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. 1998 Dec;66(12):5677–5683. doi: 10.1128/iai.66.12.5677-5683.1998

Interleukin-15 May Be Responsible for Early Activation of Intestinal Intraepithelial Lymphocytes after Oral Infection with Listeria monocytogenes in Rats

Kenji Hirose 1, Hirohiko Suzuki 1, Hitoshi Nishimura 1, Akio Mitani 1, Junji Washizu 1, Tetsuya Matsuguchi 1, Yasunobu Yoshikai 1,*
Editor: E I Tuomanen1
PMCID: PMC108717  PMID: 9826341

Abstract

Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-γ) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection with Listeria monocytogenes in rats. In in vitro experiments, invasion of L. monocytogenes into IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation of L. monocytogenes in vivo. The numbers of T-cell receptor (TCR) γδ+ T cells, NKR.P1+ cells, and CD3+ CD8+ αα cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-γ upon stimulation with immobilized anti-TCR γδ or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-γ at an early phase of oral infection with L. monocytogenes.

Interleukin-15 (IL-15) is a novel cytokine which resembles IL-2 in its biological activity (5, 16), stimulating macrophages, NK cells, T-cell receptor (TCR) αβ T cells, and B cells to proliferate, secrete cytokines, exhibit increased cytotoxicities (7, 15), and produce antibodies (Abs) (3). IL-15 mRNA is constitutively expressed in various cells and tissues such as placenta, skeletal muscle, kidney, epithelial cells, and macrophages (16). Since IL-15 expression is regulated not only at the transcriptional level but also at the translational level (36), IL-15 protein is found to be produced only by limited populations such as activated monocytes/macrophages and epithelial cells (35, 41). There have been several lines of evidence for involvement of IL-15 in infections with Salmonella sp. (35), Mycobacterium tuberculosis (14, 24), human immunodeficiency virus (10), Toxoplasma gondii (27), and hepatitis C virus (25). We have previously reported that TCR γδ T cells appearing early during the course of Salmonella infection could proliferate in response to exogenous IL-15 and IL-15 from Salmonella-infected macrophages (35). A significant number of γδ T cells, which appear in the early stage of infection, may preferentially utilize IL-15 from stimulated macrophages as a growth factor and play an important role in protection at the early phase of infection well before IL-2-producing αβ T cells appear.

Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) (1, 18, 29). i-IEL represent a unique population expressing CD8 and are able to exhibit non-major histocompatibility complex-restricted cytolytic activity. Murine i-IEL contain a large number of cells bearing TCR γδ (γδ i-IEL) (17, 31). γδ i-IEL are thought to play important roles in local immunoglobulin A response (19, 48), differentiation of i-EC (30), and surveillance against effete cells (22, 42) through cytokine production and cytotoxicity. Yamamoto et al. have reported that γδ i-IEL are stimulated to produce gamma interferon (IFN-γ) after oral infection with Listeria monocytogenes (49). We recently reported that exogenous IL-15 preferentially stimulated γδ i-IEL to proliferate and produce IFN-γ (23). It was reported elsewhere that i-EC constitutively expressed IL-15 mRNA and produced IL-15 protein (31). Taken together, it appears that IL-15 produced by i-EC is an important mediator in the intestinal immune system that serves as a primary immune barrier against microbial invasion.

To investigate whether IL-15 is involved in intestinal immune responses against intestinal infection, we examined IL-15 production by i-EC after an oral infection with L. monocytogenes in rats. In vitro experiments revealed that the invasion of L. monocytogenes into IEC-6 cells (a rat small intestine epithelial cell line) induced nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression, and consequently upregulated expression of IL-15 mRNA in IEC-6 cells. An oral inoculation with L. monocytogenes enhanced IL-15 synthesis by i-EC coincident with increases in numbers of TCR γδ+ cells, CD3+ CD8αα+ cells, and NKR.P1+ cells in i-IEL at the early phase of infection. The i-IEL exhibited an enhanced activity for IFN-γ production upon stimulation. Overall, these results suggested that IL-15 may be produced by i-EC after oral infection with L. monocytogenes and that the early IL-15 production may be involved in protection against intestinal infection through stimulation of a significant fraction of i-IEL for IFN-γ production.

