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* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture, the Ministry of Health and Welfare, and the Health Science Foundation, Japan. 2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan. E-mail address: kiyono@biken.osaka-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or TCR
and the majority of
IELs express the cytotoxic CD8+ phenotype, either
as a CD8 homodimer or a CD8 heterodimer
(1, 2, 3). Since the 1980s, several reports have suggested
that IELs possess NK-like cytotoxic activity, even without any
stimulation (4, 5, 6). It turned out that NK-like
cytotoxicity partly depends on
CD4-CD8+ and
CD4-CD8- IELs
(7). In addition, it has been shown that IELs contain not
only T cells but also TCR-negative NK cells in mice, rats, and chickens
(7, 8, 9). However, our knowledge is limited in regard to the
biological significance of TCR- NK cells in the
IEL compartment.
IL-15 is a potent T cell growth factor that uses the IL-2R chain,
-chain, and its own IL-15R chain. IL-15 shares biological
activities but no significant sequence homology with IL-2
(10). Unlike IL-2, which is secreted only by T cells,
IL-15 mRNA is expressed by non-T cells, including kidney, placenta,
skeletal muscle, macrophages, and epithelial cells (11).
IL-15 is reported to enhance the proliferation and activation of
memory type CD8+ T cells, NK cells, and IELs
(12, 13). Analysis of IL-15R-/-
and IL-15-/- mice has demonstrated a critical
role for IL-15 in regulating the development and expansion of NK cells
and IELs (14, 15).
In this study, we report that IL-15 enhanced the syngenic cytotoxicity of a specific subset of IELs whose phenotype belongs to CD4-, CD8-, TCR-, and NK marker-positive (DX5+ or NK1.1+) subsets. Moreover, in vitro culture of IELs with IL-15 induced the preferential expansion of these intraepithelial NK cells (NK IELs). These activated NK IELs can induce cell death in intestinal epithelial cell lines via a perforin-dependent pathway. These data suggest the possibility that IL-15 produced by intestinal epithelial cells (IECs) may specifically lead to the activation of NK IELs, which in turn induce the self-killing of IECs via perforin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old C3H/HeN (H-2k) and C57BL/6 (H-2b) mice were purchased from Clea Japan (Tokyo, Japan) and maintained in the animal facility of the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan) for at least 2 wk before the experiments. The murine intestinal epithelial cell line, MODE-K (H-2k), was a kind gift from Dr. D. Kaiserlian (Institute Pasteur, Lyon, France) (16). CMT-93 (H-2b) was purchased from Dainippon Pharmaceutical (Osaka, Japan).
Reagents
Human rIL-15 and murine Fas-Fc fusion protein were purchased
from R&D Systems (Minneapolis, MN) and murine rIL-7 was purchased from
PeproTech EC (London, U.K.). For the flow cytometer analysis and cell
sorting, FITC- or biotin-labeled CD4 (RM4-5), FITC- or PE-labeled
CD8 (53-6.7), PE- or biotin-labeled CD8 (53-5.8), FITC- or
PE-labeled TCR (H57-597), FITC- or PE-labeled TCR (GL3),
FITC-labeled CD3
(145-2C11), purified Fas ligand (FasL;
MFL3), biotin-labeled hamster IgG (mixture, G70-204 and G94-56),
PE-labeled NK1.1 (PK136), allophycocyanin-labeled streptavidin,
FITC-labeled Ly-49AB6 (A1), FITC-labeled Ly-49C and I (5E6),
FITC-labeled Ly-49D (4E5), FITC-labeled Ly-49G2 (4D11), FITC-labeled
NKG2A/C/E (20d5), and biotin-labeled pan-NK (DX5) Abs were all
purchased from BD PharMingen (San Diego, CA), and PE-labeled CD3 Ab
was purchased from Serotec (Oxford, U.K.). Concanamycin A (CMA) was
purchased from WAKO (Osaka, Japan) as an inhibitor for a
perforin-mediated DNA fragmentation assay.
