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Regulatory Role of Peritoneal NK1.1⁺ß T Cells in IL-12 Production During Salmonella Infection¹

Yoshikazu Naiki^*, Hitoshi Nishimura^2,*, Tetsu Kawano, Yujiro Tanaka, Shigeyoshi Itohara, Masaru Taniguchi and Yasunobu Yoshikai^*

^* Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, and Division of Molecular Immunology, Center for Biomedical Science, School of Medicine, Chiba University, Chiba, Japan; and Institute for Physical and Chemical Reseach Brain Science Institute, Saitama, Japan

	Abstract

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NK1.1⁺ß T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1⁺ß T cells during murine salmonellosis, mice lacking NK1.1⁺ß T cells by disruption of TCRß (TCRß^-/-), ß₂m (ß₂m^-/-), or J281 (J281^-/-) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCRß stimulation, whereas those in TCRß^-/-, ß₂m^-/-, or J281^-/- mice showed no IL-4 production upon the stimulation, indicating that NK1.1⁺ß T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1⁺ß T cells after Salmonella infection accompanied by increased serum IFN- level. The aberrantly increased IL-12 production in infected TCRß^-/- or J281^-/- mice was suppressed by adoptive transfer of T cells containing NK1.1⁺ß T cells but not by the transfer of T cells depleted of NK1.1⁺ß T cells or T cells from J281^-/- mice. Taken together, it is suggested that NK1.1⁺ß T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.

	Introduction

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Naive CD4⁺ T cells initially stimulated in the presence of IL-12 tend to develop into CD4⁺ Th1 cells producing IFN- (1, 2, 3), which are essential for the host defense against infection by intracellular bacteria such as Salmonella species, Listeria monocytogenes, Leishmania major, and Mycobacterium tuberculosis (4). Avirulent strains of Salmonella have a great potential not only as vaccines against virulent Salmonella infection, but also as carriers expressing foreign protein as derived from unrelated pathogens. Moreover, avirulent Salmonella strains gained increasing interest as recombinant Ag delivery systems against infectious diseases that require T cell responses for pathogen elimination. Although Salmonella species, like other intracellular parasites such as L. monocytogenes and M. tuberculosis, induce a Th1-dependent immune response, this microbe differs significantly from the well-studied L. monocytogenes model. Thus, the facts demand a more careful analysis of the host immune response against Salmonella. IL-12 is a heterodimer cytokine composed of a constitutively expressed 35-kDa (p35) subunit and an inducible 40-kDa (p40) subunit (3). IL-12 is produced by monocytes/macrophages and dendritic cells in response to bacteria, parasites, or bacterial constitutes such as LPS (3). IL-12 production is also induced on the activated T cells by interaction with APC via CD40-CD40 ligand (CD40L)³ through activation of NF-B (5, 6, 7, 8). The ability of monocyte/macrophage to produce IL-12 is regulated by several cytokines with activating or suppressing effects. IFN- and GM-CSF enhance the production of IL-12 from phagocytic cells (9), whereas IL-4, IL-10, IL-13, and TGF-ß inhibit IL-12 production when added to monocyte/macrophages simultaneously with the inducing stimulus (10, 11). Thus, the balance of these cytokines may be important for controlling of the ability of phagocytic cells to produce L-12 during course of infection.

The early IL-4 production is thought to come from NK1.1⁺ß T cells (12, 13, 14). NK1.1⁺ß T cells, first found in the thymus (15, 16, 17, 18, 19, 20), are also present in the periphery, especially in bone marrow (20), the liver (21), and the peritoneal cavity (22). A large fraction of NK1.1⁺ß T cells express an invariant TCR encoded by the V14 and J281 gene segments (23, 24, 25) and are selected by the nonpolymorphic MHC class I-like surface protein CD1d (26). This population is almost completely absent in mice deficient in ß₂-microgloblin (ß₂m^-/-) (20, 21), CD1d (27, 28), and J281 (J281^-/-) (29). We have recently reported that NK1.1⁺ß T cells in the peritoneal cavity negatively regulate the generation of Th1 cells during the course of avirulent Salmonella choleraesuis 31N-1 infection via IL-4 production in beige mice (30). However, the direct roles of NK1.1⁺ß T cells in the early phase of bacterial infection remain unknown.