MATERIALS AND METHODS

Animals.

Male F344/Slc rats, 8 weeks of age, were purchased from the Japan SLC (Hamamatsu, Japan). Rats were housed in a sterile, isolated room under specific-pathogen-free conditions.

Microorganisms.

L. monocytogenes EGD was cultured in brain heart infusion (BHI) (Difco Laboratories, Detroit, Mich.) broth at 37°C overnight, and then bacteria suspended in BHI broth containing 10% glycerol were stored at −80°C in small aliquots until use. The concentration of bacteria was quantified by plate counting.

Cell culture.

IEC-6 cells, which were established from rat small intestine cells (ATCC CRL-1592), were cultured in Dulbecco modified Eagle medium (Gibco, Grand Island, N.Y.) with 5% fetal bovine serum (FBS), penicillin (100 μg/ml), and streptomycin (100 μg/ml) at 37°C in 5% CO2.

Listeria infection assay.

L. monocytogenes was grown in BHI broth at 37°C. For each in vitro experiment, a log-phase culture of bacteria was prepared by inoculating 0.5 to 1.0 ml of an overnight culture into 4 ml of fresh BHI broth. The new culture was incubated for 3 to 5 h at 37°C with agitation to allow bacterial growth. Bacteria were washed twice by centrifugation at 12,000 × g for 3 min and then resuspended and mixed in phosphate-buffered saline (PBS). L. monocytogenes (5 × 108 CFU) was added directly to IEC-6 cells (107 cells) in complete medium without antibiotics. After the time indicated in the figure legends, cells were washed extensively with fresh medium supplemented with antibiotics to kill remaining extracellular bacteria. Then cells were incubated in fresh medium at 37°C.

Electrophoretic mobility shift assay (EMSA).

Nuclear extracts were prepared according to the modified method of Dignam et al. (12). After exposure to L. monocytogenes, IEC-6 cells (107) were washed at 4°C twice with PBS and twice with lysis buffer (10 mM Tris-HCl, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol [DTT]). Cells were homogenized with a Dounce homogenizer in lysis buffer. The homogenate was centrifuged for 10 min at 1,000 × g. The pellet was washed and centrifuged at 20,000 × g for 15 min to provide the nuclear pellet. Nuclear proteins were extracted from the pellet shaken in extraction buffer (20 mM Tris-HCl, 0.2 mM EDTA, 0.45 M NaCl, 5 mM MgCl2, 0.5 mM DTT, and 25% glycerol) for 60 min at 4°C. The supernatants containing nuclear proteins were obtained by centrifugation for 60 min at 100,000 × g. Protein concentration was normalized by bicinchoninic acid protein assay reagent (Pierce, Rockford, Ill.). The double-stranded oligonucleotides used in EMSAs were IL-15 κB (5′-TGG GAC TCC CC-3′). IL-15κB oligonucleotide was end labeled with γ-32P to be used as the probe. The binding reaction was performed on ice in a volume of 20 μl of reaction mixture (30 mM Tris-HCl, 0.6 mM EDTA, 30 mM KCl, 0.6 mM DTT, 12% glycerol), 3 × 104 cpm of γ-32P-labeled DNA probe, 5 μg of nuclear proteins, and 2 μg of poly(dI-dC). Unlabeled oligonucleotides as specific competitors (100 pmol) were added 15 min before the addition of nuclear extract. After 30 min on ice, the complexes were separated on 4% polyacrylamide gels. Densitometric analysis of DNA-protein complexes from EMSAs was performed with a Fujix BAS2000 Bio-Image analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan).

Bacterial counts in organs.

Rats were inoculated intragastrically with 3 × 109 CFU of L. monocytogenes EGD in 0.5 ml of PBS. At indicated times after inoculation, the livers, spleens, and mesenteric lymph nodes (MLN) were removed and homogenized in 5 ml of PBS. Samples were serially diluted and spread on BHI agar plates. Colonies were counted after incubation for 24 h at 37°C.

Preparation of i-IEL and i-EC.

