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+NK1.1+ T Cells for the Development of Small Intestinal Inflammation1
* Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, and Departments of
Anesthesiology, and
Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka, Japan; and
Division of Mucosal Immunolgy, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3–4 mo
of age. The numbers of CD8+ T cells, especially
CD8
+ T cells expressing NK1.1, were dramatically
increased in the lamina propria of the involved SI. The severity of
inflammation corresponded to increased numbers of
CD8+NK1.1+ T cells and levels of
production of the Th1-type cytokines IFN-
and TNF-. Locally
overexpressed IL-15 was accompanied by increased resistance of
CD8+ NK1.1+ T cells to activation-induced
cell death. Our results suggest that chronic inflammation in the SI in
this murine model is mediated by dysregulation of epithelial
cell-derived IL-15. The model may contribute to understanding the role
of CD8+ T cells in human Crohn’s disease involving the
SI. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix bundle cytokine
family, use IL-2R chain and common -chain for their action in T
cells, and have similar functional activities for the activation and
growth of T and NK cells. Despite the similarities between these two
cytokines, they differ dramatically in their expression levels at
cellular sites and the regulation levels of synthesis and secretion
(3, 4, 5, 6). IL-2 is produced by activated T cells and
controlled predominantly at the levels of mRNA transcription and
stabilization, whereas the control of IL-15 expression is much more
complex, with regulation at the levels of transcription, translation,
and intracellular trafficking (7, 8, 9).
IL-15 mRNA is widely expressed in macrophages as well as various
nonlymphoid tissues and cells, such as placenta, skeletal muscle,
kidney, lung, fibroblasts, and epithelial cells (9, 10).
Because the discovery that intestinal epithelial cells
(IECs)3 can produce
IL-15 in mice, rats, and humans, a focus of research in mucosal
immunity has been the possible role of IL-15 in the mucosal intranet
between IEC and intestinal intraepithelial lymphocytes (IEL)
(11, 12, 13). In addition, our previous study showed that
IL-15/IL-15R interaction is important in humoral aspects of mucosal
immunity such as IgA production. IL-15R chain is expressed on
mucosal B-1 but not B-2 cells, and IEC-derived IL-15 may be a selective
regulatory factor for the terminal differentiation of IgA-committed B-1
cells into IgA-producing cells (14).
IL-15 is a key regulatory cytokine, which supports innate immune cell development, activation, and homeostasis. Translation of IL-15 expression is strictly regulated at multiple distinct steps to mediate appropriate levels of the cytokine expression (4, 5, 6, 7, 8). Removal of these negative control mechanisms in an integrated fashion may lead to a major increase in IL-15 synthesis, resulting in loss of homeostasis between innate and acquired immunity. Multiple negative regulatory features controlling IL-15 expression may be required because of the potency of IL-15 as an inflammatory cytokine; if unrestrictedly expressed, IL-15, with its capacity to provide pleiotropic effects on various kinds of effector lymphoid cells, might initiate serious disorders such as autoimmune diseases.
IL-15 reportedly is overexpressed in rheumatoid arthritis (15, 16) and allograft rejection (17), and
IL-15-recruited and activated autoreactive T cells in the synovial
membrane have led to TNF- secretion in synovial fluids (15, 16). Targeted treatment for the blockade of IL-15R elements,
including IL-15R chain and the common -chain, has inhibited the
development of some experimental immunologic diseases, including
collagen-induced arthritis (18) and allograft rejection
(19, 20). Thus, IL-15 likely plays a critical role in the
immunopathology of chronic inflammation. In the mucosa-associated
inflammation of the chronic inflammatory bowel diseases, expression of
IL-15 mRNA was found to be increased in the inflamed intestinal tissues
of ulcerative colitis (21, 22, 23), and in Crohn’s disease,
expression of IL-15 was increased in intestinal macrophages
(24).
