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The Journal of Immunology, 2004, 173: 5156-5164.
Copyright © 2004 by The American Association of Immunologists

V{delta}1+ {gamma}{delta} T Cells Producing CC Chemokines May Bridge a Gap between Neutrophils and Macrophages in Innate Immunity during Escherichia coli Infection in Mice1

Tetsuzo Tagawa*,{dagger}, Hitoshi Nishimura2,*, Toshiki Yajima*, Hiromitsu Hara*, Kenji Kishihara*, Goro Matsuzaki{ddagger}, Ichiro Yoshino{dagger}, Yoshihiko Maehara{dagger} and Yasunobu Yoshikai*

* Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, and {dagger} Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and {ddagger} Division of Molecular Microbiology, University of Ryukyus Center of Molecular Biosciences, Okinawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An influx of neutrophils followed a short time later by an influx of macrophages to the infected site plays a key role in innate immunity against Escherichia coli infection. We found in this study that V{delta}1–/– mice exhibited impaired accumulation of peritoneal macrophages but not neutrophils and delayed bacterial clearance after i.p. inoculation with E. coli. Peritoneal {gamma}{delta} T cells from E. coli-infected wild-type mice produced CCL3/MIP-1{alpha} and CCL5/RANTES in response to {gamma}{delta} TCR triggering in vitro, whereas such production was not evident in {gamma}{delta} T cells from E. coli-infected V{delta}1–/– mice. Neutralization of CCL3/MIP-1{alpha} by a specific mAb in vivo significantly inhibited the accumulation of macrophages in the peritoneal cavity after E. coli infection, resulting in exacerbated bacterial growth in the peritoneal cavity. These results suggest that V{delta}1+ {gamma}{delta} T cells bridge a gap between neutrophils and macrophages in innate immunity during E. coli infection mediated by production of CC chemokines, enhancing macrophage trafficking to the site of infection.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most microorganisms are detected and destroyed within several days by pre-existing innate immunity. Cells involved in innate immunity such as neutrophils and macrophages discriminate and kill microorganisms by germline-encoded receptors that identify molecules synthesized exclusively by microbes. In microbial invasion, resident macrophages are the first to encounter pathogens in infected tissues, and then neutrophils migrate from circulation to the infected sites. They are short-lived cells, dying within several hours, and soon reinforced by the recruitment of monocytes to infected sites where they differentiate into long-lived macrophages. Conversely, humoral innate immune responses produce a variety of factors that are chemotactic for neutrophils and macrophages rapidly migrating from the blood to sites of infection. Chemokines are a large family of low-molecular-weight proteins that play important roles in leukocyte migration, activation, and degranulation (1, 2, 3). They are classified on the basis of structural features into major subclasses of CXC chemokines and CC chemokines (2). CXC chemokines predominantly target neutrophils and subsets of T cells, whereas CC chemokines target a variety of cell types, including T cells, macrophages, eosinophils, and basophils (4, 5). Chemokines can be released by many different types of cells including phagocytes, endothelial cells, keratinocytes, fibroblasts, and smooth muscle cells (1, 2, 3).

{gamma}{delta} T cells are present in small numbers in the blood and peripheral lymphoid tissues but are relatively abundant in the epithelia of the epidermis, intestine, uterus, and tongue compared with {alpha}{beta} T cells in mice (6). Most of the {gamma}{delta} T cell subsets in peripheral lymphoid tissues bear junctionally diverse TCRs, but the {gamma}{delta} T cell subsets in the epithelia, including the V{gamma}5/V{delta}1 subset in the skin and the V{gamma}6/V{delta}1 subset in the tongue and female reproductive tract, bear invariant TCRs (7, 8, 9). These two subsets differentiate in the thymus at the very early stage of ontogeny and bear truly invariant TCRs without junctional diversity (7). Such characteristics have led to the hypothesis that {gamma}{delta} T cells bearing invariant V{delta}1 TCRs represent a more primitive, first line of host defense in innate immunity. In murine models of bacterial infection, {gamma}{delta} T cells have been reported to participate in host defense against extracellular bacteria such as Escherichia coli (10, 11, 12, 13, 14, 15) and intracellular bacteria such as Listeria monocytogenes (16, 17, 18). We and others have previously reported that the number of {gamma}{delta} T cells bearing invariant V{gamma}6/V{delta}1 significantly increased in the peritoneal cavity during an i.p. infection with E. coli in mice (12, 13, 14, 15). Protection against E. coli is thought to depend mainly on recruitment of infiltrating neutrophils and macrophages (19). However, very little is known about how invariant V{gamma}6/V{delta}1 TCR-bearing {gamma}{delta} T cells function in the front line of defense against E. coli infection.