Rats were killed 1, 3, 6, and 9 days after bacterial infection. i-IEL were separated by the Percoll gradient method. Briefly, the small intestines from rats were cut into pieces less than 5 mm and stirred at 25°C for 1 h in 199 medium (Gibco) containing 10% FBS. After stirring, the cells were passed through gauze to remove debris and coarse pieces and centrifuged through a 25%-40%-75% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient at 600 × g at 20°C for 20 min. i-EC and i-IEL were obtained at the interface of 25%-40% and 40%-75%, respectively.

Ab and reagent.

Biotin-conjugated anti-CD3 monoclonal antibody (MAb) (G4.18), phycoerythrin (PE)-conjugated anti-TCR α/β MAb (R73), fluorescein isothiocyanate (FITC)-conjugated anti-TCR γ/δ MAb (V65), FITC-conjugated anti-NKR.P1 MAb (10/78), PE-conjugated anti-CD4 MAb (OX-38), FITC-conjugated anti-CD8α MAb (OX-8), PE-conjugated anti-CD8α MAb (OX-8), FITC-conjugated anti-CD8β MAb (341), and FITC-conjugated anti-CD45 MAb (OX-1) were purchased from Pharmingen (San Diego, Calif.). Anti-IL-2 polyclonal Ab (R-20) and anti-IL-15 polyclonal Ab (L-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Streptavidin-RED 670 was purchased from Gibco.

Flow cytometric (FCM) analysis.

i-IEL or i-EC were incubated with saturating amounts of FITC-, PE-, and biotin-conjugated MAbs for 30 min at 4°C. Then cells were washed and stained with streptavidin-RED 670 for 30 min at 4°C. Cells were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.). i-IEL were carefully gated by forward and side scattering. The data were analyzed with FACScan LYSIS II software (Becton Dickinson). The contamination of i-IEL in the i-EC fraction was less than 2% as assessed by FCM analysis with anti-CD45 MAb.

Cytokine ELISA.

IFN-γ levels in the culture supernatant or serum were determined with an enzyme-linked immunosorbent assay (ELISA) commercial kit provided by Toyobo (Tokyo, Japan). i-IEL were purified and suspended in complete medium at 107 cells/ml. One hundred microliters of cell suspension was distributed in each well of the microplate, which had been coated with 10 μg of anti-TCR αβ MAb, anti-TCR γδ MAb, or anti-NKR.P1 MAb per ml at 37°C for 24 h. The supernatant was used for ELISAs.

Expression of cytokine genes.

Total RNA was extracted from i-EC from rats at indicated times, basically according to the method of Chomczynski and Sacchi (11). First-strand DNA was synthesized from 2 μg of RNA by using reverse transcriptase and 20 pmol of random primer in 20 μl of reaction buffer. Synthesized cDNA was amplified by PCR with primers derived from the rat cDNAs. The specific primers were as follows: IL-15 sense (5′ GTG ATG TTC ACC CCA GTT GC 3′) and antisense (5′ TCA CAT TCT TTG CAT CCA GA 3′); β-actin sense (5′ AGA AGA GCT ATG AGC TGC CTG ACG 3′) and antisense (5′ CTT CTG CAT CCT GTC AGC CTA CG 3′). The PCR products were subjected to electrophoresis on a 1.5% agarose gel and transferred to GeneScreen Plus filter (NEN, Boston, Mass.), and probes were labeled with [γ-32P]ATP by using the Megalabel 5′-labeling kit (Takara Shuzo Co. Ltd., Kyoto, Japan). Oligonucleotide probes were as follows: IL-15 (5′ GCA ATG AAC TGC TTT CTC CT 3′) and β-actin (5′ CTA TCG GCA ATG AGC GGT TC 3′). After hybridization in 1 M NaCl–1% sodium dodecyl sulfate–10% dextran sulfate–100 μg of heat-denatured salmon sperm DNA per ml for 18 h at 60°C, the filters were washed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–1% sodium dodecyl sulfate for 15 min at 60°C. The radioactivity of each band of PCR product was analyzed with the Fujix BAS2000 Bio-Image analyzer (Fuji Photo Film Co., Ltd.).