In the present study, we have developed a novel in vivo IL-15
overexpression system for the murine intestine. Localized
overexpression of intestinal IL-15 led to the development of a unique
subset of activation-induced cell death (AICD)-resistant Th1-type
CD8+ NK1.1+ T cells
and the induction of aberrant immunologic reactions in the small
intestine (SI), but not the large intestine (LI).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To develop intestinal IL-15 transgenic (Tg) mice, a
T3b-promoter system was used (Fig. 1
A;
Ref. 25). Complementary DNAs encoding the human IL-2
signal peptide (SP), the human IL-15 mature protein (MP) coding
sequence, and the FLAG epitope tag (Eastman Kodak, Rochester,
NY) were amplified by PCR and fused by use of standard PCR-based
methods as described previously (7). The rat glucagon
promoter (RGP)-IL-2SP/IL-15 MP/FLAG plasmid contained the RGP, the
IL-2SP/IL-15/FLAG cDNA, and rabbit- globin gene sequences from the
second exon to the polyadenylation signal. The
T3b-IL-2SP/IL-15 MP/FLAG transgene was
constructed by replacing the RGP promoter of the RGP-IL-2SP/IL-15
MP/FLAG plasmid with the 2.8-kb SphI-HindIII
T3b promoter region obtained from the
T3b gene cloned into the pUC18 plasmid. Finally,
a 5.5-kb SphI-XhoI
T3b-IL-2SP/IL-15/FLAG DNA fragment was purified
and used for microinjection (Fig. 1A).
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BDF1, C57BL/6Cr, and MCH-ICR mice purchased from Japan SLC (Shizuoka, Japan) were used throughout this study. Mice were maintained under specific pathogen-free conditions in the animal facility at The Research Institute for Microbial Diseases (Osaka University, Osaka, Japan). The transgene was microinjected into the pronuclei of BDF1-fertilized eggs as described (26). For screening of founder mice, tail DNA was isolated by the SDS-proteinase K method. Founders were genotyped by PCR using specific primers for the transgene. The oligonucleotide 5'-GCTGGTTATTGTGCTGCTTC-3' was used as a forward primer, and 5'-CATCTCCGGACTCAAGTGAA-3 was used as a backward primer. The PCR conditions were initially 94°C for 3 min, and 30 cycles of amplification, each cycle consisting of 94°C for 1 min, 59°C for 1 min, and 72°C for 1 min followed by an extension for 10 min at 72°C.
Semiquantitative RT-PCR for measurement of IL-2SP/IL-15 MP/FLAG mRNA
Total RNA was extracted from various tissues by using the guanidine thiocyanate procedure. DNase digestion of extracted RNA was performed before cDNA synthesis. One microgram of total RNA was reverse transcribed into cDNA using SuperScript II RT (Life Technologies, Gaithersburg, MD). To apply the same amount of synthesized cDNA from various tissues, the amounts of synthesized cDNA labeled with digoxigenin were measured with a chemiluminescent image analyzer (Molecular Imager System; Bio-Rad, Hercules, CA). PCR amplification of 10 ng of cDNA for each sample was performed with the GeneAmp PCR System 9700 (PerkinElmer/Cetus, Branchburg, NJ). The oligonucleotide primers used for the IL-2SP/IL-15 MP/FLAG mRNA were: forward, 5'-TCCTGTCTTGCATTGCACTAAG-3'; reverse, 5'-CACATTCTTTGCATCCAGATT-3'. After heating at 94°C for 2 min, cDNA were amplified for 35 cycles, each cycle consisting of 95°C for 0 s, 55°C for 30 s, and 72°C for 30 s. The amplified products were separated by electrophoresis in 1.8% agarose gel and were visualized with ethidium bromide (1 µg/ml).
