Seeligeriolysin O, a protein toxin of Listeria seeligeri, stimulates macrophage cytokine production via Toll-like receptors in a profile different from that induced by other bacterial ligands

Yutaka Ito1,2, Ikuo Kawamura1, Chikara Kohda1, Kohsuke Tsuchiya1, Takamasa Nomura1 and Masao Mitsuyama1

1 Department of Microbiology and 2 Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan

Correspondence to: Y. Ito; E-mail: yutaka{at}kuhp.kyoto-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Seeligeriolysin O (LSO), a member of cholesterol-dependent cytolysins of Listeria seeligeri, exhibits cytokine-inducing activity. In this study, we examined the profile of cytokines expressed in macrophages of mice after stimulation with full-length form of recombinant LSO (rLSO530), C-terminal-truncated protein (rLSO483) and two authentic cytokine-inducing Toll-like receptor (TLR) ligands from bacteria, peptidoglycan (PGN) and LPS. Both rLSO530 and rLSO483 were able to induce IL-12 p40 and IL-12 p70 more strongly in macrophages than PGN or LPS. In contrast, IFN-ß and nitric oxide were induced by LPS but not by rLSO530, rLSO483 or PGN. In the presence of exogenously added IFN-ß, IL-12 p40 and IL-12 p70 production was inhibited after LSO stimulation, but IL-12 p70 production was enhanced after PGN stimulation. Although LSO signaling appeared to be associated with both TLR2 and TLR4, the profile of cytokine production by LSO stimulation was distinct from those by stimulation with PGN or LPS. Thus, it was shown that LSO is a unique bacterial ligand that induces macrophage cytokine production in a manner different from PGN or LPS.

Keywords: interferon-beta, interleukin-12, lipopolysaccharide, peptidoglycan, seeligeriolysin O, Toll-like receptor


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Listeria monocytogenes is an intracellular parasitic Gram-positive bacterium that produces various virulence factors. One of the major virulence factors listeriolysin O (LLO) is a member of cholesterol-dependent cytolysins (CDCs) and is essential for the pore formation in the phagosomal membrane enabling the bacterial escape from the phagosomal compartment into cytosolic space (15). Listeria monocytogenes has been shown to induce various inflammatory cytokines such as IL-1, IL-6, IL-12, tumor necrosis factor alpha (TNF{alpha}) and IFN-{gamma} (69). Among these cytokines, IFN-{gamma} plays the most important role in the protection of mice against L. monocytogenes infection (1013). In a series of studies, we have shown that LLO itself is able to induce IFN-{gamma} in naive spleen cells (14, 15) and that both IL-12 and IL-18 from the cells of macrophage lineage are essential for the production of LLO-induced IFN-{gamma}, which is mainly secreted from NK cells (14, 16). We have also shown that seeligeriolysin O (LSO, 59 kDa) produced by Listeria seeligeri, one of the CDCs highly homologous to LLO, is capable of inducing IFN-{gamma} more strongly than LLO (17). Therefore, it may be argued that LLO and LSO play the pivotal role in the induction of Th1-mediated immune response to listerial infection in mice.

IL-12 is a 75-kDa heterodimeric cytokine that plays a crucial role in the induction of IFN-{gamma} by NK cells and generation of Th1 cells (18, 19). This cytokine is induced by several bacterial products including LPS, un-methylated CpG motifs and intracellular parasitic bacteria. It is known that IL-12 production is regulated by several cytokines. IFN-{gamma} up-regulates, whereas IL-10, IL-4, TNF{alpha} and transforming growth factor beta down-regulate the IL-12 production (19, 20).

