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,
Immunobiology Vaccine Center, Departments of
*
Oral Biology and
Microbiology, University of Alabama Medical Center, Birmingham, AL 35294; and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and 
ß T
cells, in the gastrointestinal tract epithelium represent a major host
defense intranet that is incompletely understood. Cell-to-cell
interactions between intraepithelial lymphocytes (IELs) and intestinal
epithelial cells (IECs) comprise this intranet, and we have assessed
the role of IECs in the regulation of and ß T cell
responses. When highly purified CD3+ IEL T cells were
stimulated via the TCR-CD3 complex, high proliferative responses and
cytokine synthesis were induced. However, the addition of viable IECs
or purified IEC membranes (mIEC) down-regulated T cell proliferative
and cytokine responses. Further, the inhibitory effect of mIEC was not
restored by antibodies to TGF-ß, CD1d, E-cadherin, or MHC class I or
II. This inhibitory effect was noted for both and ß T cell
subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and
IFN-) and Th2 (IL-4 and IL-5) cytokines in and ß IELs.
In contrast, a purified membrane fraction obtained from thymocytes did
not inhibit IEL proliferative responses. Further, mIEC did not inhibit
splenic ß T cell proliferative responses. These findings show that
cell-to-cell interactions between intraepithelial and ß T
cells and IECs occur via cell surface molecules, suggesting an intranet
to prevent potential inflammatory responses at the intestinal mucosal
surface. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or ßTCR
heterodimeric chains (2, 3, 4, 5, 6).
Inasmuch as these IELs reside in the intestinal epithelium where major
Ag trafficking from the digestive tissues into the host occurs, these
IELs provide major effector functions for the mucosal immune system.
For example, IELs exhibit both Ag-specific and polyclonal cytotoxic
activities (7, 8). Further, both and ß T cells in the
intestinal epithelium provide regulatory functions for IgA immune
responses. It has been shown that T cells can abrogate systemic
unresponsiveness induced by orally administered Ags (9, 10). In
addition, the loss of T cells from the GI tract by specific
gene deletion resulted in reduced IgA responses (11). The intestinal
CD4+, CD8-, ß T cells also provide
typical helper functions in supporting Ag-specific IgA responses (10).
These findings suggest that both and ß T cells in the
intestinal epithelium can participate as regulatory T cells for the
induction of IgA immune responses.
Our recent studies have shown that although whole IEL fractions respond
poorly to mitogens or to TCR-CD3 complex-mediated stimulation signals,
purified CD3+ IELs are capable of responding to activation
signals provided via the TCR-CD3 complex (12). Thus, T cells from the
intestinal epithelium respond to environmental Ags and mitogenic
components that occur in the intestinal lumen. However, despite the
fact that and ß T cells are continuously exposed to these
stimuli, the majority of IEL T cells remain in the resting stage (13).
These findings have also suggested possible cell-to-cell communication
provided by the neighboring IECs that may arrest cell division of
and ß T cells in the intestinal epithelium.
It is well established that intestinal epithelial cells (IECs) are
capable of expressing MHC class II molecules and can act as effective
APCs for the induction and activation of T cells in vitro (14, 15, 16). In
this regard, several groups have suggested that IECs selectively
activate T cells with suppressor function that are mediated by the CD1d
molecule, which subsequently down-regulate immune responses (17, 18, 19).
Further, it was shown that IECs can provide inhibitory signals to T
cells via a PG cascade (20). In other studies, however, the IECs
induced T cell proliferative responses in intestinal lamina propria
without MHC class I or II restriction (21). Taken together, these
results suggest that IECs can provide regulatory signals for T cells to
maintain appropriate immunologic homeostasis in the GI tract; however,
no studies have directly assessed cell-to-cell interactions between
IECs and and ß IELs in the intestinal epithelium. This
study has examined the effect of IECs on intestinal epithelial
and ß T cells stimulated by signals through the TCR-CD3
complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeN mice were obtained from Harlen Sprague-Dawley, Inc. (Indianapolis, IN). All mice were received at 5 to 6 wk of age, were maintained in horizontal lamina flow cabinets, and were provided with sterile food and water ad libitum. The mice used in this study were between 8 and 12 wk of age.