Western blot analysis
The lysates of various organs were loaded directly onto 8–16% gradient gels (Tris-HCL; Bio-Rad). Proteins were electrophoresed under denaturing conditions and electroblotted to nitrocellulose membranes at 60 V for 4 h at 4°C. Membranes were blocked overnight with 1% blocking reagent (Roche Diagnostics, Mannheim, Germany) in PBS containing 0.5% Tween 20 (PBS-T) and then incubated for 2 h with mouse anti-human IL-15 mAb (clone 34593.11; R&D Systems, Minneapolis, MN) or anti-FLAG M2 mAb (Sigma-Aldrich, St. Louis, MO) diluted in PBS-T plus 2% blocking reagent. After washing of the gels with PBS-T, protein was detected by use of an ImmunoStar kit for mouse (Wako Biochemicals, Osaka, Japan) according to the manufacturer’s instructions.
Isolation of mononuclear cells
The spleen and mesenteric lymph nodes (MLNs) were aseptically removed, and single-cell suspensions were prepared by a standard mechanical disruption procedure, as described previously (27). Mononuclear cells of intestinal lamina propria (LP) were prepared by an enzymatic dissociation method using type IV collagenase (Sigma-Aldrich; Ref. 27). After removal of PP and MLN, the intestine was opened longitudinally, washed thoroughly, and cut into small fragments. Epithelial cells and IEL were removed from intestinal tissue by incubating in RPMI 1640 containing 0.5 mM EDTA and 2% FCS. The specimens were then minced and added to RPMI 1640 containing collagenase at 37°C incubation. Mononuclear cells were then isolated by use of a discontinuous density gradient procedure (40 and 75%) with Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden). For the isolation of IEC, a discontinuous density gradient (25, 40, and 75%) was also used. The cells that layered between the 40 and 25% interface were collected as IEC (28).
Abs and reagents
PE-conjugated anti-TCR (H57-597), anti-CD8
(53-6.7), anti-CD8.2 (53-5.8), anti-CD62L (MEL-14),
anti-CD44 (IM7), anti-NK1.1 (PK136), anti-CD25 (7DA), CD69
(H1.2F3), anti-CD122 (TM-1), anti-IFN- (XMG1.2),
anti-TNF- (MP6-XT22), and anti-IL-2 (JES6-5H4),
FITC-conjugated anti-CD4 (RM4-5), anti-CD8 (53-5.8),
anti-Ly-6C (AL-21), and anti-CD25 (7DA), and allophycocyanin
(APN)-conjugated anti-CD8 (53-6.7) and anti-TCR-
(H57-597) were purchased from BD PharMingen (San Diego, CA).
Biotin-conjugated anti-human IL-15 (34593.11) was obtained from R&D
Systems.
Histologic analysis
The en bloc-fixed gastrointestinal tract was dissected into stomach, jejunum, ileum, SI, LI, and rectum. Tissue specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were cut and then stained with H&E.
Flow cytometric analysis
For analysis of surface markers of lymphocytes isolated from either MLN or the LP of the SI, cells were stained with PE-, FITC-, and APN-conjugated mAbs. To block FcR-mediated binding of the mAb, anti-FcR mAb (2.4G2; BD PharMingen) was added. All incubation steps were performed at 4°C for 30 min. To detect biotin-conjugated mAb, cells were stained with APN-conjugated streptavidin after incubation with a primary mAb. The stained cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and data were analyzed by using CellQuest software (BD Biosciences). Dead cells positively stained with propidium iodide were gated out. For intracellular cytokine staining, cells from the MLN and LP were cultured with complete RPMI 1640 medium containing 10% FBS, 5 µM 2-ME, 10 U/ml penicillin, 100 µg/ml streptomycin in 12-well flat-bottom plates coated with anti-CD3 mAb (145-2C11; 10 µg/ml; BD PharMingen) for 6 h at 37°C in the presence of 5 µg/ml brefeldin A (Sigma-Aldrich). After being stained for surface Ags, cells were subjected to intracellular cytokine staining, using Cell Fixation/Permeabilization kits (BD PharMingen).
AICD
Populations of apoptotic cells in freshly isolated LP lymphocytes were detected by staining with annexin V and propidium iodide, using the Annexin V FITC Apoptosis Detection kit I (BD PharMingen) and then analyzed by flow cytometry. For flow cytometric TUNEL staining analysis, freshly isolated LP lymphocytes were analyzed by APO-DIRECT kit (BD PharMingen).
