
expression of Fas is increased on activated T lymphocytes and is thought to
correlate with, but not ensure, their susceptibility to FasL-mediated death
(19,
28). Because the majority of
intestinal T lymphocytes display markers consistent with activation, we
compared the level of Fas expression on LP T cells with activated and resting
peripheral lymphocytes (Fig.
3). Intestinal T cells expressed elevated Fas levels, comparable
with stimulated PLN T lymphocytes (Fig.
3A). Because FasL is dynamically regulated during T cell
activation and must engage Fas to induce Fas-mediated PCD
(28), we examined the
expression of FasL on LP T cells. FasL expression on LP T cells was
negligible, comparable with resting PLN T cells, and far below the levels
found on activated PLN T cells (Fig.
3B). Thus LP T cells express high levels of Fas but not
FasL.

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Fig. 3. LP T cells express elevated levels of Fas (A) but low levels of
Fas ligand (B). Histograms are gated on T lymphocytes, and numbers
show the percentages of Fas+ or Fas ligand+ cells
representative of 4 independent experiments.
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Fas receptor is constitutively expressed and does not universally dictate
sensitivity to Fas-mediated PCD. Instead, sensitivity to Fas changes during
lymphocyte activation, possibly due to altered expression or function of
death-signaling molecules (19,
28). Because FasL may induce
distinct signals from anti-Fas agonist antibodies, we used fibroblasts
expressing membrane-bound FasL to engage Fas receptors on LP T cells
(8). Approximately 70% of
thymocytes and 35% of activated PLN T cells underwent Fas-specific PCD when
exposed overnight to FasL fibroblasts, confirming the sensitivity of these
cells to Fas-mediated PCD (Table
1). In contrast, resting PLN T cells were largely resistant to
Fas-mediated PCD (Table 1).
Freshly isolated LP T cells die at a comparable rate to that of activated PLN
T lymphocytes, indicating that LP T cells exist in a Fas-susceptible state in
vivo (Table 1). Because
peripheral T cell sensitivity to Fas-induced cell death is correlated with
activation, we examined whether the degree of CD44 expression also correlated
with Fas sensitivity of LP T cells. By assaying CD44 expression on LP T cells
that had been incubated on either FasL-expressing or control fibroblasts, we
found that CD44Hi LP T cells (compared with CD44Lo LP T
cells) represent almost all of the Fas-sensitive T cells in this population
(data not shown). This indicates that LP T cells, similar to activated
peripheral T cells, express high levels of Fas and are sensitive to
FasL-induced death. These in vitro studies are somewhat compromised by the
substantial baseline death rates of T cells in culture overnight. Studies
using both normal and lpr/lpr cells in our laboratory have found that as many
as 60% of LPL and 20% of PLN T cells die from Fas-independent mechanisms in
overnight cultures. This phenomenon, which may reflect alternate death
pathways or general metabolic stress to the cells, highlights the importance
of complementary in vivo approaches to the study of cell death in the
intestinal mucosa.
MRL mice, which develop a lymphoproliferative disorder due to defective Fas
receptor function, do not accumulate LP T cells
(1). To confirm those findings
in mice from matching genetic backgrounds, we analyzed the cellularity of PLN
and lamina propria compartments from 6- to 12-wk-old lpr/lpr and control
C57BL/6J mice. The number of lpr/lpr mouse PLN T cells was significantly
greater than in normal littermates (lpr/lpr T cells = 275 ± 150% of
normal; P < 0.01, n = 8). Flow cytometric analyses of
these lymphocytes revealed that they included CD44Hi
CD3+ B220+ T cells characteristic of lpr/lpr mice (data
not shown). By contrast, there were no significant differences in the numbers
of LP T cells between lpr/lpr and littermate controls (lpr/lpr T cells = 87
± 39% of normal; P > 0.2, n = 5). The distribution
of CD3+, CD4+, and CD8+ T cells was normal
(data not shown). In addition, CD3+ B220+ T cells were
not observed in LPL populations from lpr/lpr mice, consistent with prior
studies. To further examine the role of Fas in regulating LP T cell
homeostasis, we performed BrdU labeling studies on lpr/lpr mice. A 3.2-fold
(±1.0) higher percentage of lpr/lpr PLN T cells were BrdU+
than normal. Similarly, a 1.8-fold (±0.08) higher percentage of lpr/lpr
LP T lymphocytes were BrdU+ than normal, indicating that
Fas-deficient LP T cells are proliferating cells.