Isolation and purification of IELs, IECs, splenocytes, and thymocytes
Lymphocytes and epithelial cells were isolated by a modified
protocol as described previously (13, 22). Briefly, dissected short
segments of the small intestine from five mice were stirred at 37°C
in prewarmed RPMI 1640 containing L-glutamine,
penicillin, streptomycin, and gentamicin with 2% newborn calf serum
for 10 min followed by vigorous shaking for 15 s. This process was
performed twice, and the resulting supernatants were passed through a
small cotton-glass wool column to remove cell debris and were then
separated on a Percoll density gradient (Pharmacia Fine Chemicals,
Pharmacia, Inc., Uppsala, Sweden). A discontinuous density gradient
(25, 40, and 75%) was used. The cells that layered between the 40 and
75% fractions were collected as IELs, and the cells that layered
between the 40 and 25% interface were collected as IECs, respectively.
Highly purified IECs were then obtained by flow cytometry (FACStar
Plus, Becton Dickinson Co., Sunnyvale, CA) according to cell size and
granularity (Fig. 1
A).
Following these procedures, cell yields of >96% viable cells that
possessed alkaline phosphate activity were routinely obtained (Fig. 1A). The purity of IECs was also assessed by flow
cytometry staining with FITC-labeled anti-CD3 (145-2C11,
PharMingen, San Diego, CA), FITC-anti-Ig (Southern Biotechnology
Associates, Birmingham, AL), FITC-anti-Mac 1 (M3/84, PharMingen),
and FITC-anti-H-2Kk (AF3-12.1, PharMingen; Fig. 1A). Further, purified IECs were stained with
FITC-anti-cytokeratin mAb (PCK-26, Sigma Chemical Co., St. Louis,
MO) following cytospin, and the majority (>98%) of cells were
cytokeratin positive (Fig. 1B).
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TCR (GL3, PharMingen), biotinylated
anti-ßTCR (H57-597), FITC-anti-CD4 (GK1.5), and
biotin-anti-CD8 (53.6.72, PharMingen) followed by
streptavidin-phycoerythrin (PharMingen) to obtain highly purified T
cell subsets as described previously (9, 10, 11, 13). These cells were then
sorted into specific T cell subsets according to the expression of
TCR, ßTCR, CD4, and CD8 by flow cytometry. Our previous
study showed that the highly purified T cell subsets from the
intestinal epithelium are responsible for the activation signals
provided via the TCR-CD3 complex (12). IEC cultures
IECs were resuspended in RPMI 1640 supplemented with 5 µg/ml of transferrin, 5 µg/ml of insulin, 5 ng/ml of sodium selenite, and 20 ng/ml of epidermal growth factor (23, 24). Cells were then cultured in rat collagen type I coated 24-well plates (Collaborative Biomedical Products, Becton Dickinson Labware, Bedford, MA) for 48 h at 37°C in a moist atmosphere of 10% CO2. During this incubation period, the cell viability was maintained at >60% for 24 h and approximately 40% after 48 h.
T cell proliferation
Purified T cells (2 x 106 cells/ml) were added to 96 wells precoated with 100 µl of anti-CD3 mAb (10 µg/ml; 145-2C11; PharMingen) for 48 h at 37°C in a moist atmosphere of 10% CO2. During the last 15 h of incubation, 0.5 µCi of tritiated thymidine ([3H]TdR)/well was added, the cells were harvested, and the amount of [3H]TdR incorporation was determined by scintillation counting.
Isolation of IEC membranes
A membrane preparation of IEC (mIEC) was obtained using the hypotonic lysis method as described previously (25). Briefly, cells were washed with cold PBS, and the cell pellet was mixed with lysis buffer containing mannitol (50 mM) and HEPES buffer (pH 7.4; 5 mM). Cells were then homogenized by aspiration through a 21-gauge needle and syringe. CaCl2 was added to a 10 mM final concentration followed by additional homogenization with a 25-gauge needle. The homogenized material was placed on ice for 10 min and subjected to 3,000 x g centrifugation for 15 min to remove insoluble material. The mIEC was sedimented by centrifugation at 100,000 x g for 12 min at 4°C. Purified mIEC were then resuspended in RPMI 1640 and were dialyzed against RPMI 1640. The amount of protein in mIEC was determined by bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Co., Rockford, IL).