In this study, to elucidate the roles of NK1.1⁺ß T cells during bacterial infection, we examined cytokine production in mice deficient in TCRß (TCRß^-/-), ß₂m^-/-, or J281^-/- after Salmonella infection and found that IL-12 production was aberrantly augmented in TCRß^-/-, ß₂m^-/-, or J281^-/- mice after Salmonella infection. NK1.1⁺ß T cells are mainly responsible for IL-4 production upon TCR engagement at the early phase of Salmonella infection, and anti-IL-4-neutralizing mAb significantly augmented IL-12 production in wild type (wt) mice infected with Salmonella accompanied by increased IFN- production. The aberrantly increased IL-12 production by Salmonella infection in TCRß^-/- mice was suppressed by adoptive transfer of T cells containing NK1.1⁺ T cells but not by that of T cells depleted of NK1.1⁺ T cells. Our results suggest NK1.1⁺ß T cells eliciting IL-4 function in down-regulating IL-12 production at the early phase of Salmonella infection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

TCRß^-/- mice, which lack the TCRß gene (devoid of all ß T cells but possess virtually normal T cells), and J281^-/- mice, which lack the J281 gene segment (devoid of V14⁺NKT cells while leaving the other lymphoid lineages intact), have been described (29, 31). A homogenous population was established by backcrossing heterozygotes to C57BL/6 mice more than five generations. The resultant heterozygotes were bred to obtain homozygotes. In each experiment, age- and sex-matched littermates were used. In some experiments, siblings from the same mother were used. Mice genetically deficient in ß₂m gene expression, which fail to surface express MHC class I and related molecules such as CD1 (thus devoid of conventional CD8⁺TCRß cells and CD1-dependent NK1.1⁺ß T cells), bred to the C57BL/6 background were obtained from Taconic (Germantown, NY). Female C57BL/6 mice were obtained from Japan SLC (Hamamatsu, Japan). Mice were maintained under specific pathogen-free conditions and offered food and water ad libitum. All mice were used at 6 to 8 wk of age.

Microorganisms

Salmonella subspecies choleraesuis serovar choleraesuis strain 31N-1 (30, 32, 33, 34, 35, 36, 37, 38, 39) was maintained by several passages through C57BL/6 mice. The approximate LD₅₀ was 10⁷ CFU in BALB/c mice inoculated i.p.

Antibodies

PE-conjugated anti-NK1.1 mAb (PK136), FITC-conjugated anti-CD3 mAb (145-2C11), and biotin-conjugated anti-TCRß mAb were purchased from PharMingen (San Diego, CA). Anti-TCRß mAb (H57-597) was a gift from Dr. R. Kubo (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO) and anti-TCR mAb (UC7-13D5) from Dr. J. A. Bluestone (University of Chicago, Chicago, IL). Anti-IL-4-neutralizing mAb (rat IgG1, 11B11) were purchased from PharMingen.

Cell preparation

Mice were killed 3 days after an i.p. inoculation with 2x 10⁶ CFU of avirulent strain 31N-1. The peritoneal exudate cells (PEC) were prepared by centrifuging peritoneal exudates at 110 x g for 5 min and suspended in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. Cells were plated in 60-mm tissue culture dishes and allowed to adhere for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Nonadherent cells were recovered by washing twice with HBSS, and passed over a nylon wool column (Wako Pure Chemical Industries, Osaka, Japan), which were used as enriched T cells. In some experiments, NK1.1⁺ T cell-depleted T cells, which were prepared by incubating enriched T cells with anti-NK1.1 mAb (mouse IgG2a, PK136) and rabbit complement for 60 min at 37°C, were used.

Flow cytometric analysis

Nonadherent PEC were stained with PE-conjugated anti-NK1.1 mAb, FITC-conjugated anti-CD3 mAb, and biotin anti-TCRß mAb. To block FcR-mediated binding of the mAb, 2.4G2 (anti-FcR mAb) was added. All incubation steps were performed at 4°C for 30 min. To detect biotin-conjugated mAb, cells were stained with Cy-Chrome-conjugated streptavidin (Becton Dickinson, San Jose, CA). The stained cells were analyzed by a FACScan flow cytometer (Becton Dickinson). Small lymphocytes were gated by forward and side scatter.