IFN-ß is secreted in response to viral infection and Gram-negative bacteria (21). Recently, it was reported that IFN-ß was produced in macrophages infected with L. monocytogenes expressing LLO (22, 23). IFN-{alpha}/ß modulates various immune responses including up-regulation of the inducible nitric oxide synthase (iNOS) gene (24, 25). However, the role of IFN-{alpha}/ß on IL-12 production is controversial. IFN-{alpha} suppressed IL-12 production in Staphylococcus aureus Cowan (SAC)-stimulated splenic leukocytes and in SAC- plus IFN-{gamma}-stimulated human PBMCs in an IL-10-dependent mechanism (26, 27). In contrast, IFN-ß-1b showed an additive effect on SAC-stimulated human PBMCs (28).

Toll-like receptors (TLRs) recognize bacterial products and mediate the signaling for induction of pro-inflammatory cytokines (29). Several reports have shown that different TLR agonists induce distinct cytokine production (30). TLR4 ligands, such as LPS, induced IFN-ß gene expression more strongly than TLR2 ligands, such as peptidoglycan (PGN), and the signal through TIR domain-containing adaptor inducing INF-ß (TRIF) activated IFN-ß gene expression independent of MyD88 (31, 32). The TLR9 ligand, CpG DNA, can induce the expression of IFN-ß via the MyD88-dependent pathway (33).

In the present study, we have compared the profile of cytokine expression including IFN-ß in macrophages after stimulation with LSO and other two representative bacterial ligands for TLRs, LPS and PGN. We have employed full-length recombinant LSO (rLSO530) and a C-terminal-truncated rLSO483 that we have already reported to show cytokine-inducing activity without cytolytic activity (17).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Experimental animals and preparation of cells
Female mice of C3H/HeN and TLR4-defective C3H/HeJ strain (SLC Japan, Hamamatsu, Japan), raised and maintained in a specific-pathogen-free environment, were used for experiments at 7–10 weeks of age. TLR2 knockout (KO) and TLR4 KO mice were kindly provided by S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan). Mice were intra-peritoneally injected with 3 ml of 3% thioglycolate, and peritoneal exudates cells (PECs) were harvested 3 days later from the peritoneal cavity by washing with RPMI 1640 medium (GIBCO-BRL, Rockville, MD, USA). Cells were cultured with complete medium consisting of RPMI 1640 supplemented with 10% FCS and 5 µg ml–1 gentamicin (GIBCO-BRL) for 2 h. After removal of non-adherent cells, adherent PECs were cultured with complete medium. L-929 cells were obtained from M. Kita (Department of Microbiology, Kyoto Prefectural University of Medicine, Japan). All animal experiments have been approved by the Review Board of Kyoto University Graduate School of Medicine.

Reagents
Recombinant IFN-ß (rIFN-ß) and anti-mouse IFN-ß antibody (clone 7F-D3, a rat IgG1) were obtained from KATAKURA (Tokyo, Japan) and YAMASA Syoyu Co. (Tokyo, Japan), respectively. Rat control IgG was obtained from Santa Cruz (Santa Cruz, CA, USA) and PGN from S. aureus and LPS from Escherichia coli O5 were obtained from Fluka (Tokyo, Japan) and GIBCO-BRL, respectively.

Production and purification of recombinant protein
The full-length form of rLSO530 and the truncated form of rLSO483 with deletion of C-terminal amino acids including the conserved undecapeptide were prepared as described previously (17). The level of LPS was determined by the Limulus Color KY Test (Wako Pure Chemical Industries, Osaka, Japan) and was revealed to be <5 pg ml–1 when suspended in PBS at a protein concentration of 10 µg ml–1. Before being added to PECs, rLSO530 were incubated with 40 µg ml–1 of cholesterol in order to block the cytolytic activity (17). Because of the absence of the domain required for cytolytic activity, the rLSO483 was not treated with cholesterol.

Transfection and nuclear factor {kappa}-B reporter assay
Transient transfection of RAW264.7 cells was performed using Superfect reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Transfection mixtures containing 1 µg of nuclear factor {kappa}-B (NF-{kappa}B) reporter luciferase plasmid were incubated with RAW264.7 cells for 3 h. After medium change and incubation for 18 h, the cells were stimulated with 200 nM of rLSO530, 200 nM of rLSO483, 5 µg ml–1 of PGN and 1 µg ml–1 of LPS and harvested 24 h later. Luciferase assay was performed using the Luciferase Assay Systems (Promega, Madison, WI, USA).