Data analysis
Data were expressed as mean ± SE and evaluated by the Mann-Whitney U test for unpaired samples, using a Statview II statistical program (SAS Institute, Cary, NC) designed for the Macintosh computer. Values of p < 0.05 were assumed to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human IL-15 was expressed under the control of the
T3b promoter, which supports specific
transcription in IEC (Fig. 1
A). Eight founder animals
were maintained by mating to C57BL/6 mice. For analysis of the tissue
specificity of transgene expression, total RNA was isolated from
various tissues of Tg and wild-type (WT) mice and subjected to
IL-15-specific semiquantitative RT-PCR analysis (Fig. 1B).
Tg IL-2SP/IL-15 MP/FLAG mRNA was expressed selectively in the
gastrointestinal tract, i.e., SI and LI; but not in other tissues,
i.e., MLN, spleen, liver, and lung. IL-15 production by IEC was
confirmed also at the protein level by Western blotting analysis (Fig. 1, C–E). Abundant IL-15 protein was present in the SI
and LI and in MLN. IL-15 was also detected, but in lower amounts, in
spleen, liver, and lung of the T3b-IL-15 Tg mice,
probably reflecting the presence of IL-15 in serum, at the level of
100 pg/ml. That IL-15 was most abundantly expressed in the
intestinal tract indicated that this newly established Tg animal could
be used as a model for examining the role of IL-15 in the intestinal
immune system.
Intestinal epithelium-specific overexpression of IL-15 causes intestinal inflammation
In two separate Tg lines, designated
T3b-IL-15 Tg-3-8 and
T3b-IL-15 Tg-10-7, macroscopic and histologic
signs of intestinal inflammation developed, beginning at about 3 mo of
age (Fig. 2, A and
B). In this report, the findings in the
T3b-IL-15 Tg-3-8 line are presented.
Macroscopically, the jejunum and proximal ileum were severely affected,
with swelling of the tissue (Fig. 2A) and sometimes,
hemorrhage. In contrast, the LI was not affected (Fig. 2A).
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, B and C). Massive infiltration of mononuclear
cells into the LP of the jejunum and proximal ileum (Fig. 2, B,
a and b), as well as Paneth cell deletion, hyperemia,
and crypt elongation were observed. Neither the stomach nor terminal
ileum was affected. Furthermore, no pathological changes were detected
in the LI (Fig. 2C). Infiltrated areas of the SI had
vacuolar degeneration of the epithelial cells, especially at the villus
tips, and occasionally in the crypts (Fig. 2Bc). In contrast
to the situation in many previous models of intestinal inflammation,
neither ulceration nor prominent influx of granulocytes was noted in
the Tg mice (Fig. 2Bd). We did find that the ratio of villus
to crypt height was 6.4:1 in WT compared with 2.1:1 in the Tg mice, a
finding suggesting that IL-15 can directly regulate the development of
IEC or that neighboring mucosal T cells activated by IL-15 can
subsequently affect the IEC development (29). The
pathologic changes in the SI became worse as the disease progressed (3
mo (Fig. 2B, e–g) vs 6 mo (Fig. 2B,
a–d)). In addition, the yield of cells from the MLN and
intestinal LP, but not from the IEL compartment, was significantly
increased in the Tg mice compared with the WT mice. This increase of
lymphocytes was not detected in other lymphoid tissues, such as the
cervical lymph nodes. The cumulative incidence of mucosal inflammation
in the T3b-IL-15 Tg-3-8 mice was nearly 100% at
6 mo of age, and most of the diseased mice eventually died despite
being reared under specific pathogen-free conditions. These results
indicate that the overexpression of IL-15 resulted in increased
lymphoid cell numbers selectively in the mucosa-associated tissues, and
this increase was associated with gross intestinal disease.