Cell culture

T cells enriched from PEC were cultured in 200 µl of complete culture medium in 96-well flat-bottom plates (Falcon, Becton Dickinson, Oxford, U.K.) at a density of 3x 10⁵ cells/well, with anti-TCRß mAb (100 µg/ml) that had been immobilized on the plates by prior incubation for 1 h. To estimate cytokine production, the supernatant was collected after culture for 48 h. For ELISA of monokines, macrophages (1x 10⁶ cells) were collected from mice on days 3 or 6 after infection with S. choleraesuis and cultured for 12 h, and the culture supernatants were analyzed as below.

Expression of cytokine genes

NK1.1⁺ or NK1.1^-ß T cells isolated from nonadherent PEC or liver of wt mice on day 3 after Salmonella infection by sorting using FACSvantage SE (Becton Dickinson) were applied. mRNA was extracted by QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, Milwaukee,WI), and cDNA synthesis was performed as previously described (30). Synthesized cDNA was amplified by PCR using 20 pmol each of primers specific for cytokines and ß-actin. The primers were as follows: IFN- sense (5'-AGC GGC TGA CTG AAC TCA GAT TGT AG-3'), antisense (5'-GTC ACA GTT TTC AGC TGT ATA GGG-3'); IL-4 sense (5'-CGA AGA ACA CCA CAG AGA GTG AGC T-3'), antisense (5'-GAC TCA TTC ATG GTG CAG CTT ATC G-3'); ß-actin sense (5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3'), antisense (5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'). The PCR cycles were run for 1 min at 94°C, followed by 1 min at 54°C, and 30 s at 72°C. Before the first cycle, a denaturing step for 7 min at 94°C was included, and again after 25 cycles. The PCR product was subjected to electrophoresis on a 1.0% agarose gel (Life Technologies Laboratories, Grand Island, NY), transferred to Gene Screen Plus filter (DuPont NEN, Boston, MA), and then hybridized with ³²P-labeled oligo probes: IFN- (5'-GGT CAC TGC AGC TCT GAA TG-3'), IL-4 (5'-GAG TCT CTG CAG CTC CAT GA-3'), or ß-actin (5'-TTC TGC ATC CTG TCA GCA AT-3'). After incubation for 16 h at 60°C in 1 M NaCl, 10% dextran sulfate, and 100 mg/ml heat-denatured salmon sperm DNA, the filters were washed for 30 min in 2x SSC, 1% SDS and exposed to a phosphor imaging plate for visualization on the Fujix BAS2000 Bio-image analyzer (Fuji Photo Film, Tokyo, Japan).

Cytokine ELISA

The cytokine activity in the culture supernatants was assayed using Duo Set ELISA Development system (Genzyme Diagnostics, Cambridge, MA) for IL-2, IL-4, total IL-12, or IFN-; Biotrak ELISA system (Amersham International, Buckinghamshire, England, U.K.) for IL-1, IL-10, TGF-ß, and TNF-; and Quantikine M Mouse IL-13 ELISA Kit (R&D Systems, Minneapolis, MN).

Adoptive transfer of T cells

Enriched peritoneal T cells from wt mice on day 3 after infection with S. choleraesuis were treated with anti-NK1.1 mAb (PK136, mouse IgG2a) or isotype control mouse IgG and rabbit complement for 60 min at 37°C. Whole T cells or T cells depleted of NK1.1⁺ T cells (7x 10⁵ cells) were transferred i.p. to TCRß^-/- or J281^-/- mice. In some experiments, enriched T cells from J281^-/- mice were transferred i.p. to TCRß^-/- mice. Immediately following adoptive transfer, mice were injected i.p. with 2x 10⁶ viable S. choleraesuis on day 0. Bacterial growth and serum IL-12 level were examined on day 3 after Salmonella infection.