In the present study, to elucidate potential roles of invariant V{delta}1-bearing {gamma}{delta} T cells in protection against E. coli infection, we examined bacterial growth and cellular responses in the peritoneal cavities of mice deficient in the V{delta}1 gene (V{delta}1–/–) following i.p. infection with E. coli. V{delta}1–/– mice showed severely impaired accumulation of peritoneal macrophages after E. coli infection. The peritoneal {gamma}{delta} T cells of infected wild-type mice produced large amounts of CCL as CCL3/MIP-1{alpha} and CCL5/RANTES in response to {gamma}{delta} TCR triggering in vitro, whereas there was no production of those cytokines by peritoneal {gamma}{delta} T cells of V{delta}1–/– mice. V{delta}1+ {gamma}{delta} T cells may help to serve as innate immunity by secreting chemokines for macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 mice were purchased from SLC Japan (Shizuoka, Japan). V{delta}1–/– mice were generated as previously described (20). V{delta}1–/– mice with a C57BL/6 background were backcrossed into C57BL/6 mice more than nine times. These mice were bred in specific pathogen-free conditions in our institute. Eight- to 10-wk-old male mice were used for the experiments.

Microorganisms and reagents

E. coli (no. 26; American Type Culture Collection (ATCC), Rockville, MD) grown in brain heart infusion broth (Difco, Detroit, MI) was washed repeatedly, resuspended in PBS, and stored at –80°C in small aliquots until used.

Cell preparation

Mice were i.p. inoculated with E. coli at a dose of one-fifth the LD50 (108 CFU/mouse) in 200 µl of PBS on day 0. Peritoneal exudate cells (PEC)3 were harvested on day 0, 0.25, 1, 2, 3, and 6 after inoculation by centrifugation at 110 x g for 5 min, washed twice, and resuspended at optimal concentrations in RPMI 1640 medium (Invitrogen Life Technologies, Grand Island, NY) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. Smear specimens for differential counts were stained with Giemsa solution. In some experiments, to enrich {gamma}{delta} T cells from PEC, the cells were plated in a tissue culture dish of 100 mm and allowed to adhere for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. To negatively enrich {gamma}{delta} T cells, nonadherent cells were passed over a nylon wool column (Wako Pure Chemical, Osaka, Japan), and {alpha}{beta} T cells were depleted by staining cells with an FITC-conjugated anti TCR-{beta} mAb followed by magnetic negative selection using a MACS system and anti-FITC microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s instructions. In some experiments, {gamma}{delta} T cells were positively enriched by staining nonadherent cells with FITC-conjugated anti TCR-{gamma}{delta} mAb followed by magnetic positive selection using a MACS system and anti-FITC microbeads. For liver lymphocytes, fresh liver was immediately perfused with sterile HBSS through the portal vein to wash out all remaining peripheral blood and then meshed with a stainless steel mesh. After coarse pieces had been removed by centrifugation at 50 x g for 1 min, the cell suspensions were again centrifuged, resuspended in 8 ml of 45% Percoll (Sigma-Aldrich, St. Louis, MO), and layered on 5 ml of 66.6% Percoll. The gradients were centrifuged at 600 x g for 20 min. Lymphocytes at the interface were harvested and washed twice with HBSS.

Bacterial growth

Mice were each inoculated i.p. with 108 CFU of E. coli in 200 µl of PBS. At indicated times after inoculation, the peritoneal contents were lavaged with 5 ml of HBSS and harvested after gentle massage. Samples were serially diluted with HBSS. The livers were removed and placed in homogenizers containing 5 ml of HBSS. Samples were spread on Tripto-Soya agar (Nissui Pharmaceutical, Tokyo, Japan) plates, and colonies were counted after incubation for 24 h at 37°C.

Antibodies

PE-conjugated anti-NK 1.1, anti-CD8, anti-TCR-{beta}, anti-Gr-1 mAb, FITC-conjugated anti-CD3{epsilon} mAb, CyChrome-conjugated anti-CD4, anti-F4/80 mAb, biotin-conjugated anti-TCR {gamma}{delta}, anti-B220 mAb, and CyChrome-conjugated streptavidin were purchased from BD Pharmingen (San Diego, CA). The Abs used for in vivo treatment were goat anti-mouse CCL3/MIP-1{alpha} polyclonal Ab, rat anti-mouse CCL5/RANTES mAb (53405.111), purified goat IgG, and rat IgG for use as control Abs. These Abs were purchased from R&D Systems (Minneapolis, MN). Anti-TCR-{beta} mAb (H57-597) was a gift from Dr. R. Kubo (National Jewish Center of Immunology and Respiratory Medicine, Denver, CO), and anti-TCR {gamma}{delta} mAb (UC7-13D5) was a gift from Dr. J. A. Bluestone (University of California, San Francisco, CA).

Flow cytometric analysis

Total PEC and liver lymphocytes were incubated with culture supernatant of anti-mouse Fc{gamma}R mAb-producing B cell hybridoma 2.4G2 (ATCC) for 20 min to block nonspecific binding of mAb and then washed. The cells were then stained with saturating amounts of FITC-, PE-, and biotin-conjugated mAbs for 30 min at 4°C. To detect biotin-conjugated mAb, cells were stained with CyChrome-conjugated streptavidin after incubation with a primary mAb. Cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The data were analyzed using CellQuest software (BD Biosciences).