IL-2 receptor-mediated regulation of LP T cell homeostasis. IL-2
receptor signaling primes activated T cells for Fas-mediated PCD, and studies
have suggested that a failure of FasL induction and Fas signaling on activated
T cells may be central to the pathogenesis of autoimmunity in
IL-2R
-/- mice
(19,
22,
24). However, in contrast to
lpr/lpr mice, IL-2R
-/- mice accumulate
activated LP T cells, suggesting that IL-2 receptor signaling is more
important than Fas receptor signals for the negative maintenance of LP T cell
homeostasis. To directly compare the roles of IL-2 and Fas receptors in
regulating LP T cells, we compared
IL-2R
-/- and lpr/lpr mice that were
all back-crossed to the C57Bl/6J background. Consistent with previous reports
(26,
33),
IL-2R
-/- mice possessed 3- to 10-fold
greater numbers of LP T cells than normal littermates or age- and sex-matched
lpr/lpr C57BL/6J mice. We routinely recovered 12 x 106
LPL from normal mouse colon preparations. Histologically,
IL-2R
-/- mice displayed mucosal
hypertrophy, crypt abscesses, goblet cell depletion, and edema that was scored
as severe to very severe (Fig.
4). These mice typically yielded 520 x 106
LPL. There was no difference in the disease severity index between lpr/lpr and
wild-type mice. Thus IL-2R, but not Fas, regulates inflammation and T cell
number in the intestinal lamina propria.
To address how IL-2 deficiency perturbs LPL homeostasis, we administered
BrdU to IL-2R
-/- and control mice for
4 days and studied BrdU incorporation of T cells. The proportion of
BrdU+ CD4+ T cells in
IL-2R
-/- PLNs was increased
substantially over normal (Table
2). Although the absolute number of LP T cells was greater in
IL-2R
-/- than in normal mice, the
percentage of BrdU+ LP T cells was comparable between these strains
of mice (Table 2). Thus the
rate of proliferation of PLN T cells, but not LP T cells, was increased in
IL-2R
-/- mice. This suggests that
different mechanisms might underlie the accumulation of T cells in the PLN vs.
the intestinal lamina propria of
IL-2R
-/- mice.
To better understand why IL-2R signals maintain normal numbers of LP T
cells and because prior studies suggested that IL-2R signals predispose
activated PLN T cells to undergo Fas-mediated PCD
(18,
28), we asked whether IL-2
deficiency reduces the sensitivity of LP T cells to undergo Fas-mediated PCD.
Freshly harvested LP T cells from
IL-2R
-/- and control mice were
incubated with FasL-expressing fibroblasts overnight. Comparable Fas-specific
killing was observed in LP T cells freshly harvested from all strains
(Table 1). Thus IL-2R signals
are not necessary for the acquisition of Fas sensitivity by LP T cells.
 |
DISCUSSION
|
---|
The inflammation that occurs in
IL-2R
-/- mice is thought to be due, in
part, to the reduced ability of
IL-2R
-/- T cells to undergo
Fas-induced cell death (24).
This relationship between IL-2 and Fas is supported by the observation that
peripheral lymphadenopathy occurs in IL-2-/-,
IL-2R
-/-, and Fas-deficient mice.
However, spontaneous IBD occurs in IL-2-/-
and IL-2R
-/- but not Fas-deficient
mice. We therefore examined the possibility that Fas and/or IL-2 play roles in
the homeostatic regulation of LP T cells that are distinct from their roles in
peripheral T cell homeostasis. In this report, we show that although LP T
cells are Fas sensitive, Fas signals contribute only minimally to their basal
homeostatic regulation, possibly because of a paucity of FasL expression in
activated LP T cells. We also show that LP T cells are cycling in lpr/lpr mice
and also in IL-2R
-/- mice but that
these cells accumulate only in the intestinal lamina propria of the latter.
Finally, we demonstrate that FasL sensitivity is not diminished in
IL-2R
-/- LP T cells, suggesting that
the accumulation of T lymphocytes in IL-2-/-
animals is not due to a diminution of their sensitivity to Fas killing.
Our data showing that the intestinal LP T cell population includes a
significant proportion of cycling cells that rapidly turn over in vivo suggest
that this population must be subject to negative homeostatic regulation,
because the size of the LP compartment remains relatively constant. We
therefore investigated the possible role of Fas-mediated cell death as a
mechanism for negative homeostatic regulation of LP T cells. Although LP T
cells expressed high levels of Fas and were sensitive to FasL-induced killing
ex vivo, there was no increase in the number of LPLs of lpr/lpr mice compared
with littermate controls in the same genetic background. This confirms prior
studies showing no intestinal inflammation in lpr/lprmrl mice
(1). Unlike activated
peripheral T cells, the activated T cells of the gut do not display increased
FasL expression. Thus Fas may not play significant a role in intestinal T cell
homeostasis because LP T cells do not upregulate FasL. To dispel the
possibility that lpr/lpr LPLs do not accumulate owing to a lack of cycling in
the gut, we demonstrated that these cells indeed do incorporate BrdU in vivo
and that the rate of cycling was somewhat higher than that of control
littermates. The slightly higher percentage of BrdU+ cells seen in
lpr/lpr LP T cells suggests that Fas deficiency allows a greater proportion of
proliferating LP T cells to survive in vivo, even in the C57BL/6J background.