Statistics

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peritoneal exudate NK1.1⁺ß T cells are a main source of IL-4 production during Salmonella infection

To determine whether the NK1.1⁺ß T cells appear in the peritoneal cavity of wt mice during salmonellosis, we followed the appearance of NK1.1⁺ cells in TCRß⁺ fraction in the peritoneal cavity after an i.p. inoculation with avirulent S. choleraesuis. An appreciable number of NK1.1⁺ß T cells were increased in the peritoneal cavity as early as on day 3 after infection (Fig. 1).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Kinetics of NK1.1+ß T cells in the peritoneal cavity after an i.p. challenge with S. choleraesuis 31N-1. The nonadherent cells were stained with FITC-anti-CD3 mAb, PE-anti-NK1.1 mAb, and biotin anti-TCRß mAb on day 3, 6, or 10 after an i.p. inoculation with S. choleraesuis (2 x 106 CFU) 31N-1. The analysis gate was set on TCRß+ T cells, and a typical single profile is shown. Data are representative of three independent experiments using pooled cells of five C57BL/6 mice.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. IL-4 production by NK1.1+ß T cells after an i.p. challenge with S. choleraesuis (2 x 106 CFU) (A). The expression of mRNAs specific for IL-4 and IFN- in freshly isolated NK1.1+ß or NK1.1-ß T cells by means of cytokine RT-PCR. Each ß T cell population was sorted by FACSsorting system from the peritoneal cavity or liver of mice 3 days after an i.p. challenge with S. choleraesuis 31N-1. ß-actin was coamplified with a known amount of specific competitor "mimic" fragment. cDNA was coamplified in a series of three reactions with a 2-fold dilution series of mimic concentrations. The products were separated on 1% agarose gels. Synthesized cDNA was amplified by PCR using each of the primers specific for IL-4 and IFN-. The PCR products of IL-4 and IFN- were subjected to electrophoresis on a 1.0% agarose gel and transferred to Gene Screen Plus filter, and then hybridized with 32P-labeled oligo probes. After incubation, the filters were exposed to a phosphor imaging plate for visualization on the Fujix BAS2000 Bio-image analyzer (B). Cytokine production by the T cells from wt mice infected with Salmonella in response to immobilized anti-TCRß mAb. The enriched T cells (3 x 105 cells) from the peritoneal cavity or the liver of mice on day 3 after an i.p. challenge with 2 x 106 CFU of S. choleraesuis were cultured with immobilized anti-TCRß mAb for 2 days at 37°C. Thereafter, the supernatants were collected, and cytokine activity was determined by ELISA (C). FACS profiles of the peritoneal nonadherent cells from NK1.1+ß T cell-deficient mice on day 3 after an i.p. challenge with 2 x 106 CFU of S. choleraesuis. The cells were stained with FITC-anti-CD3 mAb (145-2C11) and PE-anti-NK1.1 mAb (PK136), and are analyzed by FACScan. The analysis gate was set on small lymphocytes by forward and side scattering (D). IL-4 production by the peritoneal exudate T cells from NK1.1+ß T cell-deficient mice infected with Salmonella in response to immobilized anti-TCRß mAb. The enriched T cells (3 x 105 cells) from wt, TCRß-/-, ß2m-/-, or J281-/- mice on day 3 after an i.p. challenge with 2 x 106 CFU of S. choleraesuis were cultured with immobilized anti-TCRß mAb for 2 days at 37°C. The supernatants were collected, and cytokine activity was determined by ELISA. Columns are mean values ±SD of five mice. Data are represented of three independent experiments. Statistical significance was determined by the Student’s t test (*, p < 0.005). ND, not detectable.

View this table: [in this window] [in a new window]   Table I. Absolute cell numbers of lymphocyte subpopulations in the peritoneal cavity of NK1.1+ß T cell-deficient mice on day 3 after infection with S. choleraesuis1