Chemokine ELISAs

Tissue culture 96-well plates were incubated for 1 h at 37°C with 100 µg/ml anti-TCR {gamma}{delta} mAb. The plates were then washed thoroughly and incubated for 1 h at 37°C with RPMI 1640 medium containing 10% FCS. The negatively enriched {gamma}{delta} T cells (3 x 104 cells/well), as previously described, were incubated in the 96-well plates for 42 h with or without immobilized anti-TCR {gamma}{delta} mAb in the presence or absence of LPS (10 µg/ml; Sigma-Aldrich). CCL2/MCP-1, CCL3/MIP-1{alpha}, CCL5/RANTES, and CCL8/eotaxin levels in the culture supernatants were determined by using an ELISA kit (Genzyme Techne, Minneapolis, MN). Levels of CCL3/MIP-1{alpha} and CCL5/RANTES were measured in the lavage fluid of peritoneal cavity of wild-type or V{delta}1–/– mice on day 2 after E. coli infection by using an ELISA kit.

Expression of chemokine genes

To synthesize cDNA, total RNA was extracted from positively enriched {gamma}{delta} T cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. RNA was then reverse transcribed to synthesize cDNA using Superscript reverse transcriptase (Invitrogen Life Technologies) and random hexamers (Invitrogen Life Technologies) according to the manufacturer’s instructions. The cDNA was amplified with sense and antisense primer pairs and AmpliTaq Gold TaqDNA polymerase (PerkinElmer, Norwalk, CT) using a thermal cycler (Takara Shuzo, Tokyo, Japan). PCR cycles were run for 1 min at 94°C, 1 min at 54°C, and 2 min at 72°C. Before the first cycle, a denaturation step for 1 min at 94°C was included, and after 33 cycles the extension was prolonged for 10 min at 72°C. The PCR products were subjected to electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. The amount of RNA in each sample was standardized by the preliminary amplification for {beta}-actin and readjusting the sample concentration according to the comparison of {beta}-actin bands. The adjusting systems were repeated until the {beta}-actin bands were equalized in serially diluted samples. The specific primers used are as follows: mouse {beta}-actin sense, 5'-GGAATCCTGTGGCATCCATGAAAC-3'; mouse {beta}-actin antisense, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; mouse CCL3/MIP-1{alpha} sense, 5'-AACATCATGAAGGTCTCCAC-3'; mouse CCL3/MIP-1{alpha} antisense, 5'-CCAAGACTCTCAGGCATTCA-3'; mouse CCL5/RANTES sense, 5'-GGTACCATGAAGATCTCTGCA-3'; mouse CCL5/RANTES antisense, 5'-AAACCCTCTATCCTAGCTCAT-3'.

In vivo administration of anti-chemokine Abs

Wild-type mice were each injected i.p. with 100 µg of neutralizing anti-CCL3/MIP-1{alpha} polyclonal Ab, anti-CCL5/RANTES mAb or together with both Abs of 100 µg each at 1 h before E. coli infection and injected again with 150 µg at 24 h after infection. Control mice were injected with control goat IgG or rat IgG for anti-CCL3/MIP-1{alpha} Ab or anti-CCL5/RANTES mAb, respectively or together with both Abs at doses comparable to those used in the experimental animals. On day 2 after infection, the numbers of bacteria in the peritoneal cavity and liver were determined by colony formation assays on Tripto-Soya agar, cell numbers in the peritoneal cavity were counted, and the populations of PEC were judged by morphologic characteristics after staining with Giemsa solution.

Statistics

The statistical significance of the data was determined by Student’s t test. The value of p < 0.05 was taken as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of bacterial growth in organs of V{delta}1–/– mice after i.p. inoculation with E. coli

Wild-type mice and V{delta}1–/– mice were inoculated i.p. with E. coli at a dose of 108 CFU (one-fifth the LD50/mouse), and the kinetics of bacterial growth in the peritoneal cavity and liver were examined. As shown in Fig. 1, the numbers of bacteria in the peritoneal cavity and liver had decreased by day 6 of infection in both mouse strains. However, on day 3, the number of bacteria in the peritoneal cavity in wild-type mice was significantly smaller than that in V{delta}1–/– mice (Fig. 1, p < 0.05). There was no difference between the numbers of bacteria in the livers of the two strains of mice at any stage after E. coli infection.



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  		FIGURE 1. Kinetics of bacterial growth in the peritoneal cavity and liver after i.p. inoculation with E. coli. Wild-type and V{delta}1–/– mice were inoculated i.p. with 108 CFU of E. coli (0.2 x LD50). Bacterial growth in the peritoneal cavity and liver of each mouse was measured at indicated days after infection using wild-type (•) and V{delta}1–/– ({circ}) mice. Day 0 means data from mice without infection (before infection). Each point and vertical bar indicate the mean ± SD of five mice for each group. The data are representative of three separate experiments; *, significant differences from the values for wild-type mice (p < 0.05).