Measurement of IL-15 protein synthesis.

i-EC (5 × 105 cells/well) on day 1 after infection were cultured in 100 μl of complete medium for 24 h in 96-well microplates, and the culture supernatants were collected and tested for the presence of IL-15 by its ability to support the proliferation of the IL-2- and IL-15-responsive CTLL-2 cell line. CTLL-2 cells were dispensed into 96-well flat-bottom plates containing 100 μl of medium. Duplicate cultures were assayed in the presence of anti-IL-2 Ab (2 μg/well) or anti-IL-15 Ab (1 or 2 μg/well) to ensure that the effect was due to IL-15. One hundred microliters of culture supernatant was dispensed in each well and incubated at 37°C in 5% CO2 for 18 h before the addition of 1 μl of [3H]thymidine containing 0.25 μCi to each well. Plates were incubated for an additional 6 h. Then cells were harvested onto glass fiber filter paper, and proliferation was assessed by [3H]thymidine incorporation as determined with a scintillation counter.

Statistical analysis.

The statistical significance of the data was determined by Student’s t test. A P value of less than 0.05 was taken as significant.

RESULTS

IL-15 mRNA was induced by Listeria invasion of IEC-6 cells.

In order to investigate whether L. monocytogenes induces IL-15 mRNA expression in i-EC after invasion, IEC-6 cells (107), which had been established from rat small intestine epithelial cell lines, were exposed to L. monocytogenes (5 × 108 CFU) in Dulbecco modified Eagle medium containing 5% FBS for 1 h and then washed and incubated in fresh medium containing antibiotics to kill extracellular remaining bacteria. We confirmed by light microscopy that more than 30% of IEC-6 cells were invaded by L. monocytogenes (data not shown). After incubation for indicated times, IL-15 mRNA expression was detected by reverse transcriptase PCR. As shown in Fig. 1, IL-15 mRNA expression was upregulated 6 and 18 h after infection, followed by a rapid decrease at 24 h. Thus, L. monocytogenes invasion can induce the expression of IL-15 mRNA.

FIG. 1.

FIG. 1

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Expression of IL-15 mRNA in IEC-6 cells. IEC-6 cells were exposed to L. monocytogenes for 60 min. After infection, IEC-6 cells were incubated in fresh medium. Then mRNAs were extracted from IEC-6 cells incubated for 1, 6, 18, 24, and 72 h. IL-15 mRNA was detected by reverse transcriptase PCR by using specific primers for the rat IL-15 gene, and Southern hybridization was carried out with specific probes.

We have recently found that the NF-κB binding site is essential for the transcriptional activation of IL-15 in lipopolysaccharide (LPS)-stimulated macrophages (47). To examine whether L. monocytogenes activates NF-κB after invasion, nuclear extracts were prepared for NF-κB DNA binding in an EMSA. The activation of NF-κB in the IEC-6 cells reached a maximum level by 30 min after L. monocytogenes invasion, and the Listeria-induced protein-DNA complex was completely inhibited by the specific competitor, which is a nonradiolabeled probe (data not shown). From these findings, it is suggested that L. monocytogenes invasion can induce the expression of IL-15 mRNA in correlation with NF-κB activation.

Bacterial load in liver, spleen, and MLN after an oral infection with L. monocytogenes.

To investigate in vivo roles of IL-15 in intestinal infection, we tried to establish a rat model for oral infection with L. monocytogenes. First, we counted the bacterial numbers in liver, spleen, and MLN after an oral inoculation with 3 × 109 L. monocytogenes bacteria. As shown in Fig. 2, the numbers of bacteria in liver, spleen, and MLN increased by day 3 and then gradually decreased by day 9 after the oral infection with L. monocytogenes. These results suggested that L. monocytogenes invaded from the intestine and translocated to the organs.

FIG. 2.

FIG. 2

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Kinetics of bacterial growth in liver, spleen, and MLN after oral infection with L. monocytogenes. Rats were inoculated intragastrically with 3 × 109 CFU of L. monocytogenes EGD. Data were obtained from five separate experiments and were expressed as the means ± SDs at each point.

IL-15 synthesis in i-EC is upregulated by L. monocytogenes.