We examined the kinetics of IL-4 production in serum after Salmonella infection but detected no IL-4 activity in the serum of wt mice at any stage after infection. To obtain evidence for early IL-4 production in Salmonella infection, wt mice were treated i.p. with anti-IL-4-neutralizing mAb (11B11) at 2 h before or at 48 h after Salmonella challenge, and the bacterial number in the peritoneal cavity was counted on day 3 after infection. As shown in Fig. 3, the number of bacteria was significantly reduced in the peritoneal cavity of mice treated with anti-IL-4 mAb at 2 h before infection compared with that in mice treated with isotype control rat IgG (p < 0.005). However, anti-IL-4 mAb treatment at 48 h after infection had no effect (Fig. 3). To determine the effects of neutralization of endogenous IL-4 on cytokine production, we next examined IFN-, IL-1, IL-6, IL-10, IL-12, and TNF- in serum of mice treated with anti-IL-4 mAb or control IgG during salmonellosis. Notably, the serum IL-12 and IFN- were significantly augmented in mice treated with anti-IL-4 mAb at 2 h before infection compared with those in control IgG-treated mice (Fig. 3). The peritoneal macrophages from the anti-IL-4-mAb-treated mice on day 3 after Salmonella infection produced significantly higher level of IL-12 than those from mice treated with control IgG (Fig. 3). On the other hand, anti-IL-4 mAb treatment at 48 h after infection had no effect on IL-12 production (Fig. 3). These results suggest that an early IL-4 production may be induced in vivo by Salmonella infection and plays an important role in down-regulation of IL-12 and IFN- production during Salmonella infection.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Effect of in vivo administration of anti-IL-4 mAb to wt mice infected with S. choleraesuis on the production of IL-12. Anti-IL-4-neutralizing mAb (11B11, rat IgG; 500 µg) or isotype control rat IgG were administered i.p. to wt mice at 2 h before or on day 2 after an i.p. challenge with S. choleraesuis (2 x 106 CFU). Three days later, the number of bacteria in the peritoneal cavity, and IL-12 and IFN- levels in serum or in culture supernatants of macrophage for 24 h, were determined. Columns are mean values ±SD of five mice in each group. Data are represented of three independent experiments. Statistical significance was determined by Student’s t test (*, p < 0.005; **, p < 0.05).

Aberrantly increased IL-12 production in NK1.1⁺ß T cell-deficient mice after Salmonella infection

We showed that the peritoneal NK1.1⁺ß T cells were the main source of early IL-4 production after Salmonella infection and that IL-4 was suggested to regulate IL-12 production by macrophages. To obtain direct evidence for the aberrantly increased production of IL-12 during salmonellosis in NK1.1⁺ß T cell-deficient mice, we investigated the IL-12 production induced by Salmonella infection in TCRß^-/-, ß₂m^-/-, and J281^-/- mice. IL-12 and IFN- levels in the serum were significantly higher in J281^-/- and ß₂m^-/- mice than in wt mice on day 3 (Fig. 4A) and on day 6 (data not shown) after Salmonella infection. Similarly, the IL-12 levels produced by the peritoneal macrophages were significantly higher in J281^-/- and ß₂m^-/- mice on day 3 after infection than in wt mice (p < 0.005, Fig. 4A). The kinetics of IL-12 production by macrophages of TCRß^-/- mice showed that aberrantly increased production of IL-12 was detected as early as on day 3 after Salmonella infection (Fig. 4B). Thus, aberrantly increased IL-12 production was evident in mice deficient in NK1.1⁺ß T cells bearing V14/J281 at the early phase of Salmonella infection.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. IL-12 production in mice lacking TCRß, ß2m, or J281 gene (A). Serum IFN- or IL-12, or IL-12 production of macrophages in ß2m-/- or J281-/- mice on day 3 after infection with S. choleraesuis (2 x 106 CFU). For IL-12 production by macrophages, 24-h culture supernatants of the peritoneal macrophages were collected, and the IL-12 protein levels were determined by ELISA (B). Kinetics of IL-12 production by the peritoneal macrophages of TCRß-/- mice were determined after an i.p. infection with a sublethal dose (2 x 106 CFU) of S. choleraesuis 31N-1. The peritoneal macrophages from TCRß-/- () or wt () mice were recovered on indicated days after infection with S. choleraesuis, and the 24-h culture supernatants of the macrophages were collected. The IL-12 protein levels in supernatants were determined by ELISA. Statistical significance was determined by the Student’s t test (*, p < 0.005; **, p < 0.05). Columns are mean values ±SD of five mice in each group. Data are represented of three independent experiments.

Adoptive transfer of NK1.1⁺ß T cells restores the down-regulation of IL-12 production after Salmonella infection

To further elucidate the potential role of NK1.1⁺ß T cells in controlling IL-12 production after Salmonella infection, adoptive transfer experiments were conducted in TCRß^-/- mice, using the peritoneal T cells with NK1.1⁺ß T cells or those depleted of NK1.1⁺ß T cells. NK1.1⁺ß T cells were almost depleted by treatment with anti-NK1.1 mAb plus complement (Fig. 5A). The serum IL-12 levels were determined on day 3 after Salmonella infection in TCRß^-/- or J281^-/- mice transferred with 7 x 10⁵ peritoneal T cells containing NK1.1⁺ß T cells or those depleted of NK1.1⁺ß T cells. The serum IL-12 levels were significantly suppressed in TCRß^-/- or J281^-/- mice transferred with T cells containing NK1.1⁺ß T cells but not in those transferred with T cells depleted of NK1.1⁺ß T cells (Fig. 5, B and C). Similarly, adoptive transfer of peritoneal T cells from J281^-/- mice to TCRß^-/- mice did not affect the excessive IL-12 production in the TCRß^-/- mice after Salmonella infection (Fig. 5B).