 
Cell influx in the peritoneal cavity of V{delta}1–/– mice after E. coli infection

We first examined the kinetics of {gamma}{delta} T cells in the peritoneal cavity and liver of V{delta}1–/– mice after E. coli infection. Flow cytometry analysis of the expression of TCR-{alpha}{beta} and TCR-{gamma}{delta} was conducted with total PEC and liver lymphocytes from both strains of mice on day 0, 0.25, 1, 2, 3, and 6 of infection. Representative results from three independent experiments are shown in Fig. 2A. The proportion of {gamma}{delta} T cells in the PEC of wild-type mice increased, accounting for >41.8 ± 5.5% of CD3+ cells on day 2 after E. coli inoculation, whereas the proportion of {gamma}{delta} T cells in the PEC of V{delta}1–/– mice was only 21.7 ± 4.8% at this stage of infection. No significant difference between the proportions of {gamma}{delta} T cells in livers of wild-type and V{delta}1–/– mice was observed at any stage of infection (Fig. 2A). The proportion of {alpha}{beta} T cells in the liver changed little and that in the peritoneal cavity decreased after E. coli infection in wild-type mice. The kinetics of the absolute numbers of peritoneal and liver {gamma}{delta} T cells after i.p. inoculation with E. coli are shown in Fig. 2B. The absolute number of {gamma}{delta} T cells in the peritoneal cavity was significantly increased 2 days after E. coli infection in wild-type mice and V{delta}1–/– mice, whereas the absolute number of {gamma}{delta} T cells in V{delta}1–/– mice was significantly lower than that in wild-type mice (p < 0.05). No significant difference between the numbers of {gamma}{delta} T cells in the livers of wild-type mice and V{delta}1–/– mice was found.



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  		FIGURE 2. Kinetics of {gamma}{delta} T cells in the peritoneal cavity and liver after E. coli infection. Wild-type and V{delta}1–/– mice were inoculated i.p. with 108 CFU of E. coli. A, Total PEC or liver lymphocytes were stained with anti-CD3{epsilon}, anti-TCR {beta}, and anti-TCR {gamma}{delta} mAb and analyzed by FACSCalibur. Analysis gates were set on CD3+ cells. The number at the bottom right indicates the percentage of the cells in CD3+ cells. B, Kinetics of the absolute numbers of peritoneal and liver {gamma}{delta} T cells after E. coli infection. The number of {gamma}{delta} T cells was calculated from the percentage of the cells between total PEC or liver lymphocytes among wild-type (•) and V{delta}1–/– ({circ}) mice. Each point and vertical bar indicate the mean ± SD of five mice for each groups. The data are representative of three separate experiments; *, significant differences from the values for wild-type mice (p < 0.05).

 
To examine the cell influx in the peritoneal cavity after E. coli infection, we analyzed the kinetics of PEC from wild-type mice and V{delta}1–/– mice inoculated i.p. with 108 CFU of E. coli. As shown in Fig. 3A, the absolute numbers of PEC in both mouse strains had increased by day 1 and then gradually decreased by day 6 after E. coli infection. However, on day 2 after infection, the absolute number of PEC in V{delta}1–/– mice was much lower than that of PEC in wild-type mice (p < 0.05). The number of polymorphonuclear leukocytes (PMN, Gr-1+F4/80–) in the peritoneal cavity had increased at 6 h after infection, peaked on day 1, and then gradually decreased by day 6 (Fig. 3, B and C). The numbers of PMN in V{delta}1–/– mice were slightly smaller than those in wild-type mice on days 1 and 2. In contrast, the number of macrophages (Gr-1+F4/80+) in the peritoneal cavity began to increase following the increase in the number of PMN on day 1. On day 2, the number of macrophages in the peritoneal cavity was significantly smaller in V{delta}1–/– mice than that in wild-type mice (Fig. 3, B and C, p < 0.05). The number of macrophages in the peritoneal cavity in V{delta}1–/– mice increased by day 6 to almost the same level as that in wild-type mice. There was no significant difference between the absolute numbers of lymphocytes in the peritoneal cavities of the two strains of mice at any stage after E. coli infection (Fig. 3B).



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  		FIGURE 3. Kinetics of PEC after E. coli infection. Wild-type and V{delta}1–/– mice were inoculated i.p. with 108 CFU of E. coli. A, PEC were obtained at indicated days after infection and the absolute numbers of cells were calculated among wild-type (•) and V{delta}1–/– ({circ}) mice. Each point and vertical bar indicate the mean ± SD of five mice for each group. The data are representative of three separate experiments; *, Significant differences from the values for wild-type mice (p < 0.05). B, Populations of PEC obtained from wild-type or V{delta}1–/– mice at indicated days after E. coli infection. PMN, macrophages, and lymphocytes were judged by morphologic characteristics after staining with Giemsa solution in wild-type (•) and V{delta}1–/– ({circ}) mice. Each point and vertical bar indicate the mean ± SD of five mice for each group. The data are representative of three separate experiments; *, Significant differences from the values for wild-type mice (p < 0.05). C, PEC were obtained at indicated days after infection and were stained with anti-Gr-1 and anti-F4/80 mAb and analyzed by FACSCalibur. The number in each panel indicates the percentage of the cells in whole peritoneal cells. The data are representative of three separate experiments.