Measurement of pro-inflammatory cytokine
Adherent PECs were plated at 2 x 105 per well in a 96-well flat-bottom tissue culture plate and were stimulated with rLSO530, rLSO483, PGN or LPS. In some experiments, rIFN-ß, anti-IFN-ß antibody or control IgG was added to the culture. The culture supernatant was collected after incubation for 24 h at 37°C in 5% CO2. The titers of various cytokines were determined by a two-site sandwich ELISA. ELISA kits for IL-12 p70 were from R&D Systems (Minneapolis, MN, USA), IL-6, IL-12 p40 and TNF{alpha} from Pharmingen (San Diego, CA, USA). To determine the nitric oxide (NO) production, the concentration of nitrite in the culture supernatant was measured by Griess reaction by using modified Griess reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA).

Reverse transcription–PCR
Adherent PECs were stimulated with each ligand for 6 h. Total cellular RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was reverse transcripted from 0.2 µg of total RNA using a random primer as described previously (16, 27, 28). PCR was performed by using KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan) and primer sets specific for each cytokine, iNOS and ß-actin. The PCR cycle consisted of 94°C for 15 s, 60°C for 30 s and 68°C for 60 s. The samples were amplified for 22–30 cycles. The most appropriate number of amplification cycles for each cytokine was determined by preliminary experiments. The reaction was terminated by incubation at 68°C for 7 min. The sequence of the oligonucleotide primers used were as follows: 5'-CTCTAGAGCACCATGCTACAGAC-3' and 5'-TGGAATCCAG-GGGAAACACTG-3' for IL-1{alpha}, 5'-AAGCTCTCCACCTCAATGGACAG-3' and 5'-CTCAAACTCCAC-TTTGCTCTTGA-3' for IL-1ß, 5'-CTGCATCAGCTCATCGATGG-3' and 5'-CAG-AAGCTAACCATCTCCTGGTTT-3' for IL-12 p35, 5'-TCCGGAGTAATTTGGTGC-TTCACA-3' and 5'-ACTGTACAACCGCAGTAATACGG-3' for IL-12 p40, 5'-ACT-GTACAACCGCAGTAATACGG-3' and 5'-AGTGAACATTACAGATTTATCCC-3' for IL-18, 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' and 5'-ACATTCGAGGCTC-CAGTGAATTCCA-3' for TNF{alpha}, 5'-AAACAATTTCTCCAGCACTG-3' and 5'-AT-TCTGAGGCATCAACTGAC-3' for IFN-ß, 5'-CCCTTCCGAAGTTTCTGGCAGCA-GC-3' and 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3' for iNOS and 5'-TGGAAT-CCTGTGGCATCCATGAAAC-3' and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3' for ß-actin. The PCR products (5 µl) were electrophoresed on a 2% agarose gel in 0.5 M Tris–acetate–EDTA buffer and stained with 0.005% ethidium bromide. The bands of the PCR product were visualized on a UV transilluminator.

Real-time quantitative reverse transcription–PCR
A quantitative analysis of expression of IL-12 p35 and IL-12 p40 was done by real-time PCR assay for cDNAs by using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The reaction mixture was incubated for 2 min at 50°C, denatured for 10 min at 95°C and subjected to 40 cycles of two-step amplification with annealing/extension at 60°C for 1 min followed by denaturation at 95°C for 15 s. Primers for 12 p40 and ß-actin were the same as indicated previously. Primers for IL-12p35 were 5'-GCAGACCCTTACAGAGTGAAAATG-3' and 5'-GATAGCCCATCAC-CCTGTTGA-3'. The values were expressed as arbitrary units relative to ß-actin.