Cell preparations
IELs were isolated by a modified method that has been described elsewhere (17). Briefly, short segments of small intestine were stirred in RPMI 1640 with 2% FBS and 0.5 mM EDTA. After a 15-min incubation, segments were vigorously shaken and mononuclear cells were collected. To obtain lymphocyte-enriched fractions, mononuclear cells were subjected to the Percoll density gradient separation containing 40 and 75% fractions (Pharmacia Fine Chemicals, Uppsala, Sweden). This procedure has been shown to remove intestinal epithelial cells for the enrichment of IELs (18).
Culture conditions
IELs were cultured for 3–7 days in complete RPMI 1640 with 10% FBS, 50 µg/ml gentamicin, 100 µg/ml streptomycin, 100 U/ml penicillin, 50 µM 2-ME, 2 mM glutamate, 100 µM nonessential amino acid (Life Technologies, Tokyo, Japan), 25 mM HEPES buffer, and 50 ng/ml human IL-15. In some experiments, 50 ng/ml murine IL-7 instead of IL-15 was added. This dose of IL-7 and IL-15 has been shown to be optimal for the activation of IELs (19). MODE-K was precultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 50 µg/ml gentamicin, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% FBS (16).
Isolation of IEL subsets by magnetic cell sorting
IELs cultured for 3 days with IL-15 were separated into three
subsets (e.g., CD4+ and
CD4-CD8+ mixed
fraction, CD4-CD8+
fraction, or CD4- CD8-
fraction) by magnetic cell sorting (auto-MACS; Miltenyi Biotec,
Bergisch, Germany). First of all, IELs were harvested from a culture
flask and then dead cells were removed using a Dead Cell Removal kit
(Miltenyi Biotec). IELs were stained with anti-CD4-biotin and
anti-CD8-biotin for 30 min at 4°C. After being washed, IELs
were stained with streptavidin-microbeads for 15 min at 4°C. These
cells were then subjected to auto-MACS. The positive fraction contained
CD4+ and
CD4-CD8+ mixed
subsets. The negative fraction was further separated into
CD4-CD8+ and
CD4-CD8- lymphocytes by
the treatment with CD8-microbeads (53-6.7; Miltenyi Biotec).
Furthermore, CD4-CD8-
lymphocytes were separated into
CD4-CD8-
TCR+ fractions and
CD4-CD8-
TCR- fractions by auto-MACS with biotin-TCR
and biotin-TCR Ab followed by streptavidin-microbeads
incubation.
DNA fragmentation assay
A DNA fragmentation assay was performed by using a modified protocol described previously (20). Briefly, MODE-K, which was cultured for 20 h before use in a 96-well culture plate, was pulsed with 10 µCi/well [3H]thymidine for 2 h. Unincorporated [3H]thymidine was removed with PBS containing 2% FBS. MODE-K was incubated with effector cells (e.g., IELs) at various concentrations in flat-bottom 96-well plates in the presence of 50 ng/ml IL-15 or IL-7. After incubation for 6 h, cells were washed with PBS and detached from the culture plate by 0.05% trypsin-EDTA (Life Technologies) to harvest adherent cells. Incorporated [3H]thymidine was quantified using a scintillation counter. [3H]Thymidine-labeled unfragmented DNA was calculated as follows: percent DNA fragmentation = 100 x (1 - cpm experimental group/cpm control group).