Ab inhibition studies
The anti-CD1d (1B1), anti-H-2Kk (AF3-12.1), anti-H-2Dk (15-5-5), and anti-I-Ak (10-3.6) mAbs were purchased from PharMingen. Anti-E-cadherin was purchased from Zymed Laboratories, Inc. (South San Francisco, CA). Anti-human TGF-ß that recognizes murine TGF-ß (26) was purchased from Genzyme (Cambridge, MA). Ten micrograms of mIEC was preincubated with 100 µg of mAbs for 30 min at 4°C. The membranes were washed with PBS to remove unbound mAbs. The mAb-treated or untreated mIEC was then incubated with 2 x 106 cells/ml of intraepithelial T cells in the wells precoated with anti-CD3 mAb.
Cytokine-specific reverse transcriptase-PCR (RT-PCR)
The Th1 and Th2 cytokine-specific RT-PCR was performed as
previously described (12, 13, 27). Briefly, RNA were extracted by the
acid guanidinium thiocyanate phenol chloroform procedure, and RNA
preparations from IEL T cells were subjected to the IFN--, IL-2-,
IL-4-, and IL-5-specific RT-PCR. Total RNA from T cells was added to a
reaction mixture containing MgCl2 (Perkin-Elmer/Cetus,
Norwalk, CT), PCR buffer II (Perkin-Elmer Cetus), dNTPs
(Perkin-Elmer/Cetus), RNase inhibitor (Promega, Madison, WI), Moloney
murine leukemia virus RT (Life Technologies, Grand Island, NY), and
oligo(dT)16 (Perkin-Elmer). Samples were reverse
transcribed, and the RT products were added to each tube with AmpliTaq
DNA polymerase, 5' primer, 3' primer, MgCl2, and PCR buffer
II (Perkin-Elmer/Cetus) and were amplified for 35 cycles. After RT-PCR,
the products were stored at 4°C until analyzed.
Capillary electrophoresis analysis of RT-PCR products
The capillary electrophoresis with the laser-induced fluorescence detection system (LIF-P/ACE, Beckman Instruments, Fullerton, CA) was applied to RT-PCR products to quantitate the relative changes in the levels of cytokine-specific mRNA as described previously (28, 29, 30). Briefly, the analysis of cytokine-specific RT-PCR products was conducted using coated capillary tubes (Beckman Instruments) in Tris-borate EDTA-containing replaceable linear polyacrylamide gel and the fluorescent intercalator. RT-PCR products were run for 25 min at 200 V/cm. The level of cytokine-specific mRNA was normalized to the corresponding ß-actin signal (30).
Statistical analysis
Results are expressed as the mean ± 1 SD of the mean, and statistical significance (p < 0.05) was analyzed by the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although freshly isolated IELs respond poorly to stimulation
signals provided by T cell mitogens or TCR-CD3 complex-specific mAbs
(4, 31, 32), our previous studies have shown that highly purified
CD3+ IEL T cells respond to stimulation via the
TCR-CD3 complex (12). Since most procedures for isolation of IELs from
the murine GI tract generally result in fractions containing
approximately 10% enterocytes (5, 13), we considered the possibility
that enterocytes actually induce hyporesponsiveness of intraepithelial
T cells stimulated with anti-CD3, anti-, or
anti-ß in vitro, effects that could mimic the in vivo
situation. As expected, highly purified CD3+ T cells
isolated from murine intestinal epithelium responded well to activation
via solid phase anti-CD3, anti-, or anti-ß mAbs
(Fig. 2). In contrast, when different
ratios of IECs were added to CD3+ IEL T cell cultures
(1:100, 1:50, 1:10, and 1:5), ratios of 1:5 and 1:10 of IEC:T cells
inhibited proliferative responses (Fig. 2), suggesting that IECs
dispatch inhibitory signals to neighboring and ß T cells in
the intestinal epithelium.
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). On the contrary, when IEC culture
supernatants were added to the CD3+ IEL cultures without
other stimulation, low, but significant, proliferation was induced
(data not shown). Taken together, these findings suggested that IECs
did not spontaneously produce inhibitory cytokines that down-regulated
IEL T cell responses.