To determine whether L. monocytogenes delivery to the small intestine induces IL-15 expression by i-EC in vivo, we prepared i-EC from rats orally infected with L. monocytogenes and examined IL-15 expression at transcriptional and protein levels. The freshly isolated i-EC constitutively expressed IL-15 mRNA without infection, and the level of IL-15 mRNA increased on day 1 after infection and rapidly disappeared thereafter, similar to the results in in vitro experiments with the IEC-6 cell line (data not shown). The culture supernatants of i-EC from rats orally infected with L. monocytogenes 1 day previously significantly induced CTLL-2 proliferation, but those from normal rats did not (Fig. 3). The proliferation was inhibited by the addition of neutralizing anti-IL-15 MAb in a dose-dependent manner but not significantly inhibited by anti-IL-2 MAb. These results suggested that i-EC apparently produced a significant amount of IL-15 protein after Listeria infection.

FIG. 3.

FIG. 3

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Proliferation of CTLL-2 cells stimulated with culture supernatant of i-EC after infection with L. monocytogenes. i-EC (5 × 105) on day 1 after infection were recovered and cultured in 100 μl of culture medium for 24 h. CTLL-2 cells were stimulated by culture supernatant with 2 μg of anti-IL-2 Ab or 1 or 2 μg of anti-IL-15 Ab or without Ab for 24 h. The data were obtained from three separate experiments and were expressed as the means ± SDs. ∗, significantly different from values for control without Ab (P < 0.05).

Monitoring of the i-IEL populations following oral infection with L. monocytogenes.

FCM analyses for the expression of CD3, TCR αβ, TCR γδ, CD4, CD8α, CD8β, and NKR.P1 were carried out on i-IEL on days 1, 3, 6, and 9 after the oral infection with L. monocytogenes. Typical results of FCM analysis are shown in Fig. 4, and the absolute numbers of each population from five rats are summarized in Table 1. The absolute numbers of total i-IEL were (1.4 ± 0.9) × 106 (mean ± standard deviation [SD] of five rats) on day 0, (2.5 ± 0.2) × 106 on day 1, (1.9 ± 1.3) × 106 on day 3, (1.5 ± 0.7) × 106 on day 6, and (0.8 ± 0.6) × 106 on day 9 after the infection. The numbers of TCR αβ+, TCR γδ+, CD3+ NKR.P1+, and CD3− NKR.P1+ cells significantly increased on day 1 after infection. CD3+ CD8αα+ cells, which included both TCR αβ+ and TCR γδ+ T cells, also increased at this stage, whereas CD3+ CD8αβ+ cells, most of which expressed TCR αβ, only marginally increased after oral infection. Taken together, these results indicated that γδ T cells, αβ T cells bearing CD8αα, and NKR.P1+ cells preferentially increased in the i-IEL at the very early phase of oral infection with L. monocytogenes.

FIG. 4.

FIG. 4

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Surface markers of i-IEL were analyzed by FCM analysis. Expression of CD3, TCR αβ, TCR γδ CD4, CD8α, CD8β, and NKR.P1 on i-IEL on indicated days after infection with L. monocytogenes is shown. CD3+ cells were gated and analyzed except for the analysis of CD3/NKR.P1. The absolute numbers of total i-IEL, which were calculated by multiplying the total numbers of the recovered cells by the percentages of lymphocytes gated in the FCM analysis, are as follows: day 0, (1.4 ± 0.9) × 106; day 1, (2.5 ± 0.2) × 106; day 3, (1.9 ± 1.3) × 106; day 6, (1.5 ± 0.7) × 106; day 9, (0.8 ± 0.6) × 106 (means ± SDs of five rats).

TABLE 1.

Absolute cell number of each i-IEL subset after oral infection with L. monocytogenesa