Bioassay for type I IFN
Type I IFN in culture supernatant was determined by a bioassay based on previous reports (34, 35). In short, the supernatants from adherent PECs after stimulation with rLSO530, rLSO483, PGN and LPS were serially diluted with complete medium. L-929 cells (2.5 x 104 per well) were incubated with the diluted supernatants on the 96-well flat-bottom tissue culture plate for 6 h, then the cells were infected with 500 plaque-forming units per well of vesicular stomatitis virus. The IFN titer was read 18 h later as the reciprocal of dilution. One unit per milliliter was defined as the amount that inhibits the cytopathic effect by 50%.

Statistical analysis
A Student's t test or one-way analysis of variance was used to examine for significant effects of culture condition on cytokine or NO production. Variation among culture conditions was examined with a Scheffe test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
LSO-induced TNF{alpha} production and NF-{kappa}B activation in macrophages
We previously reported that LSO induced IFN-{gamma} production and expression of various cytokine genes in spleen cells (17). When adherent PECs of C3H/HeN mice were stimulated by the full-length form of the recombinant protein (rLSO530) and the C-terminal-truncated form (rLSO483) for 24 h, TNF{alpha} was produced in the culture supernatant (Fig. 1A). The level of contaminating LPS in rLSO530 and rLSO483 was <5 pg ml–1. A possible involvement in TNF{alpha} response of this minute amount of LPS in recombinant preparations could be ruled out because the same amount of LPS alone never induced the response (data not shown). Moreover, addition of polymyxin B (PMB) did not affect cytokine response induced by rLSO530 or rLSO483, whereas the same amount of PMB completely abolished the LPS-induced TNF{alpha} production (Fig. 1A). Both cholesterol-treated rLSO530 and cholesterol-non-treated C-terminal-truncated rLSO483 were not cytolytic, but were able to induce TNF{alpha} production. Therefore, it was confirmed that LSO-induced cytokine production from macrophages did not depend on cytolytic activity or cholesterol treatment. This result was consistent with our previous experimental data in mouse spleen cells (17).



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Fig. 1. (A) TNF{alpha} production induced by rLSO530 and rLSO483 and effect of PMB. Adherent PECs were stimulated with rLSO530, rLSO483 (200 nM) or LPS (10 pg ml–1) for 24 h in the presence or absence of PMB (0.5 µg ml–1). The level of TNF{alpha} was measured by ELISA. A representative result was shown. Similar results were obtained in three independent experiments. Data are means ± SE for three determinations. Asterisks indicate that the value is significantly different from the untreated group as determined by the Student's t test (P < 0.05). LSO induced activation of NF-{kappa}B. RAW267.4 cells were transfected with NF-{kappa}B reporter luciferase plasmid. The cells were stimulated with rLSO530 (200, 100 and 50 nM) and rLSO483 (400, 200 and 100 nM) (B) and with rLSO530 (200 nM), rLSO483 (200 nM), PGN (5 µg ml–1) and LPS (1 µg ml–1) (C). Similar results were obtained in three independent experiments and a representative result was shown. Data are means ± SE for three determinations. Asterisks indicate that the value is significantly different from the culture containing PBS as determined by one-way analysis of variance and the Scheffe test (P < 0.05). No statistical significance is detected among cultures containing rLSO530, rLSO483, PGN and LPS.

 
Among several transcription factors, NF-{kappa}B is an important molecule in the transcription of various pro-inflammatory cytokine genes including TNF{alpha} (36). In order to assess NF-{kappa}B activity of LSO, we measured the NF-{kappa}B promoter activity of RAW264.7 cells transfected with the plasmid-harboring NF-{kappa}B-binding promoter site and downstream luciferase reporter. NF-{kappa}B was activated by rLSO530 and rLSO483 in a dose-dependent manner (Fig. 1B). When stimulated with 200 nM of rLSO530 and rLSO483, 5 µg ml–1 of PGN and 1 µg ml–1 of LPS for comparison, almost the same level of NF-{kappa}B activation was induced (Fig. 1C).