Quantitative RT-PCR for measurement of perforin mRNA levels
To measure perforin-specific mRNA levels of freshly isolated and cultured IELs, quantitative RT-PCR was adapted using LightCycler (Roche Diagnostics, Mannheim, Germany) technology as described previously (21). IELs were collected and total RNA was extracted by TRIzol reagent (Life Technologies). To ensure that the same amount of synthesized cDNA was applied, the amount of cDNA labeled with digoxigenin was determined by a chemiluminescent image analyzer (Molecular Imager System; Bio-Rad, Hercules, CA). A detailed protocol for the synthesis of cDNA was previously reported by our laboratory (21). For the amplification of cDNA, 20 µl of PCR mix was added to each tube to give a final concentration of 50 µM 5' primer, 50 µM 3' primer, 200 µM FITC-labeled probe, 200 µM LightCycler Red-labeled probe, 4 mM MgCl2, and 1x LightCycler-Fast Start DNA Master Hybridization Probe Mix (Roche Diagnostics). The oligonucleotide primers specific for the perforin (sense, 5'-GACCGCACCTGCACCCTCTGT-3'; antisense, 5'-TGAAGTCAAGGTGGAGTGGAG-3'), perforin detection FITC-labeled probe (5'-CAGGACCAGTAC AACTTT AATAGCGACA-3'), and LightCycler Red 640-labeled hybrid probe (5'-AGTAGAGTGTCGCATGTACAGTTTTCG-3') were designed and produced by Nihon Gene Research Laboratories (Sendai, Japan). After being heated at 94°C for 10 min, cDNA was amplified for 45 cycles, each cycle consisting of 95°C for 15 s, 62°C for 20 s, and 72°C for 20 s. RT-PCR products of IEL using the primers above were used as an external control. After PCR was completed, LightCycler software converted the raw data into amoles per applied cDNA (1 µg) concentration of target molecules (21, 22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-7 and IL-15 produced by IECs can provide a stimulation signal
for the proliferation and survival of IELs (23). At first,
our investigation was aimed to test whether these cytokines influenced
the killing activity of IELs against IECs. Following the incubation of
the cytokine-treated IELs isolated from C3H/HeN
(H-2k) mice along with the syngenic IEC line,
MODE-K (H-2k), the level of DNA fragmentation in
MODE-K was assessed to determine the extent of cell death. Although
freshly isolated IELs showed no killing activity, IL-15-pretreated IELs
induced significantly increased levels of DNA fragmentation of MODE-K
(Fig. 1
). On the other hand, IL-7
pretreatment resulted in only a minimal increase in cytotoxic activity
(Fig. 1). These data demonstrate that IL-15 could much more
dramatically enhance the in vitro cytotoxic activity of IELs against
IECs than could IL-7.
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Because IELs contain both thymus-dependent and -independent
lymphocytes, we next sought to determine which subset of IELs possessed
the most potent IL-15-induced killing activity. Following 3 days of
culture with IL-15, IELs were separated by magnetic cell sorting into
three fractions, including the thymus-dependent
CD4+ and
CD4-CD8+ mixed
fraction, thymus-independent
CD4-CD8+ fraction,
and thymus-independent
CD4-CD8- fraction. These
three fractions were then used as effector cells in the DNA
fragmentation assay. Among these three fractions,
CD4-CD8- IELs showed the
most killing function (Fig. 2A).
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T cells)
and TCR- fractions (data not shown)
(24),
CD4-CD8- IELs were
further separated into TCR+ or
TCR- fractions by auto-MACS to
investigate which of the two fractions was the more potent killer cell
subset. Due to the limited number of cells recovered from the
CD4-CD8- fraction after
auto-MACS cell separation, IELs were initially cultured with IL-15 for
>3 days. TCR- IELs increased up to 
30% of
the entire cell population after 7 days of culture (data not shown).
DNA fragmentation assays of 7-day pretreated IELs revealed that the
CD4-CD8-TCR-
fraction is the far more potent killer fraction than the
CD4-CD8-TCR+
fraction (Fig. 2
B).
The IL-15-induced IEC killing activity of
CD4-CD8-TCR-
IELs was also demonstrated by the use of the C57BL/6 strain. CMT-93,
derived from rectal carcinoma of C57BL mice, were used as target cells.
IL-15-pretreated
CD4-CD8-TCR-
IELs of C57BL/6 mice showed far more killing activity than any other
fractions (Fig. 2C).