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Effect of adoptive transfer of the peritoneal T cells with or without NK1.1+ß T cells into TCRß-/- mice on IL-12 production after Salmonella infection. Enriched peritoneal T cells from wt mice on day 3 after infection with S. choleraesuis were treated with anti-NK1.1 mAb and rabbit C. Before transfer, the treated T cells were stained purified-anti-NK1.1 mAb (PK136) followed by FITC anti-mouse IgG Ab and analyzed by FACScan. Single profiles from expression of NK1.1 are shown after the gate was set on TCR ß+ T cells (A). Whole T cells or T cells depleted of NK1.1+ population (7 x 105 cells) were transferred i.p. to TCRß-/- (B) or J281-/- mice (C). The enriched peritoneal T cells from J281-/- mice on day 6 after an i.p. challenge with 2 x 106 CFU of S. choleraesuis were cultured and were transferred i.p. to TCRß-/- mice (B). Immediately following transfer, mice were injected i.p. with 2 x 106 viable S. choleraesuis on day 0. IL-12 protein levels in serum were determined on day 3 after infection. Columns are mean values of five mice in each group. Data are representative of two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-12 is produced by monocytes/macrophages and dendritic cells in response to infection with bacteria or to bacterial constitutes such as LPS (3). The production of proinflammatory cytokines such as IL-12 in monocytes/macrophages is regulated by deactivating cytokines such as IL-4, IL-10, IL-13, and TGF-ß (10, 11). We observed no remarkable differences in IL-10, IL-13, and TGF-ß production between wt and TCRß^-/- mice after Salmonella infection (data not shown). Although IL-4 production was not detected in the serum of wt mice infected with Salmonella, in vivo administration of anti-IL-4 mAb enhanced IL-12 production in wt mice after infection. IL-4 may be secreted in a very low amount, thus acting in a narrow intracellular range during Salmonella infection. IL-4 production was detected in the peritoneal T cells stimulated in vitro with immobilized anti-TCRß mAb, whereas it was significantly impaired in NK1.1⁺ß T cell-deficient mice (Fig. 2). Therefore, we speculate that IL-4 may down-regulate the IL-12 production by the macrophages during Salmonella infection and that impaired IL-4 production may be responsible for aberrantly increased IL-12 production in NK1.1⁺ß T cell-deficient mice after Salmonella infection. Several mechanisms controlling IL-12 production by early IL-4 production come to mind. IL-4 is known to suppress lL-12 production by macrophages at the transcriptional level (11), suggesting that IL-4 may act directly on infected macrophages. IFN- is known as a potent stimulator for IL-12 production by macrophages, raising another possibility that an early IL-4 production may regulate IFN- production by NK and T cells appearing after Salmonella infection and in turn may lead to down-regulation of IL-12 production by macrophages during infection (30, 33, 34, 35, 36, 37, 38, 39). In fact, our current results revealed that serum IFN- levels were increased in NK1.1⁺ß T cell-deficient mice in correlation with the level of IL-12 production after Salmonella infection. An early IFN- production by NK and T cells may up-regulate the IL-12 production in NK1.1⁺ß T cell-deficient mice after Salmonella infection. IL-12 production is also induced by the interaction between activated T cells and APC via CD40-CD40L through activation of NF-B (5, 6, 7, 8). Therefore, it is also possible that the frequency of activated T cells expressing CD40L may differ between wt and NK1.1⁺ß T cell-deficient mice following Salmonella infection. However, up-regulation of IL-12 production was observed in NK1.1⁺ß T cell-deficient mice at the early stage (day 3) of Salmonella infection, well before ß T cells expressing CD40L clonally expanded. Hence, it is unlikely that the up-regulation of IL-12 production in NK1.1⁺ß T cell-deficient mice after Salmonella infection is due to the increase in the number of the specialized Th1 cells producing IFN- and expressing CD40L. Since NK1.1⁺ß T cells are an essential target of IL-12 (29), it is alternatively possible that IL-12 may not be consumed in NK1.1⁺ß T cell-deficient mice, resulting in accumulation of IL-12 in serum, after Salmonella infection. However, we observed an increased ability of macrophages to produce IL-12 in NK1.1⁺ß T cells infected with Salmonella, suggesting that early IL-4 production by NK1.1⁺ß T cells down-regulates IL-12 synthesis following Salmonella infection.