 
Production of chemokines by {gamma}{delta} T cells induced by E. coli infection in the peritoneal cavity of V{delta}1–/– mice

As stated earlier, recruitment of macrophages to the peritoneal cavity was significantly reduced in V{delta}1–/– mice compared with that in wild-type mice on day 2 after E. coli infection. There is a possibility that E. coli infection induces production by {gamma}{delta} T cells of chemotactic factors such as CC chemokines to recruit macrophages to the peritoneal cavity. We examined the production of chemokines by enriched {gamma}{delta} T cells from wild-type mice infected with E. coli 2 days previously. The {gamma}{delta} T cells (90% < purities) were incubated for 42 h on anti-TCR {gamma}{delta} mAb-coated dishes. Fig. 4 shows that {gamma}{delta} T cells in the peritoneal cavities of E. coli-infected mice produced significantly high levels of CCL3/MIP-1{alpha} and CCL5/RANTES in response to TCR triggering. However, the {gamma}{delta} T cells produced, if any, only small amounts of CCL2/MCP-1 and CCL8/eotaxin in response to TCR triggering (Fig. 4). Conversely, negatively enriched {alpha}{beta} T cells in the peritoneal cavities of wild-type mice infected with E. coli 2 days previously did not produce any chemokines at all upon TCR engagement (data not shown).



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  		FIGURE 4. Chemokine production by {gamma}{delta} T cells induced by E. coli infection. Purified {gamma}{delta} T cells (3 x 104 cells) from the peritoneal cavities of wild-type mice infected with E. coli 2 days previously were cultured in a 96-well culture plate with or without immobilized anti-TCR {gamma}{delta} mAb for 42 h. Culture supernatants were subjected to ELISAs. The data are representative of three separate experiments and are expressed as the means of triplicates ± SD; *, significant differences between the values for TCR stimulation and nonstimulation (p < 0.05).

 
To determine whether V{delta}1+ {gamma}{delta} T cells produce these chemokines, we compared production of chemokines by {gamma}{delta} T cells of wild-type mice with that by {gamma}{delta} T cells of V{delta}1–/– mice after E. coli infection. Fig. 5A shows that {gamma}{delta} T cells of wild-type mice produced large amounts of CCL3/MIP-1{alpha} and CCL5/RANTES, whereas {gamma}{delta} T cells of V{delta}1–/– mice did not produce those chemokines at all upon TCR engagement. These results suggest that peritoneal {gamma}{delta} T cells bearing V{delta}1 induced by E. coli infection produce CC chemokines in response to TCR triggering.



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  		FIGURE 5. Production of chemokines by {gamma}{delta} T cells from V{delta}1–/– mice infected with E. coli. A, Purified {gamma}{delta} T cells (3 x 104 cells) were cultured in a 96-well culture plate with or without immobilized anti-TCR {gamma}{delta} mAb in the presence or absence of LPS (10 µg/ml) for 42 h. Culture supernatants were subjected to ELISAs. The data are representative of three separate experiments and are expressed as the means of triplicates ± SD; *, Significant differences between the values for wild-type mice and V{delta}1–/– mice (p < 0.05). B, Semiquantitative RT-PCR analysis of chemokine mRNA in peritoneal {gamma}{delta} T cells of wild-type or V{delta}1–/– mice infected with E. coli. Total RNA extracted from positively enriched peritoneal {gamma}{delta} T cells of wild-type or V{delta}1–/– mice infected with E. coli 2 days previously was reverse-transcribed into cDNA and amplified by PCR with specific primers for CCL3/MIP-1{alpha} and CCL5/RANTES. PCR products were electrophoresed on 1% agarose gel and then stained with ethidium bromide. The amount of RNA in each sample was standardized by preliminary amplification for {beta}-actin. The data are representative of three separate experiments. C, In vivo production of chemokines in wild-type or V{delta}1–/– mice infected with E. coli. Wild-type or V{delta}1–/– mice were inoculated i.p. with 108 CFU of E. coli. Lavage fluid of peritoneal cavity of wild-type or V{delta}1–/– mice were recovered on day 2 after infection and subjected to ELISAs. Each column and vertical bar indicate the mean ± SD of three mice for each group. The data are representative of three separate experiments; *, significant difference between the values for wild-type mice and V{delta}1–/– mice (p < 0.05).

 
We previously reported that {gamma}{delta} T cells induced by E. coli infection in C3H/HeN mice produced large amounts of IFN-{gamma} in response to LPS even in the absence of APC upon TCR engagement. Therefore, we examined the production of chemokines by {gamma}{delta} T cells induced by E. coli infection in wild-type and V{delta}1–/– mice in response to LPS. As shown in Fig. 5A, no production of chemokines in response to LPS was observed. The production of chemokines in response to TCR engagement was not augmented even in the presence of LPS. These results suggest that invariant TCR and TLR4 signaling play differential roles in the function of peritoneal V{delta}1+ {gamma}{delta} T cells induced by E. coli i.p. infection in mice.

To confirm that V{delta}1+ {gamma}{delta} T cells in the peritoneal cavity expressed these chemokines in vivo after E. coli infection, we examined gene expression of CCL3/MIP-1{alpha} and CCL5/RANTES in positively enriched peritoneal {gamma}{delta} T cells of wild-type and V{delta}1–/– mice infected with E. coli 2 days previously by semiquantitative RT-PCR. As shown in Fig. 5B, the peritoneal {gamma}{delta} T cells of wild-type mice infected with E. coli expressed CCL3/MIP-1{alpha} and CCL5/RANTES more abundantly than did those of V{delta}1–/– mice.