Infiltrating cells in small intestinal inflammatory lesions consist
of both CD8+ and CD8+ T cells, but
the increase in the former T cells corresponded to disease activity in
the small intestinal LP
As shown in Fig. 3A, the
numbers of CD8+ T cells, i.e.,
CD8+ and CD8+ T
cells, in the small intestinal LP of T3b-IL-15 Tg
mice were significantly increased compared with the numbers in WT
controls (9.40 ± 1.23 x 107
(T3b-IL-15 Tg) vs 0.706 ± 0.173 x
107 (WT)). Also, the degree of intestinal
pathology was proportional to the abundance of
CD8+ T cells in the diseased mucosa. As the
disease activity became more severe with age, the proportion of
CD8+ T cells profoundly increased, so that
at 6 mo of age, the proportion of CD8+ T
cells was >80% of small intestinal LP mononuclear cells (Fig. 3A). In contrast, CD8+ T cells
dominated when compared with CD8+ T cells
among the fraction of CD8+ T cells in
T3b-IL-15 Tg mice that had no disease or only
mild intestinal pathology (at 2–3 mo; Fig. 3A).
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B). In contrast, an early activation marker, CD69, was
up-regulated in T3b-IL-15 Tg mice when compared
with WT controls (Fig. 3B). It was important to note that
the IL-2R chain, but not the IL-2R chain, was up-regulated in
CD8+ T cells of
T3b-IL-15 Tg mice when compared with WT controls
(Fig. 3B). In contrast, IL-2R chain was not up-regulated
in CD8+ T cells of
T3b-IL-15 Tg mice when compared with WT controls
(Fig. 3B)
The infiltrating cells in draining lymph nodes of the intestine
consist mainly of memory-type CD8+
TCR+ T cells in the T3b-IL-15 Tg mice
Because increased numbers of activated
CD8+ T lymphocytes were selectively present
in small intestinal LP of the Tg mice (Fig. 3), we next tried to
elucidate the phenotype of the infiltrating cells in the MLN.
CD8+ cells in the MLN of
T3b-IL-15 Tg mice which were greatly increased
compared with the numbers in WT controls (46.3 ± 20.1 x
107 (T3b-IL-15 Tg) vs
0.861 ± 0.173 x 107 (WT)).
Mononuclear cells isolated from the enlarged MLN consisted exclusively
of CD8+ T cells (Fig. 4A). The usage of TCR by these
CD8+ T cells was restricted to and
heterodimers, and high expression of CD44 and Ly-6C Ags was noted
(Fig. 4A). When these
CD44highCD8+ T cells were
stained for an early activation marker, CD25, no expression was
detected (Fig. 4A). Up-regulation of CD122 (IL-2R chain),
but not CD25 (IL-2R), was observed in the
CD8+ T cells of the MLN from
T3b-IL-15 Tg mice compared with those from WT
controls (Fig. 4A). Thus, the pattern of cell surface
markers suggests that these expanded
CD44highCD8+ T cells were
resting memory-type T cells.
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+ T cells in T3b-IL-15 Tg mice
coexpressed NK1.1 molecules
Because IL-15 is important in the development and propagation of
NK and NK-T cells (30, 31), it was important to elucidate
whether the expanded CD8+ T cells express
the NK marker. In the MLN and small intestinal LP of the
T3b-IL-15 Tg mice, T cells expressing both NK1.1
and CD8 were increased (Fig. 4B). Populations of
CD8+ T cells expressing the NK marker were
not detected in the WT controls (Fig. 4B). An increase of
the classical CD4+ NK-T cells and NK cells was
not observed by flow cytometric analysis in the
T3b-IL-15 Tg mice (data not shown). Thus, the
overexpression of IL-15 induced a unique subset of
CD8+ NK-T cells rather than
CD4+ NK-T cells.