Rapid IL-4 secretion by NK1.1⁺ T cells has been demonstrated in mice treated with anti-CD3 mAb or superantigens (13). NK1.1⁺TCRß T cells express the canonical TCR encoded by V14 and J281 segments (23, 24) and rapidly produce large amounts of IL-4 upon binding of the ß₂m-associated nonclassical MHC class I b protein CD1d, a homologue of human CD1b (26). Our present studies with ß₂m^-/- or J281^-/- mice revealed that the peritoneal NK1.1⁺ß T cells are responsible for early production of IL-4, which consequently regulates the IL-12 production by macrophages during Salmonella infection. We have recently reported that NK1.1⁺ß T cells specialize in recognizing glycosylceramides containing -anomeric sugar with a longer fatty acyl chain and sphingosine base, which are detected in certain bacteria but rarely detected in normal mammalian tissues (40). Although it is not known whether Salmonella species have the relevant Ags, NK1.1⁺ T cells may respond to the related Ags derived from Salmonella and produce IL-4 rapidly, which in turn down-regulates IL-12 production by macrophages after Salmonella infection. In contrast to the peritoneum, NK1.1⁺ß T cells from the liver expressed only IFN-. NK1.1⁺ß T cells in the liver are reported to be an essential target of IL-12 (29). Intracellular bacteria such as S. choleraesuis preferentially induce IL-12 production by macrophage/dendritic cells. Therefore, it can be speculated that IL-12 may dominantly activate NK1.1⁺ß T cells to produce IFN-, which inhibit IL-4 production (41, 42). Further analysis at the clonal level of NK1.1⁺ß T cells is required to clarify this possibility.

Biological significance for controlling of IL-12 production by NK1.1⁺ß T cells remains unknown. IL-12 induces IFN- production by resting and activated NK cells, T cells, and Th1 cells, which are essential for the host defense against intracellular bacteria such as Salmonella species (30, 33). However, excessive IL-12 production induces tissue damage including liver and colitis (43, 44, 45). Therefore, it is conceivable that IL-4 produced by NK1.1⁺ß T cells may regulate such excessive inflammatory response after bacteria infection. We do not have evidence for severe liver injury in NK1.1⁺ß T cell-deficient mice following Salmonella infection as assessed by serum alanine amino-transfected activity. This can be explained as a result of lack in NK1.1⁺ß T cells of the liver, which are an essential target of IL-12 and presumably effector cells for liver injury. Additional experiments are required to elucidate the roles of NK1.1⁺ß T cells producing IL-4 in controlling excessive inflammation during salmonellosis.

In conclusion, the peritoneal NK1.1⁺ß T cells eliciting IL-4 may play an important role in down-regulation of IL-12 production by macrophages during the course of bacterial infection.

	Acknowledgments

 
We thank Drs. J. A. Bluestone and R. T. Kubo for providing the hybridoma mAbs. We also thank Mrs. Itano for providing excellent technical support.

	Footnotes

 
¹ This work was supported in part by a grant from the Ministry of Education, Science and Culture of the Japanese Government (JSPS-RFTF97L00703), by Ohyama Health Foundation, by Inoue Foundation for Science, by the Center of Excellence, and by the Core Research for Evolutional Science and Technology (CREST) Project.

² Address correspondence and reprint requests to Dr. Hitoshi Nishimura, Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan. E-mail address:

³ Abbreviations used in this paper: CD40L, CD40 ligand; ß₂m^-/- mice, ß₂-microgloblin-deficient mice; J281^-/- mice, J281-deficient mice; TCRß^-/- mice, TCRß-deficient mice; wt, wild type; PEC, peritoneal exudate cell; MNC, mononuclear cell.

Received for publication January 26, 1999. Accepted for publication June 2, 1999.

	References

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