Different profile of macrophage cytokine response to LSO, PGN and LPS
We next compared the profile of various cytokines (TNF{alpha}, IL-6, IL-12 p40 and IL-12 p70) and NO production after stimulation with rLSO530, rLSO483, PGN and LPS. All bacterial ligands induced the production of cytokines and NO in a dose-dependent manner, but the profiles of the produced cytokines were different. The levels of TNF{alpha} production were also almost the same at the concentration of bacterial ligands which induced a similar level of NF-{kappa}B. In the same condition, a very high level of IL-12 p40 and p70 production was induced by both rLSO530 and rLSO483, whereas the highest level of IL-6 and NO was induced by PGN and LPS, respectively, but not by rLSOs (Fig. 2).



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Fig. 2. A different profile of cytokine and NO production induced by bacterial ligands. Adherent PECs were stimulated for 24 h with rLSO530 (200, 100 and 50 nM), rLSO483 (400, 200, 100 and 50 nM), PGN (5, 2.5, 1 and 0.1 µg ml–1) and LPS (1, 0.1, 0.01 and 0.001 µg ml–1). The levels of TNF{alpha}, IL-6, IL-12 p40 and IL-12 p70 in culture supernatant were measured by ELISA. The level of NO was measured by the Griess assay. Similar results were obtained in three independent experiments and a representative result was shown. Data are means ± SE for three determinations. When compared with control value obtained after stimulation with rLSO530 (200 mM), each asterisk indicates the significant difference as determined by one way analysis of variance and the Scheffe test (P < 0.05).

 
Cytokine gene expression in macrophages
To assess the different profile of cytokine production induced by the bacterial ligands, gene expression for macrophage-derived cytokine was examined by reverse transcription (RT)–PCR. Total RNA was extracted at 2, 4 and 6 h after stimulation, and cytokine-specific mRNA was examined by RT–PCR (Fig. 3A). Expression of IL-1{alpha}, IL-1ß, IL-6, IL-18 and TNF{alpha} was induced similarly by all the ligands tested. Production of IL-12 p35 and IL-12 p40 induced by rLSO530, rLSO483 and PGN was dependent on the time of stimulation. In contrast, IL-12 p40 production was induced immediately after stimulation with LPS followed by gradual decrease. The expression of IL-12 p35 and IL-12 p40 was further analyzed by real-time quantitative PCR (Fig. 3B). Both rLSO530 and rLSO483 induced these cytokines more strongly than PGN and LPS. This result was consistent with the production of IL-12 p40 and IL-12 p70 measured by ELISA (Fig. 2). It was shown that LPS strongly induced the expression of IFN-ß and iNOS genes whereas PGN did poorly (Fig. 3A), and this observation was consistent with a previous report (31). The pattern of IFN-ß and iNOS gene expression induced by rLSO530 and rLSO483 was similar to that induced by PGN.



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Fig. 3. Expression of mRNA for various cytokines and iNOS after stimulation with rLSO530, rLSO483, PGN and LPS. PECs were stimulated with rLSO530 (200 nM), rLSO483 (200 nM), PGN (5 µg ml–1) and LPS (1 µg ml–1) for 6 h. Total RNA was extracted and subjected to RT–PCR for detection of cytokine mRNA for IL-1{alpha}, IL-1ß, IL-6, IL-12 p35, IL-12 p40, IL-18, TNF{alpha}, IFN-ß, iNOS and ß-actin. Lanes: 1, PBS; 2, LSO530; 3, LSO483; 4, PGN; 5, LPS. (A) PCR products were run on a 2% agarose gel and visualized using ethidium bromide. (B) cDNA was analyzed for the expression of IL-12 p35 and IL-12 p40 by the quantitative PCR assay. Values were expressed as arbitrary units (relative to ß-actin). A representative result was shown. Similar results were obtained in two independent experiments.