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Thus far, our findings suggested that direct cell-to-cell
interactions between IECs and IEL T cells down-regulate T cell
activation. To directly address this issue, mIEC from intestinal
epithelium were added to anti-CD3 mAb-stimulated IEL T cell
cultures (Fig. 4). The addition of mIEC
to anti-CD3-stimulated IEL T cell cultures resulted in inhibition
of T cell proliferation (Fig. 4). As little as 5 µg of mIEC inhibited
intraepithelial T cell proliferative responses in vitro. Further,
complete inhibition of IEL T cell proliferative responses was seen with
10 µg of mIEC, whereas membrane fractions from thymocytes did not
affect the activation of intraepithelial T cells (Fig. 4). These
results showed that the mIEC could inhibit neighboring intraepithelial
CD3+ T cells in response to stimulation signals
provided through the TCR-CD3 complex. We also examined the possibility
that mIEC competitively bound to the TCR-CD3 complex with subsequent
blocking of T cell activation. To this end, CD3+ T cells
were preincubated with immobilized anti-CD3 mAb, and after 2
h, the mIEC was then added to T cell cultures. The mIEC inhibited IEL T
cell proliferation (Fig. 5), indicating
that the inhibitory effect was not due to blocking of the TCR-CD3
complex on intraepithelial T cells by mIEC.
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In the next series of studies, we tested whether the inhibitory
signals from IECs to IEL T cells were provided via the known cell
surface molecules, since several molecules and cytokines have been
shown to be involved in cell-to-cell interactions between IECs and T
cells. Thus, mIEC were incubated with anti-TGF-ß anti-CD1d,
anti-E-cadherin, anti-H-2Kk,
anti-H-2Dk, or anti-I-Ak mAbs before
cultivation with anti-CD3 stimulated IEL T cells. None of these Abs
blocked inhibitory signals of mIEC for IEL T cells (Fig. 6). Further, the addition of these mAbs
to individual wells containing anti-CD3-stimulated IEL T cells and
mIEC did not block the inhibitory effect of mIEC (data not shown).
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and ß T cells, but
not splenic T cells
Inasmuch as the study described above demonstrated that the mIEC
inhibited the activation of CD3+ IEL T cells, it was
important to examine whether the mIEC specifically inhibited a subset
of T cells, since IEL T cells contain multiple phenotypes based on the
expression of CD4, CD8, TCR, and ßTCR (5). Initially,
CD4+ and CD8+ T cells isolated from IELs or
spleen were stimulated via the TCR-CD3 complex in either the presence
or the absence of mIEC. Both purified IEL CD4+ and
CD8+ T cell subsets resulted in elevated T cell
proliferation in response to activation signals provided by
anti-CD3 mAb. The proliferative response in both IEL T cell subsets
was inhibited by mIEC (Fig. 7). In
contrast, mAb anti-CD3-activated CD4+ and
CD8+ T cells isolated from spleen were not inhibited by
mIEC (Fig. 7). These findings suggested that IECs can deliver
inhibitory signals to both CD4+ and CD8+ T
cells in the intestinal epithelium, but not to splenic T cells, via
specific cell-to-cell interactions.
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and ß T
cell subsets, since a unique feature of IEL CD3+ T cells is
an enrichment (up to 50%) of CD3+ T cells that express
chains of TCR (2, 3, 4, 5, 6). The stimulation of both intraepithelial
and ß T cells via the TCR-CD3 complex resulted in high
proliferative responses (Fig. 8), as our
previous studies have shown (12). The addition of mIEC markedly
inhibited anti-CD3-stimulated and ß T cell
proliferation; however, the proliferative responses of splenic ß T
cells remained normal, even in the presence of higher concentrations of
mIEC (Fig. 8). These findings further suggest that the IECs can
specifically down-regulate both intraepithelial and ß T
cells, but not splenic T cells, via their specific cell-to-cell
interactions.
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Our previous studies have shown that freshly isolated IELs
spontaneously produce IFN- and IL-5 in both (Fig. 9, A and B)
and ß (Fig. 9, C and D) T cell
subsets (5, 13, 33). Further, when IEL T cells were stimulated via the
TCR-CD3 complex in vitro, these T cells also produced IL-2 and IL-4
(Fig. 9, A–D) (33). Therefore, we next examined the
effect of mIEC on subsequent cytokine synthesis by anti-CD3
mAb-stimulated intraepithelial and ß T cells.