However, the maintenance of normal LP T cell numbers in lpr/lpr mice argues
that alternative cell death mechanisms compensate for the absence of
Fas-mediated PCD in these mice. Such pathways may include those mediated by
other death-inducing receptors. TNF receptors are unlikely to play this role,
because TNF-/- and
TNFR1-/- and
TNFR2-/- mice do not develop intestinal
inflammation (11,
25) and do not have increased
numbers of LP T cells (D. Boone, unpublished observations). It is possible
that LP T cell homeostasis does not require the active induction of cell death
but instead is accomplished through "death by neglect." This might
occur if LP T cells are inappropriately driven to cycle by bystander
mechanisms or bacterial induction of monocyte/macrophage cytokine elaboration
and then die as a result of no TCR ligation with their cognate antigen.
Although T cells die by neglect in the thymus, a similar process has not yet
been described in the gut. These findings do not preclude a more significant
role for Fas-mediated PCD during grossly inflammatory states. For example,
FasL expression in intestinal tissues may be induced as a result of systemic
staphylococcal enterotoxin exposure and may play a role in the death of T
cells in the intestine following their peripheral expansion
(3).
IL-2 receptor signaling primes activated T cells for Fas-mediated PCD, and
some studies have suggested that a failure of FasL induction and Fas signaling
on activated T cells may be central to the pathogenesis of autoimmunity in
IL-2-/- mice
(24). In contrast to lpr/lpr
mice, both IL-2-/- and
IL-2R
-/- mice accumulate activated LPL
T cells, suggesting that IL-2 receptor signaling may be more important than
Fas receptor signals for the negative maintenance of LP T cell homeostasis.
Our finding that IL-2R
-/-/LP T cells
are highly sensitive to FasL-induced death supports the idea that defects in T
cell Fas sensitivity are not the cause of IBD seen in
IL-2R
-/- mice. Despite this, our
observation that IL-2R
-/- LP T cells
do not cycle more rapidly than wild-type LP T cells suggests that defective
cell death processes may underlie the IBD of
IL-2R
-/- mice. In addition, we
observed a higher rate of BrdU incorporation in the PLN of
IL-2R
-/- mice and cannot discount the
possibility that the increased cell numbers in the LP of these mice was a
consequence of the peripheral expansion and recruitment of T cells into the
gut. In either case, novel IL-2-dependent death molecules that regulate the LP
T cell population may yet be discovered.
In addition, the role of regulatory cells in IBD has recently received
renewed interest (13,
29). One such population,
identified phenotypically as CD4+ CD25+ regulatory cells, has been implicated
in the SCID transfer model of colitis. These CD25+ cells produce significant
immunoregulatory transforming growth factor-
(TGF-
) and can
prevent colitis in some models of IBD
(30). Mice with targeted
disruption of TGF-
signaling in T cells develop intestinal inflammation
(17,
27). Significantly, Ehrhardt
et al. (10) showed that
treatment of mice with colitis-inducing trinitrophenol-keyhole limpet
hemocyanin causes significant TGF-
production in wild type but not
IL-2-/- mice. The absence of these
TGF-
-producing regulatory cells rather than defective Fas signaling may
underlie the IBD seen in IL-2R
-/-
mice.
 |
DISCLOSURES
|
---|
This work was supported by National Institute of Diabetes and Digestive and
Kidney Diseases Grants RO1-DK-52751 and DDRCDK-42086, National Cancer
Institute Center Grant CA-14599, National Sciences and Engineering Research
Council Canada postdoctoral fellowship (to D. L. Boone), Crohn's and Colitis
Foundation (to D. L. Boone), and the Gastrointestinal Research Foundation.
 |
ACKNOWLEDGMENTS
|
---|
We thank R. Hingorami and N. Crispe for the generous gift of
FasL-expressing 3T3 fibroblasts. We thank J. Tschopp for the generous gift of
anti-FasL antibody. We thank M. R. Binder and M. Binder for generous support
of the IBD transgenic facility. We thank M. Fitterman and S. Fitterman and A.
Edelstein and M. Edelstein for generous support of the IBD flow cytometry
Facility.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: A. Ma, Univ. of
Chicago, Medicine Dept., Committee on Immunology, MC 6084, 5841 S. Maryland
Ave., Chicago, IL 60637 (E-mail:
ama{at}medicine.bsd.uchicago.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
* D. L. Boone and T. Dassopoulos contributed equally to this work. 
 |
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