These results suggest that V{delta}1+ {gamma}{delta} T cells in the peritoneal cavity actually express CCL3/MIP-1{alpha} and CCL5/RANTES after E. coli infection. To determine in vivo production of these chemokines, we next examined the levels of CCL3/MIP-1{alpha} and CCL5/RANTES in the lavage fluid of peritoneal cavity in wild-type or V{delta}1–/– mice on day 2 after E. coli infection. As shown in Fig. 5C, the levels of CCL3/MIP-1{alpha} production in wild-type mice was significantly higher than that of V{delta}1–/– mice (p < 0.05), whereas CCL5/RANTES level did not differ in V{delta}1–/– mice from that in wild-type mice. Taken together, these results suggest that V{delta}1+ {gamma}{delta} T cells in the peritoneal cavity are main source of at least CCL3/MIP-1{alpha} following E. coli infection.

Involvement of CCL3/MIP-1{alpha} in the recruitment of macrophages after infection with E. coli

The {gamma}{delta} T cells that appear during the course of infection with E. coli produce CC chemokines, including CCL3/MIP-1{alpha} and CCL5/RANTES, after TCR stimulation. The recruitment of macrophages to the peritoneal cavity was significantly reduced in V{delta}1–/– mice compared with that in wild-type mice on day 2 after E. coli infection. We therefore examined whether CCL3/MIP-1{alpha} and CCL5/RANTES produced by {gamma}{delta} T cells are involved in the accumulation of macrophages during E. coli infection. To elucidate the role of CCL3/MIP-1{alpha} and CCL5/RANTES in the defense against E. coli infection, we examined the effects of in vivo administration of anti-CCL3/MIP-1{alpha} Ab or anti-CCL5/RANTES mAb on the accumulation of macrophages and the eradication of E. coli in wild-type mice after infection. Wild-type mice were injected i.p. with anti-CCL3/MIP-1{alpha} neutralizing Ab (100 µg), anti-CCL5/RANTES neutralizing mAb (100 µg), both Abs or isotype control Abs at 1 h before E. coli challenge and were injected again with each Ab (150 µg) i.v. at 24 h after infection with E. coli. The numbers of bacteria in the peritoneal cavity and the populations of PEC were determined 24 h later. The number of peritoneal {gamma}{delta} T cells in anti-CCL3/MIP-1{alpha} Ab or anti-CCL5/RANTES mAb-treated mice was almost the same as that in control mice at that stage of E. coli infection (data not shown). There was no difference between the appearance of lymphocyte subpopulations, including CD4, CD8, NK, NKT, and B cells, in neutralizing Ab-treated mice and that in control Ab-treated mice (data not shown). As shown in Fig. 6A, a significant increase was found in the number of E. coli cells in the peritoneal cavities of anti-CCL3/MIP-1{alpha} Ab-treated mice compared with that in control Ab-treated mice. Conversely, the number of bacteria in the peritoneal cavities of anti-CCL5/RANTES mAb-treated mice was not different to that in control Ab-treated mice. Notably, the numbers of Gr-1+F4/80+ macrophages in the peritoneal cavities of anti-CCL3/MIP-1{alpha} Ab-treated mice were smaller than those in control Ab-treated mice (Fig. 6, B and C, p < 0.05), whereas the numbers of Gr-1+F4/80– PMN and lymphocyte did not differ from those in control Ab-treated mice. No differences of PEC populations between anti-CCL5/RANTES mAb and control Ab administrated mice were found. Simultaneous administration of anti-CCL3/MIP-1{alpha} and anti-CCL5/RANTES Abs did not result in synergistic effect on inhibition of macrophage accumulation and bacterial eradication. These results suggest that CCL3/MIP-1{alpha} produced by V{delta}1+ {gamma}{delta} T cells play a key role in the early protection against E. coli infection via recruitment of macrophages.



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  		FIGURE 6. Effects of chemokine neutralizations on bacterial clearance and cell influx in the peritoneal cavity after E. coli infection. Wild-type mice were injected i.p. with 100 µg of neutralizing anti-CCL3/MIP-1{alpha} Ab, anti-CCL5/RANTES Ab, both Abs or comparable doses of isotype control Ab at 1 h before E. coli challenge and were injected again with 150 µg at 24 h after infection. On day 2 after infection, the numbers of bacteria in the peritoneal cavity (A) and the populations of PEC judged by morphologic characteristics after staining with Giemsa solution (B) were counted. Each column and vertical bar indicate the mean ± SD of three mice for each group. The data are representative of three separate experiments; *, Significant differences between the values for wild-type mice and V{delta}1–/– mice (p < 0.05). C, PEC were stained with anti-Gr-1 and anti-F4/80 mAb and analyzed by FACSCalibur. The number (top right) indicates the percentage of the cells in whole PEC. The data are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We showed a potential role of V{delta}1+ T cells in recruitment of macrophages during E. coli infection via CC chemokine production. V{delta}1–/– mice exhibited an impaired accumulation of peritoneal macrophages after i.p. inoculation with E. coli, resulting in delayed bacterial clearance. Peritoneal {gamma}{delta} T cells from infected wild-type mice but not those from infected V{delta}1–/– mice produced CCL3/MIP-1{alpha} in response to {gamma}{delta} TCR triggering in vitro. Furthermore, in vivo CCL3/MIP-1{alpha} production at the infected site in V{delta}1–/– mice was severely impaired as compared with that in wild-type mice. Neutralization of CCL3/MIP-1{alpha} by a specific Ab in vivo significantly inhibited accumulation of macrophages and bacterial clearance in the peritoneal cavity after E. coli infection. These results suggest that V{delta}1+ {gamma}{delta} T cells play a novel role in innate immunity against E. coli infection mediated by production of CC chemokines enhancing macrophage trafficking to the site of infection.