Interestingly, mIEC inhibited de novo production of IL-2- and
IL-4-specific mRNA; however, mIEC was less effective on existing mRNA
levels of IL-5 and IFN- (Fig. 9, A–D). When 10
µg/well of the mIEC fraction was added to anti-CD3-stimulated
intraepithelial and ß T cell cultures, mRNA levels for both
Th1 (IL-2 and IFN-) and Th2 (IL-4 and IL-5) cytokines were reduced
in these T cell cultures. It should be pointed out that mRNA for IL-5
and IFN- were still detected in anti-CD3 mAb-stimulated
and ß T cells even in the presence of mIEC. On the other hand,
IL-2- and IL-4-specific mRNA was not detected in these and
ß T cell cultures treated with mIEC (Fig. 9, A–D). These results showed that mIEC down-regulated
not only cell proliferation but also the expression of Th1 (IL-2) and
Th2 (IL-4) cytokine-specific mRNA, which most likely serve as T cell
growth factors within the epithelium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß T cells.
Further, this inhibitory signal was delivered from IECs to T cells via
cell surface molecules, since mIEC, but not culture supernatants,
inhibited anti-CD3 mAb-stimulated IEL and ß T cells. To
place these findings in perspective, other studies should also be
discussed. For example, it has been shown that whole IEL preparations
obtained from the murine small intestine respond poorly to T
cell-specific activation signals (4, 12, 31, 32). However, it should be
noted that most IEL preparations obtained by published procedures,
which consist of mechanical dissociation followed by discontinuous
Percoll gradient centrifugation, normally contain approximately 10%
enterocytes (4, 5, 31). On the other hand, it was shown that highly
purified CD3+ IEL T cells respond to stimulation
signals transduced via the TCR-CD3 complex (12). Taken together, these
findings suggested the possibility that even small numbers of IEC could
transduce inhibitory signals to adjacent intraepithelial and
ß T cells, which led to the present investigation.
To address the possible effects of IECs, our initial experiments were
aimed at testing whether IEC could down-regulate the activation of
intraepithelial CD3+ T cells. When IEC were added to
anti-CD3 mAb-stimulated IEL CD3+ T cell cultures at
ratios of 1:5 or 1:10, the reduction of CD3+ IEL
proliferative responses to mAb specific for CD3, , and ßTCR
were observed (Fig. 2
). In this regard, IECs have been shown to inhibit
proliferative responses of rat lymph node cells stimulated by either Ag
and/or mitogens, an effect attributable to PG secretion (20). An
additional study has shown that rat IEC culture supernatants contain an
inhibitory factor that associated with a protein with an approximate
molecular mass of 32 kDa and down-regulated proliferative responses of
lymphocytes to Con A, IL-2, or Ag (34). In human studies, it has been
shown that pretreatment of peripheral T cells with intestinal mucosa
supernatants results in the reduction of proliferative responses to
protein kinase C activators (e.g., phorbol 12,13-dibutyrate and
ionomycin) (35). In addition, several studies have reported that IECs
were capable of producing inhibitory cytokines such as TGF-ß
(36, 37, 38, 39). In the present study, however, the addition of IEC culture
supernatants did not inhibit T cell proliferative responses in mAb
anti-CD3-stimulated IEL T cells (Fig. 3). Furthermore, treatment of
mIEC with anti-TGF-ß mAb did not block inhibitory signals from
IECs (Fig. 6). These results indicated that IEC did not spontaneously
produce inhibitory molecules such as TGF-ß and/or the membrane form
of TGF-ß for CD3+ IEL proliferative responses in vitro.
However, after appropriate stimulation of IECs, these cells may produce
inhibitory cytokines. Instead, it was interesting to note that addition
of IEC culture supernatants induced CD3+ IEL proliferative
responses in the absence of any other stimulus (data not shown). In
this regard, our separate study has shown that IECs express mRNA for
IL-7, and this cytokine has been shown to induce the proliferation of
CD3+ IEL T cells (22).