Inasmuch as the TCR- fraction showed
cytotoxicity against IECs, it was interesting to examine whether this
fraction expressed the NK marker. According to flow cytometric
analysis, IL-15 but not IL-7 increased the number of
TCR- IELs and these TCR-
IELs are NK marker (DX5) positive (Fig. 3A). In the case of C57BL/6
mice, we investigated the expression of NK1.1. As one might expect, the
treatment of IELs from C57BL/6 mice with an optimal concentration of
IL-15 resulted in the increase of
TCR-NK1.1+ cells (Fig. 3B). We next examined the expression of NK receptors of
IL-15-treated TCR-NK1.1+
IELs. When the expression of Ly-49 molecules and NKG2A/C/E was analyzed
by flow cytometry,
TCR-NK1.1+ IELs of C57BL/6
mice expressed Ly-49C, D, G2, and NKG2A/C/E but not Ly-49A (Fig. 3C). IL-15 treatment enhanced the expression of Ly-49C, D,
G2, and NKG2A/C/E but not Ly-49A (Fig. 3C). NKG2D mRNA was
also detected by RT-PCR both in freshly isolated and IL-15-pretreated
TCR- IEL fractions (data not shown). These
results indicate that the TCR-NK marker
+ IEL fraction is responsible for the
IL-15-mediated killing of IECs. Inasmuch as mRNA specific for CD3
and pre-TCR were not detected by RT-PCR (data not shown) but NK
receptors are expressed in this TCR-NK
marker+ IEL fraction (Fig. 3C), we
referred to this IEL subset as NK IELs.
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In general, perforin/granzyme and FasL are major molecules which
are involved in the cytotoxicity of NK cells (25). To
evaluate the role of perforin in the cytotoxic activity of NK IELs
against MODE-K, the level of perforin-specific mRNA expressed by
IL-15-treated IELs was examined by real-time quantitative RT-PCR. The
IL-15 treatment significantly increased the level of perforin-specific
mRNA expression by IELs when compared with the IL-7 treatment (Fig. 4A). When perforin expressions
by the thymus-dependent CD4+ and
CD4-CD8+ mixed
fraction, thymus-independent
CD4-CD8+ fraction,
and thymus-independent
CD4-CD8- fraction were
compared after IL-15 treatment for 3 days, the level of perforin mRNA
was the highest in the
CD4-CD8- fraction (data
not shown). Furthermore, the level of perforin mRNA was higher in
CD4-CD8-TCR-
NK IELs than in
CD4-CD8-TCR+
fractions after 7 days of treatment (Fig. 4B). In contrast,
it was interesting to note that IL-15 did not induce FasL expression in
the NK IEL subset (Fig. 4C).
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). We next assessed the role of Fas/FasL
signaling for the IL-15-induced IEC killing by IELs. When 20 µg/ml
Fas-Fc fusion protein, which has been reported to completely inhibit
the Fas/FasL signaling of IELs (20), was added to the
culture wells containing MODE-K- and IL-15-pretreated IELs, Fas-Fc
fusion protein did not prevent the death of MODE-K induced by
IL-15-stimulated IELs (Fig. 5). These findings indicate that IL-15
preferentially up-regulates perforin-mediated cytotoxic activity of NK
IELs for the induction of IEC death.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TGF-, monocyte
chemoattractant protein 1, TNF-, and GM-CSF, which act as
communication tools for the mucosal intranet (27, 28, 29, 30, 31, 32).
Bacterial or viral invasion at the intestinal mucosa results in
induction and/or up-regulation of cytokine and chemokine expression
(31, 32, 33). These biologically active factors are critical
for the regulation of both innate and acquired immunity in mucosa.