Dominant production of Th1-type cytokines by the increased
CD8+ T cells in small intestinal LP of
T3b-IL-15 Tg mice
Cell-size analysis revealed that mononuclear cells isolated from
the small intestinal LP of T3b-IL-15 Tg mice had
larger and more blastic features compared with cells from WT mice (Fig. 5A). Even though the
expression of CD25 and CD44 by CD8+ T cells
revealed a similar profile between the Tg and WT mice (Fig. 3B), the cell-sizing analysis suggested that a population of
CD8+ T cells in the
T3b-IL-15 Tg mice possesses effector function
rather than resting-memory function. This notion was further supported
by the analysis of Th1- and Th2-type cytokine synthesis (Fig. 5B), where CD8+ T cells in the
small intestinal LP of the Tg mice contained a significantly higher
percentage of the Th1-type (IFN- and TNF-) cytokine-producing
cells than did those of WT mice. However,
CD8+ T cells, another increased population
of CD8+ T cells, did not produce Th1-type
cytokines. There was no difference in IL-2 production by
CD8+ and CD8+ T
cells between T3b-IL-15 Tg and WT mice. In the
diseased T3b-IL-15 Tg mice, the degree of
intestinal pathology paralleled the levels of production of Th1-type
cytokines from the IL-15-induced CD8+ T
cells in LP mononuclear cells (3 vs 6 mo; Fig. 5B). At 3 mo
of age, T3b-IL-15 Tg mice with mild pathology
showed low levels of Th1-type cytokine production (Fig. 5B),
but when the disease became more severe, at 6 mo of age, abundant
Th1-type cytokines were produced by the IL-15-induced
CD8+ T cells (Fig. 5B). In
contrast, no synthesis of the Th2-type cytokines IL-4, IL-5, IL-6, and
IL-10 was detected from CD8+ T cells isolated
from the small intestinal LP and MLN of T3b-IL-15
Tg or WT mice (data not shown). CD8+ T
cells in the MLN secreted low amounts of IFN- and TNF- in
comparison to the amounts secreted by small intestinal LP cells.
Furthermore, to support this finding, the size of mononuclear cells
isolated from MLN was smaller than that of intestinal LP cells (data
not shown). The fact that increased levels of IFN- and TNF-
production by small intestinal CD8+ T cells
paralleled the progression of inflammation suggests that these Th1-type
cytokine-producing
CD8+NK1.1+ T cells are
important in the development of intestinal inflammation.
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Because IL-15 has been shown to extend the survival of lymphocytes
by inhibiting anti-Fas- or dexamethasone-mediated apoptosis
(32), a possible explanation for the preferential
expansion of CD8+
NK1.1+ T cells in small intestinal LP and MLN
could be the anti-apoptotic activity of IL-15. Therefore, we
examined apoptotic activity of lymphocytes in inflammatory lesions of
T3b-IL-15 Tg mice by FACS analysis, using annexin
V and propidium iodide. The overproduction of IL-15 in the SI caused a
significant (p < 0.05) decrease in the
percentage of annexin V-positive and propidium iodide-negative
apoptotic LP cells in comparison to the situation in littermate
controls (Fig. 6A). A similar
finding was generated also by flow cytometric TUNEL analysis (Fig. 6B). These results suggest that locally overexpressed IL-15
induced resistance to AICD in
CD8+NK1.1+ T cells in
the inflamed SI.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the T3b-IL-15 Tg mice, both
CD8+ and CD8+ T
cells in the small intestinal LP were dramatically increased under the
influence of overexpressed IL-15. Moreover, the increased numbers of
CD8+ T cells, but not
CD8+ T cells corresponded to disease
severity. This latter finding raised the possibility of a pathogenic
role for CD8+ T cells in the intestinal
inflammation of the T3b-IL-15 Tg mice. Relevant
to this possibility, an association of CD8+
T cells with autoimmune intestinal pathology has been reported
(34, 35). A heat shock protein 60-specific
CD8+ T cell clone (34) or
OVA-specific CD8+ T cells (35) were
associated with the development of intestinal inflammation on an
autoimmune basis. Also, in inflammatory cutaneous lesions in
IRF-2-/- mice, expansion of
CD44high, Ly-6C+, and
CD8+ T cells was found responsible, and
expression of IL-15 mRNA was increased in the skin (36).