Innate immunity characterized by the establishment of chemotactic gradients redirecting normally circulating phagocytes into the sites of infection takes place immediately after introduction of an infectious agent into the host environment (3). The accumulation of circulating cells at the site of infection appears to be mediated by the localized release of chemokines that are produced by a variety of cell types (1, 2, 3). Given the fact that these chemokines were induced early, they are probably critical for the initiation of cellular infiltration in the peritoneal cavity. During the course of i.p. infection with E. coli, neutrophils are the first cells to migrate from circulation to the peritoneal cavity, with monocytes/macrophages being recruited later. Although bacterial products such as N-fMLP, complement activation of fragments such as C5a, C3a, and C4a, and CXC chemokines produced by cells resident in the peritoneal cavity may be involved in early infiltration of neutrophils, the vast majority of chemokine expression for monocyte/macrophage infiltration appears to be associated with the infiltrating cells (1, 2, 3). However, it remains to be determined which cells are involved in macrophage migration following infiltration of neutrophils. We found in the previous and present studies that invariant V{delta}1+ {gamma}{delta} T cells were present in the peritoneal cavity on day 2 after infection, concurrent with infiltration of macrophages in the peritoneal cavity of wild-type mice. Although the early cellular infiltration of neutrophils was intact on day 1, the following infiltration of peritoneal macrophages was severely impaired in V{delta}1–/– mice on day 2 after infection. The chemokine profile of V{delta}1+ {gamma}{delta} T cells induced by E. coli infection and in vivo neutralization of these chemokines revealed that CCL3/MIP-1{alpha} produced by V{delta}1+ {gamma}{delta} T cells is strongly involved in the infiltration of macrophages in the peritoneal cavity during E. coli infection. In contrast, early infiltration of neutrophils normally occurs in V{delta}1–/– mice after E. coli infection, suggesting that CXC chemokines and other chemoattractants predominantly targeting neutrophils are normally produced in the absence of V{delta}1+ T cells. It can be speculated that bacterial products such as N-fMLP, complement activation of fragments such as C5a, C3a, and C4a and CXC chemokines produced by cells resident in the peritoneal cavity that are the first to encounter the pathogen induce the initial phase of migration of neutrophils from circulation to the infected sites, whereas V{delta}1+ {gamma}{delta} T cells play a critical role in second phase of recruitment of monocytes to the infected sites. Thus, V{delta}1-bearing {gamma}{delta} T cells, which are thought to be more primitive than other T cells, may bridge a gap between host defense mechanisms by neutrophils and macrophages in innate immunity during E. coli infection.

Similar to {alpha}{beta} T cells, {gamma}{delta} T cells secret various cytokines and have cytolytic functions. Most {gamma}{delta} T cells appearing after infection with intracellular bacteria have been reported to produce Th1-type cytokines, particularly IFN-{gamma} (18, 21, 22, 23), whereas {gamma}{delta} T cells during infection with helminth, Nippostrongylus brasiliensis, preferentially produced Th2-type cytokines, mostly IL-4 (23). Furthermore, {gamma}{delta} T cells, especially those in the epithelium, produce TGF-{beta} for immunoregulation and/or IgA production (24, 25, 26). In a murine model for neurocysticercosis by Mesocestoides corti infection, {gamma}{delta} T cells produced chemokines, including CCL3/MIP-1{alpha} and CCL5/RANTES, in the CNS immune response, enhancing leukocyte trafficking to the brain during neurocysticercosis (27). In simian immunodeficiency virus infection, mucosal {gamma}{delta} T cells produced CCL3/MIP-1{alpha}, CCL4/MIP-1{beta}, and CCL5/RANTES upon TCR triggering (28). Boismenu et al. (29) reported that a dendritic epidermal T cell line (DECT7-17) bearing invariant V{gamma}5/V{delta}1 TCRs expressed CCL3/MIP-1{alpha}, CCL4/MIP-1{beta}, and CCL5/RANTES but not CCL2/MCP-1. Dorner et al. (30) have recently reported that CCL3/MIP-1{alpha}, CCL4/MIP-1{beta}, CCL5/RANTES, and ATAC/lymphotactin functioned together with IFN-{gamma} as type 1 cytokines. In murine listeriosis, NK cells on day 1 or day 2 after infection in innate immunity and Th1 cells on day 7 after infection in adaptive immunity produced CCL3/MIP-1{alpha}, CCL4/MIP-1{beta}, CCL5/RANTES, and ATAC/lymphotactin, which are not only chemoattractants but also coactivators of macrophages together with IFN-{gamma}. Our previous results also revealed that {gamma}{delta} T cells accumulating in the peritoneal cavity after E. coli infection produced IFN-{gamma} in the presence of LPS under TCR triggering (12, 15). We speculate that V{delta}1+ {gamma}{delta} T cells produce these chemokines together with IFN-{gamma} in response to E. coli and are involved in the attraction and activation of macrophages, which consequently eliminate the E. coli. The early production of Th1-type cytokines in innate immunity is thought to play an important role in subsequent differentiation of Th1 cells in adaptive immunity. Canonical V{gamma}6/V{delta}1 {gamma}{delta} T cells are abundant in the female reproductive tract. We have recently found that V{delta}1–/– mice had impaired CD4 Th1 responses against intravaginal infection with herpes simplex type 2 (31). Although CD4 Th1 response is not apparent in E. coli infection, V{delta}1+ {gamma}{delta} T cells may also play a role in bridging the innate and adaptive immunity in mucosal infection via production of type 1 cytokines, including CCL3/MIP-1{alpha}, CCL4/MIP-1{beta}, CCL5/RANTES, and ATAC/lymphotactin.