IELs, which consist of thymus-dependent and -independent T cells, have
been shown to contain subsets which possess cytotoxicity against
bacteria and viruses (3, 34, 35). It should be noted that
this cytotoxic activity is provided by both non-MHC-restricted and
MHC-mediated Ag-specific manners, corresponding to innate and acquired
immunity, respectively (5, 7, 35). For the appropriate induction and regulation of innate and acquired immunity, the mucosal intranet formed by IECs and IELs utilizes a wide variety of regulatory and inflammatory cytokines and chemokines. In particular, stem cell factor, IL-7, and IL-15 secreted by IECs are key cytokines for the development and stimulation of IELs, especially for thymus-independent IELs (19, 36). In the present study, we have provided new evidence that the cell death program of IECs is preferentially regulated by the self-production of IL-15, which activates perforin-mediated killing provided by CD4-CD8-TCR- DX5+(or NK1.1+) IELs (NK IELs).
It has been reported that IELs are capable of providing the death
signal to IECs in vivo and in vitro. Freshly isolated IELs with high
density spontaneously killed freshly isolated syngenic Fas-positive
IECs through the FasL-mediated pathway in vitro (37). It
was shown that the activation signal provided via the CD3-TCR
complex resulted in the augmentation of the FasL expression of
IELs. In the case of the mouse graft-vs-host disease model, donor
allogenic splenic cells migrated to the IEL fraction and killed
recipient IECs through the FasL-mediated pathway (20).
TCR+CD4-CD8+
IELs in particular were increased and possessed killing activity
through the FasL-dependent pathway in graft-vs-host disease. In
addition to the Fas/FasL-mediated cell killing process between IECs and
IELs, our present findings demonstrate that the death signal for IECs
is also provided via the perforin-dependent system. Our observation
that treatment with CMA but not with Fas-Fc fusion protein completely
blocked the cytotoxic activity of NK IELs clearly indicates that the
killing of IECs by IELs depends on perforin but not on FasL (Fig. 5).
Quantitative RT-PCR data further showed that IL-15 enhances the mRNA
level of perforin (Fig. 4B). The FasL expression of
TCR-DX5+ is not
up-regulated after cocultivation with IL-15 (Fig. 4C). Thus,
the IEC death process is regulated via a redundant mechanism of
Fas/FasL signaling and a perforin-mediated pathway by two distinct
subsets of IELs. TCR+ IELs with high density
express FasL for the induction of apoptosis in IECs, whereas
TCR- NK IELs are capable of inducing apoptosis
of IECs via perforin following the stimulation signal provided by
epithelial cell-derived IL-15. It might be interesting to speculate
that these two phases of apoptosis may correspond to acquired and
innate immunity, respectively.
Our data suggested that IL-15 can provide both growth- and
effector-promoting signals for the TCR- NK IELs
(
Figs. 1–3). On the other hand, IL-15 was reported to be a growth- but
not effector-promoting cytokine for murine
CD8+TCR+ IELs
(38). In humans, IL-15 was reported to be the most
effective cytokine for the enhancement of cell proliferation, IFN-
synthesis, and killing by IELs (23). From these results,
we suggest that IL-15 works in a different manner (e.g., growth- and/or
effector-promoting effects) among different subsets of effector cells
(e.g., NK, T, and T cells) even in the same epithelial
compartment.
It was shown that IL-15 enhanced the cytotoxic activity of human IELs
and made them more potent killers of the human epithelial cell line
(HT-29) (23). The study provided evidence that NK-type
IELs are not involved in this cytotoxicity since the numbers of CD16-
and CD56-positive NK cells did not change following the stimulation
with IL-15. In contrast, our finding provided new evidence that IL-15
can induce cytotoxic activity against IECs in
CD4-CD8-TCR-
IELs (Fig. 2). These triplet negative lymphocytes possess the most
potent killer activity and this fraction expressed an NK marker, DX5 or
NK1.1 (Fig. 3). This fraction did not have either CD3 or pre-TCR
mRNA. To further support this finding, our recent and separate study
showed that IL-15-treated splenic NK cells
(TCR-DX5+ cells) deliver a
cell death signal to the IEC line (data not shown). T cell-deficient
mice, both nude and SCID, have been shown to possess
CD3- NK marker-positive IELs (7).