These reports support our finding that overexpression of IL-15 induced
immunopathologic CD8+ T cells in
experimental intestinal disease.
Although we observed an increase of CD8+ T
cells in diseased T3b-IL-15 Tg mice, we noted
such an increase of CD8+ T cells in mice
that had no intestinal pathology as well. Because the number of
CD8+ T cells was decreased in the diseased
mice, CD8+ T cells could be a subset of
regulatory T cells that can inhibit disease development. The increase
of CD8+ T cells is compatible with the
reported appearance of CD8+ T cells in the
livers of IL-15 Tg mice that had no immunopathologic change despite
having high levels of IL-15 expression (37). Furthermore,
T cells, which constitute a major fraction of
CD8+ T cells, reportedly inhibited the
development of airway hypersensitivity in the lung (38, 39). These findings, together with our observations in
T3b-IL-15 Tg mice, suggest that two distinct
subsets of CD8+ T cells, expressing or
dimers, exert opposing effects (pathologic vs inhibitory) in the
intestinal inflammatory process. This interesting possibility is
currently under intensive investigation in our laboratory.
IL-15 promotes the growth of IEL (11). However, in T3b-IL-15 Tg mice we found no increase of any fraction of IEL despite the increased expression of IL-15 protein and mRNA. In contrast, there was an increase in the numbers of T lymphocytes isolated from the intestinal LP and MLN of the mice. IEC or IEC membranes can down-regulate the proliferative and cytokine responses of IEL (28). Thus, our results suggest that IEL and LPL CD8+ T cells respond differently to the regulatory effects of IEC.
Several murine models of CD4+ T cell-mediated
colitis have been attributed to an exaggerated Th1-type response
manifested by extensive production of IFN- and TNF-
(40, 41, 42); neutralization of IFN- and TNF- with mAbs
substantially improved intestinal inflammation in several of these
models (40, 43). It has been reported also that the
involvement of IFN- and TNF- in small intestinal pathology by a
heat shock protein 60-specific CD8+ clone
(34) supported the role of Th1-type cytokines produced by
CD8+ T cells in the pathogenesis of
inflammation. Together with the Th1-type cytokine profiles, our results
in the cell-sizing analysis and CD69 expression of the IL-15-induced
CD8+ T cells further indicate that the
locally expanded intestinal CD8+ T cells
have effector functions.
Recent studies have shown that CD8+ T cells could
acquire NK1.1 and NK cell-associated molecules in both in vitro and in
vivo situations (44, 45, 46). IL-2, IL-4, and IL-15 can induce
the expression of NK1.1 and NK cell-associated molecules on
CD8+ T cells after in vitro culture under
the dependent manner of cytokine signaling via IL-2R chain.
Furthermore, influenza viral infections and lymphocytic
choriomeningitis virus induced virus-specific effector
CD8+ T cells expressing NK1.1 markers (45, 46). Our present findings directly demonstrated that
overexpression of mucosal IL-15 created an immunologic environment
favorable for the development of CD8+ T
cells expressing NK1.1. Although the biological significance of NK1.1
expression by pathologic CD8+ T cells is
unknown, the development and activation of
CD8+NK1.1+ T cells in
response to the abrogation of the negative regulatory influence of
IL-15 accompanied the development of small intestinal inflammation.
The intestinal inflammation in our T3b-IL-15 Tg
mice was not accompanied by an increased production of IL-2 or
expression of IL-2R by
CD8+NK1.1+ T cells.
This finding suggests that the Th1-type
CD8+NK1.1+ T cells
were not driven by the growth and activation signals of IL-2.