The mIEC obtained from intestinal epithelium showed inhibitory effects
on intraepithelial T cell proliferative responses induced via the
TCR-CD3 complex. Further, different T cell subsets of activated IEL T
cells, including both and ß T cells as well as
CD4+ and CD8+ T cell subsets, were inhibited by
mIEC. However, this effect was not seen with splenic T cells. These
findings suggest two related possibilities: 1) that specific
cell-to-cell interactions occur between IECs and IELs to provide
down-regulatory signals from the former to the latter cell population;
and 2) that the inhibitory signal was provided by novel and undefined
cell surface molecules, including adhesion and/or ligand molecules.
Since we did not examine the effect of cell membrane molecules from
other epithelial cells or cell lines, it will be important to further
examine cell membrane fractions from epithelial cells of different
tissues to elucidate the latter possibility. A recent study has
provided supportive evidence that specific IEC and IEL cross-talk
occurs in the intestinal epithelium, where the removal of TCR gene
(e.g., lack of IEL) results in a reduction in the generation and
differentiation of IECs (40). Together with the results of our current
study, it is now clear that a intranet between IEL and IEC is an
important cellular and molecular cross-talk for the maintenance of an
appropriate immnologic homeostasis in the GI tract. An interesting
aspect of a present study is the possibility of a novel and undefined
cell surface molecule(s) expressed on IEC that may contribute to this
mucosal intranet.
Others have reported that the inhibitory effect of IECs on the
lymphocyte proliferative response is related to the activation of
CD8+ T cells (18, 41). In the rat system, these
IEC-activated CD8+ T cells from lymph nodes have been shown
to suppress Ag-specific proliferative responses (41). In contrast,
human IEC-stimulated peripheral blood CD8+ T cells were
found to down-regulate proliferative responses in a polyclonal fashion
(18). Thus, based on previous studies the inhibitory mechanisms
provided by IECs could be explained by the induction of
CD8+ T cells with suppressor functions. For cellular
interactions between IECs and CD8+ T cells, it has been
shown that CD1d, a class I-like molecule expressed on murine and human
epithelial cells (42, 43), may play an important role, since Abs to
CD1d were capable of inhibiting proliferation of CD8+ T
cells in these cocultures (19). However, a recent study demonstrated
that CD1d transfectants did not activate CD8-associated
p56lck, a src-like tyrosine kinase
that is necessary for activation of CD8+ T cells (44).
Further, anti-CD1d mAb did not block the inhibitory effects of
mIEC. In this regard, Abs to MHC class I or II also failed to block
inhibitory signals from IECs (Fig. 6). Eß7
integrin has been shown to be expressed on IEL T cells, but not on
peripheral T lymphocytes (45), and mediates specific adhesive
interactions between IELs and IECs (46, 47). Further, it has been
reported that heterotypic adhesion between IECs and IELs is mediated by
E-cadherin and Eß7 integrin (48, 49).
Thus, an alternative possibility would be that signals through
E-cadherin may inhibit the activation of T cells expressing
Eß7 integrin. However,
anti-E-cadherin-treated mIEC still inhibited IEL T cell
proliferative responses. Taken together, these results suggest that
novel and yet undefined molecule(s) expressed on IEC may provide
inhibitory effects for intraepithelial T cells, and this inhibition is
not due to CD8+ T cells. However, our results have not
excluded the possibility that other known cell surface molecules may
indeed contribute to the inhibition of intraepithelial T cell
responses.
From an anatomical and histologic standpoint, the intestinal epithelium
contains a large number of CD3+ T cells that are located
adjacent to IECs (7, 8). Interestingly, approximately 80% of these
CD3+ IELs are CD8+ (2, 3, 4, 5, 6, 7, 8). Therefore, it was
natural to assume that IECs may provide activation signals to
intraepithelial CD8+ T cells that lead to active
suppression. However, our results showed that IECs dispatch inhibitory
signals to neighboring intraepithelial CD4+ T cells,
CD8+ T cells, T cells, and ß T cells via direct
cell-to-cell contact. Possible explanations for these findings could be
that compared with the peripheral T cells, which are anatomically and
completely isolated from the direct and continuous exposure to
environmental Ags, IELs are chronically exposed to microenvironmental
Ags and mitogens from the intestinal epithelium. If IELs chronically
responded to these Ags, it would create a hyper-T cell-reactive region
in the intestinal epithelium that would lead to severe inflammation.