These CD3-NK+ IELs showed
Ab-dependent cell-mediated cytotoxicity as well as cytotoxicity against
YAC-1 cells (7). To this end, our recent separate study
demonstrated that IL-15-treated NK IELs possess cytotoxic activity
against YAC-1 cells (data not shown). According to these data, it is
likely that IL-15 is capable of inducing and stimulating NK cells in
the IEL compartment for the induction of apoptosis in neighboring
IEC.
In general, NK cells do not kill syngenic cells that express the class
I MHC molecule (39), probably because inhibitory NKR
recognize the class I MHC molecules (40). IL-15 has been
reported to participate in the expression of inhibitory NKR, CD94/NKG2A
on human CD8+ T lymphocytes (41).
After stimulation by IL-15,
TCR-NK+ IELs showed
enhanced expression of Ly-49 and NKG2 molecules, which include both
activating and inhibitory receptors (Fig. 3C). Among murine
NK receptors, Ly-49D and NKG2D are activating NKR (42, 43). We observed that anti-Ly49D Ab and NKG2D monomer, which
are shown to block the binding of these molecules to their ligands,
only partially blocked the death of MODE-K induced by IL-15-pretreated
IELs (data not shown). These data may suggest the possibility that
unknown activating NKR may be up-regulated by IL-15 and may have a
critical role in the cytotoxicity of NK IELs.
NK IELs appear to be heterogeneous in view of the expression of NKR
after the treatment with IL-15 (Fig. 3C). It has been shown
that murine NK cells commonly coexpress at least two or three Ly-49 and
NKG2 receptors (44). Furthermore, NK cells expressing one
receptor are capable of expressing other NKR, while maintaining
expression of the initially expressed receptor (44).
Together with our results, it may be possible that IL-15 regulates the
expression of different NKR in a nonspecific and cumulative manner.
The production of IL-15 has been shown to be up-regulated after infection with Salmonella choleraesuis. (45), Mycobacterium tuberculosis, Toxoplasma gondii (46), Listeria monocytogenes (47), HIV (48, 49), or hepatitis C virus (50). It was reported that after oral infection of rats with L. monocytogenes, the level of IL-15 production by IECs was up-regulated and the numbers of CD3-NK+ IELs also were increased (47). Our data also demonstrate that IL-15 can expand TCR- NK IELs for the subsequent induction of cell death in IECs. Taken together, these data may also suggest the possibility that up-regulation of IL-15 synthesis enables TCR- NK IELs to kill infected IECs. Removal of infected IECs through the perforin-mediated pathway by IL-15-stimulated NK IELs may represent an important weapon in the mucosal defense system against microbial infection.
In summary, our data demonstrate that 1) IL-15 induces murine intestinal IELs to provide a killing signal to the syngenic IEC line, 2) the NK IEL fraction is the most responsible for the IL-15-induced cytotoxicity, and 3) the IEC killing provided by IL-15-stimulated NK IELs is dependent on perforin. Taken together, these findings suggest an interesting possibility that apoptosis in IECs, at least in part, is induced in a self-regulated way whereby production of IL-15 by IECs itself leads to the activation of perforin-mediated cytotoxicity of NK IELs.
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Acknowledgments |
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Footnotes |
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Division of Mucosal Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 
2 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; IEC, intestinal epithelial cell; CMA, Concanamycin A.
Received for publication July 1, 2002. Accepted for publication October 1, 2002.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) from
C3H/HeN mice and IELs were pretreated for 3 days with 50 ng/ml IL-15
(
) or IL-7 (
) as effector cells in the presence of 50 ng/ml IL-15
or IL-7 at various E:T ratios, respectively. These data are
representative of three independent experiments.
) and
CD4-CD8-TCR- (