Group CD3+ TCR αβ+ CD3+ TCR γδ+ CD3+ CD8αα+ CD3+ CD8αβ+ CD3− NKR.P1+ CD3+ NKR.P1+
No infection (8.9 ± 0.3) × 105 (2.7 ± 0.3) × 105 (4.2 ± 0.3) × 105 (4.3 ± 0.6) × 105 (2.7 ± 1.0) × 105 (2.0 ± 0.6) × 105
Day 1 (17.3 ± 2.0) × 105* (6.7 ± 1.8) × 105* (10.9 ± 2.1) × 105* (6.1 ± 2.2) × 105 (6.8 ± 1.5) × 105* (5.6 ± 0.4) × 105*
Day 3 (12.2 ± 0.5) × 105* (3.4 ± 0.5) × 105 (5.3 ± 1.2) × 105 (6.2 ± 1.1) × 105 (4.0 ± 0.5) × 105* (3.7 ± 1.3) × 105
Day 6 (9.7 ± 1.1) × 105 (3.5 ± 1.2) × 105 (5.3 ± 1.4) × 105 (5.0 ± 1.8) × 105 (3.4 ± 0.6) × 105 (3.3 ± 0.7) × 105
Day 9 (5.3 ± 1.1) × 105 (2.0 ± 0.4) × 105 (3.9 ± 0.4) × 105 (2.7 ± 0.2) × 105 (2.0 ± 0.4) × 105 (1.2 ± 0.4) × 105
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a

The data were obtained from five rats and are expressed as the means ± SDs. The numbers represent absolute numbers of each subset in i-IEL per mouse. ∗, significantly different from the values for noninfection controls (P < 0.05). 

IFN-γ production by i-IEL after oral infection with L. monocytogenes.

To compare the activity of i-IEL for cytokine production before and after oral infection with L. monocytogenes, i-IEL from naive rats or rats orally infected with L. monocytogenes were collected and cultured on anti-TCR αβ MAb-, anti-TCR γδ MAb-, or anti-NKR.P1 MAb-coated plates. i-IEL from naive mice produced an appreciable level of IFN-γ upon stimulation with immobilized anti-TCR αβ MAb, anti-TCR γδ MAb, or anti-NKR.P1 MAb. IFN-γ production by i-IEL from rats orally infected with L. monocytogenes 1 day previously was significantly higher after stimulation with anti-TCR γδ MAb and anti-NKR.P1 MAb but not after stimulation with anti-TCR αβ MAb (Fig. 5). These results suggest that γδ T cells and NKR.P1 cells in i-IEL are selectively activated at the early phase of oral Listeria infection.

FIG. 5.

FIG. 5

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IFN-γ production of in vitro-cultured i-IEL with Ab stimulation. i-IEL were separated from the rats inoculated with L. monocytogenes orally on day 3 and cultured with 10 μg of either anti-TCR αβ MAb, anti-TCR γδ MAb, or anti-NKR.P1 MAb for 24 h. IFN-γ concentrations in 100 μl of the culture supernatants were determined by ELISA. The data are obtained from three separate experiments and are expressed as means ± SDs. ∗, significantly different from the values for i-IEL from naive rats (P < 0.01).

Serum IFN-γ level during listerial infection was also determined with rat IFN-γ ELISA kits. IFN-γ concentration increased on days 1 and 3 and reached approximately 9,000 pg/ml on day 3. Then it gradually decreased and returned to the noninfection level on day 9 (Fig. 6).

FIG. 6.

FIG. 6

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Transient presence of IFN-γ in the serum following oral L. monocytogenes delivery. Sera from rats inoculated with L. monocytogenes orally were recovered on indicated days. The data are obtained from three separate experiments and are expressed as means ± SDs.

DISCUSSION

We here show that L. monocytogenes infection induced early activation of NF-κB in rat i-EC in vitro, resulting in upregulation of IL-15 gene expression. Coincident with early IL-15 production by i-EC, γδ T cells and NKR.P1+ cells in i-IEL increased in number and became activated for IFN-γ production at the early stage after oral infection with L. monocytogenes. It is conceivable that IL-15 produced by the infected i-EC may play an important role in the early activation of a significant fraction of i-IEL, which provide the first line of host defense against intestinal infection with microbes.