Therefore, specific inhibitory signals generated by IECs to neighboring
intraepithelial and ß T cells would be an important
mechanism for the maintenance of immunohomeostasis in the intestinal
environment. To support this view, our previous results have shown that
although freshly isolated IELs contain some CD3+ T cells
that spontaneously secrete Th1- and Th2-type cytokines, >90% of T
cells were in the inactive cell cycle stage of G0 to
G1 (13).
Freshly isolated intraepithelial and ß T cells expressed
IL-5 and IFN- mRNA, but not IL-2 and IL-4. When these
CD3+ IELs were stimulated by mAb anti-CD3, IL-2- and
IL-4-specific mRNA were induced in addition to the enhanced IL-5- and
IFN--specific mRNA expression (Fig. 9, A-D), in
accord with our earlier findings (5, 13, 33). It was important to note
that mIEC inhibited the expression of these Th1 and Th2
cytokine-specific mRNA in both and ß T cells. These
observations have further shown that mIEC provide inhibitory signals
that down-regulate the expression of Th1 and Th2 cytokine-specific mRNA
in intraepithelial and ß T cells. It is well known that Th1
and Th2 cells down-regulate each other via specific cytokines (e.g.,
IFN- and IL-10, respectively) (50). However, mIEC inhibited both Th1
and Th2 cytokines simultaneously. This interesting finding provided two
possibilities. The first is that new or known inhibitory substances
(e.g., TGF-ß) that down-regulate both Th1 and Th2 responses are
produced by IELs following the interaction with cell surface molecules
on IECs. Alternatively, a unique and new signal through a surface
molecule on IEC may directly inhibit both Th1 and Th2 cytokine
activation pathways. These issues are currently under investigation by
our group.
It is interesting to note that although the levels of elevated IFN-
and IL-5 mRNA were decreased following treatment of activated
and ß T cells with mIEC-treated IELs, these cytokine-specific mRNA
were still detected. On the other hand, the expression of IL-2- and
IL-4-specific mRNA was completely inhibited by the presence of mIEC.
Thus, the profile of Th1 and Th2 cytokines seen in the mIEC-treated
intraepithelial and ß T cells (e.g., IFN- and IL-5, but
not IL-2 and IL-4) that had been activated via the TCR-CD3 complex is
similar to that seen in the freshly isolated and ß IELs
(Fig. 9, A-D). Thus, it appeared that expression of
cytokine-specific mRNA by IELs is down-regulated by IECs in vivo. Since
intraepithelial and ß T cells are continuously exposed to a
wide variety of intestinal Ags and stimulatory molecules,
down-regulatory signals provided by IECs would be necessary for the
maintenance of an appropriate cytokine balance in the GI tract
environment. Thus, these IECs down-regulate quiescent IEL T cells that
are otherwise responsive to exogenous and pathogenic agents, including
bacteria, virus, and parasites.
In summary, our study is the first to show that IECs directly inhibit
proliferative responses as well as cytokine synthesis in neighboring,
activated intraepithelial and ß T cells induced by
stimulation signals through the TCR-CD3 complex. However, IECs did not
produce inhibitory molecules spontaneously. Further, the inhibitory
signals from IECs were not blocked by Abs to TGF-ß, CD1d, E-cadherin,
or MHC class I or II, suggesting that specific molecules expressed by
IECs may provide an inhibitory signal(s) to intraepithelial and
ß T cells. Our current effort is focused on the isolation and
characterization of this unknown molecule for the understanding of the
precise mechanisms for inhibitory signal transduction from IECs to
IELs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, 3–1 Yamada-oka, Suita, Osaka 565, Japan.
3 Abbreviations used in this paper: GI, gastrointestinal; S-IgA, secretory immunoglobulin A; IELs, intraepithelial lymphocytes; IECs, intestinal epithelial cells; mIEC, intestinal epithelial cell membranes; RT-PCR, reverse transcriptase-polymerase chain reaction.
Received for publication February 25, 1997. Accepted for publication November 11, 1997.
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) or CD8+ (
) T cells were
isolated from IELs or spleen and incubated with mIEC in the presence of
immobilized anti-CD3 mAb. This result is representative of four
separate experiments. * p < 0.05 compared with
cultures stimulated with anti-CD3 only. **Control indicates culture
containing cells only.