The V{gamma}6/V{delta}1 {gamma}{delta} T cells induced by E. coli infection bear truly invariant TCRs, even to the nucleotides in the TCR gene junction (7, 8, 9). The canonical sequence is very simple, with no apparent N region contribution (7). Such a characteristic has led to the hypothesis that {gamma}{delta} T cells represent a preprogramm to recognize a limited set of Ags in E. coli or self Ag induced by E. coli infection. At present, the specificity of {gamma}{delta} T cells remains unknown. {gamma}{delta} T cells can directly recognize Ags in the form of intact proteins or nonpeptide compounds, unlike {alpha}{beta} TCRs, which only recognize Ags bound to MHC molecules (32). A number of murine {gamma}{delta} T cell clones have been reported to recognize MHC molecules or MHC-related gene products such as TL and Qa in a manner quite different from the Ag recognition shown by {alpha}{beta} T cells (33, 34, 35, 36). Human {gamma}{delta} T cells are stimulated by apparently nonproteinous low-molecular-weight ligands, including isopentenyl pyrophosphate, which represents a ubiquitous metabolite of various vitamins, lipids, and steroids both in prokaryotic and eukaryotic cells (37, 38, 39). Using genetically engineered E. coli knockout strains, Altincicek et al. (40) demonstrated that the ability of E. coli extracts to stimulate human {gamma}{delta} T cell proliferation is abrogated when genes coding for essential enzymes of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome. Recently, Feurle et al. (41) reported that the structures of the two compounds responsible for the human {gamma}{delta} T cell-stimulating capacity of E. coli are similar to those of mycobacterial phosphoantigens 3-formyl-1-butyl-pyrophosphate and its Mr 275 homologue TUBag2. They showed that the deoxy-D-xylulose 5-phosphate pathway is essential for the biosynthesis of the phosphoantigens in E. coli. Because this pathway is absent in human cells, it proves an ideal target for efficiently focusing the antimicrobial electivity of human {gamma}{delta} T cells. Therefore, it is of interest to elucidate whether the murine {gamma}{delta} T cells induced by E. coli recognize such unique Ags in a manner different from that of {alpha}{beta} T cells.

In conclusion, we have shown that V{delta}1+ {gamma}{delta} T cells induced in the peritoneal cavity after E. coli infection produce Th1-type chemokines in response to TCR triggering. V{delta}1+ {gamma}{delta} T cells may participate in attraction and activation of macrophages after E. coli infection, thus bridging protection mechanisms by neutrophils and macrophages during E. coli infection.


   Acknowledgments
 
We thank Dr. R. Kubo and Dr. J. A. Bluestone for providing anti-TCR-{beta} mAb (H57-597) and anti-TCR-{gamma}{delta} mAb (UC7-13D5), respectively. We also thank K. Kaneda for preparing the manuscript and Y. Kobayashi for technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grant-in-aid for Scientific Research on Priority Areas, Japan Society for the Promotion of Science, and by grants from the Japanese Ministry of Education, Science, and Culture (to Y.Y.), Yakult Bioscience Foundation (to Y.Y.), Uehara Memorial Foundation (to Y.Y.), Nakamura Jishirou Foundation (to H.N.), Kurozumi Medical Foundation (to H.N.), Kanzawa Medical Research Foundation (to H.N.), Japan Rheumatism Foundation (to H.N.), and Kudo Research Foundation (to H.N.). Back

2 Address correspondence and reprint requests to Dr. Hitoshi Nishimura, Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: nishihit{at}bioreg.kyushu-u.ac.jp Back

3 Abbreviations used in this paper: PEC, peritoneal exudate cell; PMN, polymorphonuclear leukocyte. Back

Received for publication April 23, 2004. Accepted for publication August 11, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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