Furthermore, IL-2 and IL-15 seem to differently affect the survival and
death of memory-type CD8+ T cells under the
condition of AICD (37, 47). IL-2 can promote AICD of T
cells, while IL-15 inhibits the AICD pathway. The independence of
CD8+ NK1.1 T cells from the effects of IL-2
may further promote the avoidance of AICD-associated apoptosis by
pathogenic CD8+ T cells. The balance between the
expansion of autoreactive T cells and their rapid elimination by AICD
is disturbed in intestinal inflammatory diseases (49, 50, 51, 52).
In T3b-IL-15 Tg mice, we found that intestinal
CD8+NK1.1+ T cells,
under the influence of overexpressed IL-15, circumvented AICD-induced
apoptosis and thus, continuously expanded in the intestine. In
addition, the IL-2/IL-2R-independent nature of
CD8+NK1.1+ T cells
further contributed to the avoidance of the AICD pathway. Thus, it
appears that locally produced IL-15 disrupted the balance between
proapoptotic IL-2 and anti-apoptotic IL-15, thus favoring the
propagation of pathogenic
CD8+NK1.1+ T
cells.
The restriction of inflammation to the SI in our model contrasts with the selective involvement of the LI in various other intestinal disease models (42, 48, 49, 50, 51, 52). In the colitis models, a role has been suggested for the bowel microflora (52, 53), but this does not seem to be a plausible explanation for small intestinal disease in our model. In fact, when we decontaminated the digestive tract with broad spectrum antibiotics, we found that the bacterial flora were diminished while the immunopathologic changes in the jejunum of T3b-IL-15 Tg mice remained, suggesting that bacterial microflora have a very minimal role in intestinal disease in this murine model.
Involvement of the SI is one characteristic feature of Crohn’s
disease. Thus, the preferential expansion and activation of
CD8+ NK1.1+ T cells
in the SI of T3b-IL-15 Tg mice may be relevant to
the pathology of this human inflammatory bowel disease. However, the
complexity of the biological effects of IL-15 prevents us from
ascribing the intestinal inflammation and death in our Tg mice to a
single factor. Although abundant Th1-type cytokine synthesis was noted
in intestinal CD8+
NK1.1+ T cells of the diseased
T3b-IL-15 Tg mice, low levels of Th1-type
cytokine production were observed from expanded
CD8+ NK1.1+ T cells
in MLN of the same mice. Analysis of the TCR V repertoire usage of
the CD8+ T cells in MLN and small
intestinal LP did not show any drastic skewing, but enhancement of
selective TCR V usage was noted by FACS analysis (our unpublished
observation). This result excludes the possibility that IL-15
production stimulated simple polyclonal expansion of
CD8+ NK1.1+ T cells
and the subsequent production of IFN- and TNF-. Instead, the
induction and expansion of CD8+
NK1.1+ T cells by Ags of unknown specificity may
be involved.
In summary, we have established a new and novel model of small
intestinal inflammation by the selective overexpression of IL-15 in the
murine gastrointestinal tract, using the transgene construct
T3b-hIL-2–15 FLAG. IL-15-induced
CD8+ T cells expressing
NK1.1+, CD69+, and CD122
(IL-2R)+ appear to be critical in the
pathogenesis of the small intestinal lesions in the
T3b-IL-15 Tg mice. Furthermore, this unique
subset of CD8+ T cells preferentially
produced Th1-type cytokines, and the preferential expansion of these
Th1-type CD8+ NK1.1+
T cells could be partly attributed to the anti-apoptotic activity
of IL-15. The finding of small intestinal inflammation in
T3b-IL-15 Tg mice suggests that
CD8+ T cells and IL-15 are potential targets of
therapy in chronic inflammatory diseases of the SI, such as Crohn’s
disease.
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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 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: kiyono{at}biken.osaka-u.ac.jp
3 Abbreviations used in this paper: IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte; AICD, activation-induced cell death; SP, signal peptide; MP, mature protein; RGP, rat glucagon promoter; MLN, mesenteric lymph node; LP, lamina propria; APN, allophycocyanin; Tg, transgenic; WT, wild type; SI, small intestine; LI, large intestine.
Received for publication December 27, 2001. Accepted for publication April 29, 2002.
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