We have recently reported that 5′ upstream sequence of mouse IL-15 genomic DNA contained an NF-κB binding site and that this binding site was essential for the transcriptional activation of the IL-15 gene in macrophages stimulated with LPS (47). We here show that L. monocytogenes invasion of a rat small intestine cell line induced early activation of NF-κB and subsequently upregulated IL-15 mRNA expression. NF-κB is a transcription factor and a pleiotropic mediator of the inducible and tissue-specific gene control and is involved in the transcription of a variety of genes such as IL-1, IL-6, IL-8, and tumor necrosis factor alpha (4). NF-κB is activated upon stimulation by a large variety of pathogenic agents including LPS. The released NF-κB dimer rapidly translocates to the nucleus and activates the transcription of target genes (4). Several bacterial species without LPS have been reported to activate NF-κB (34). Similarly, L. monocytogenes is a gram-positive bacterium that does not have LPS on its outer membrane. Mengaud et al. reported that Listeria invasion of epithelial cells was mediated by Listeria surface proteins internalin A and B (33). One of the receptors for the internalins is E-cadherin, which is expressed on the nonphagocytic cells (33). E-cadherin is present only on the basolateral surface of the differentiated enterocytes (6, 9, 20). The entry processes used by L. monocytogenes for i-EC may be associated with the signal transduction cascades involving NF-κB, because recent data indicate direct links between receptors for internalins and some signaling pathways such as activation of phospholipase Cγ (28, 32).

IL-15 has a stimulatory activity for NK cells, γδ T cells, and NK1.1+ αβ T (NKT) cells via β and γ chains of IL-2 receptor (IL-2R) (7, 8, 15, 39, 46). The majority of NK and NKT cells constitutively express an intermediate-affinity IL-2R, which is composed of β and common γ subunits (39, 45, 46). Development of NK cells and NKT cells is impaired in mice lacking IL-2Rβ or IL-2Rγ chains, which are shared by IL-2 and IL-15 (13, 43, 44). Ogasawara et al. reported that interferon-regulatory factor 1 (IRF-1)-deficient mice lacked IL-15 mRNA expression after stimulation with IFN-γ and LPS (38). In the IRF-1-deficient mice, development of NK cells and NKT cells was remarkably impaired (38, 40). Thus, it is possible that IL-15 may be a key cytokine in development of NK cells and NKT cells. We previously reported that CD8αα+ T cells including γδ T cells in i-IEL preferentially proliferated in response to exogenous IL-15 (23). Ohteki et al. have recently demonstrated that i-IEL bearing CD8αα were selectively reduced in IRF-1-deficient mice (40). In our present study, CD8αα+ T cells, in addition to γδ T and NK cells, were increased in number in rat i-IEL on day 1 after infection with L. monocytogenes. These findings suggest that IL-15 produced by i-EC plays an important role in the increase of CD8αα+, NK, and NKT cells in the intestine after oral infection with L. monocytogenes.

Besides the Th1 type of αβ T cells capable of producing IFN-γ, NK cells, NKT cells, and γδ T cells are thought to play protective roles in infection with L. monocytogenes (21, 26, 37). Yamamoto et al. have reported that γδ i-IEL in mice infected orally with L. monocytogenes produced a large amount of IFN-γ, suggesting the contribution of γδ i-IEL to the local resistance against listeriosis (49). In our experiments, stimulation of i-IEL from the infected rats with anti-TCR γδ MAb induced a higher level of IFN-γ secretion than did stimulation with anti-TCR αβ MAb. Cross-linking of NK1.1 antigen is known to induce IFN-γ production by NK and NKT cells in mice (2). Consistent with this, stimulation with anti-NKR.P1 MAb evoked IFN-γ production by the i-IEL from infected rats (Fig. 6). IL-15 can stimulate NK cells and γδ T cells including i-IEL for IFN-γ production (7, 23, 35). Taken together, it appears that a significant fraction of i-IEL may be activated by IL-15 derived from infected i-EC to produce IFN-γ and to contribute to the clearance of Listeria. However, we do not have direct evidence for the protective roles of IL-15 against oral infection with L. monocytogenes. Further studies with mice lacking IL-15 may provide a direct role for IL-15-dependent i-IEL in the protection against oral infection with L. monocytogenes.

In conclusion, IL-15 is secreted from i-EC in response to L. monocytogenes invasion and γδ T cells and NK cells in i-IEL are activated to produce IFN-γ at the early stage of oral infection with L. monocytogenes. Our findings suggest that IL-15 produced by i-EC in response to Listeria invasion may have a role in the early activation of i-IEL in the intestine, which contributes to the first immune barrier of host defense against oral infection by invasive bacteria.

ACKNOWLEDGMENTS

This work was supported in part by grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan (to Y.Y.) and a Searle Scientific Research Fellowship (to H.N.).

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