 
Type I IFN production and the effect of IFN-ß-specific neutralizing antibody on LPS-induced cytokines and NO production
As an ELISA kit for mouse IFN-ß was not commercially available, IFN-ß production was determined by a bioassay based on the cytopathic effect. Just like the RT–PCR result of IFN-ß gene expression, production of type I IFN was observed after stimulation with LPS but not with rLSO530, rLSO483 or PGN (Fig. 4A). NO production induced by LPS was reported to be regulated by autocrine/paracrine secreted IFN-{alpha} (24, 25). One possibility for the different profile of cytokine production was due to a strong expression of IFN-ß by LPS. In order to assess the effect of IFN-ß on the production of NO, TNF{alpha}, IL-12 p40 and IL-12 p70, IFN-ß-specific neutralizing antibody was added to adherent PECs upon stimulation with LPS. As expected, NO production was inhibited in the presence of antibody to IFN-ß, whereas the neutralizing antibody did not affect the production of TNF{alpha}, IL-12 p40 and IL-12 p70 (Fig. 4B). Therefore, it was suggested that LPS-induced IFN-ß production was involved in a large amount of NO production by LPS stimulation.



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Fig. 4. (A) Type I IFN production from adherent PECs stimulated by bacterial ligands. The culture supernatants obtained in Fig. 2 were serially diluted with complete medium. After L-929 cells (2.5 x 104 per well) were incubated with the diluted supernatants for 6 h, the cells were infected with 500 plaque-forming units per well of vesicular stomatitis virus. The IFN titer was read as the reciprocal dilution 18 h later. One unit per milliliter was defined as the amount necessary for cytopathic effect by 50%. (B) Effect of anti-IFN-ß polyclonal antibody on cytokines and NO production induced by LPS. PECs were stimulated with LPS (1 µg ml–1) for 24 h in the presence of anti-IFN-ß polyclonal antibody or control rabbit IgG (5 µg ml–1). The level of TNF{alpha}, IL-12 p40 and IL-12 p70 was measured by ELISA and the level of NO was measured by the Griess assay. A representative result was shown. Similar results were obtained in two independent experiments. Data are means ± SE for three determinations. The single asterisk indicates that the value is significantly different from the culture containing PBS as determined by one-way analysis of variance and the Scheffe test (P < 0.05). Double asterisks indicate that the value is significantly different from the culture containing control IgG as determined by the Student's t test (P < 0.05).

 
Effect of exogenous addition of IFN-ß on cytokine and NO production
Next, we evaluated the effect of addition of rIFN-ß exogenously on the cytokine and NO production. PECs were stimulated with rLSO530, rLSO483, LPS and PGN in the presence of varying doses of rIFN-ß. Addition of rIFN-ß did not show any effect on TNF{alpha} and IL-6 production, whereas NO production was strongly enhanced. This result was compatible with that obtained by using IFN-ß-specific neutralizing antibody in LPS stimulation (Fig. 4B). An interesting finding was the effect of rIFN-ß on IL-12 p70 production. When stimulated with rLSO530 or rLSO483 in the presence of rIFN-ß, IL-12 p70 production was suppressed depending on the dose of rIFN-ß. In contrast, IL-12 p70 production was enhanced by addition of rIFN-ß to the culture stimulated with PGN (Fig. 5).



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Fig. 5. Effect of additional IFN-ß on cytokines and NO production induced by bacterial products. PECs were stimulated with the same dose of bacterial ligands as in Fig. 1 for 24 h in the presence of several doses of rIFN-ß. The level of TNF{alpha}, IL-6, IL-12 p40 and IL-12 p70 was measured by ELISA and the level of NO was measured by the Griess assay. A representative result was shown. Similar results were obtained in two independent experiments. Data are means ± SE for three determinations. Asterisks indicate that the value is significantly different from the culture of cells stimulated without rIFN-ß as determined by one-way analysis of variance and the Scheffe test (P < 0.05).

 
Involvement of TLRs in the LSO-induced cytokine response
The expression of IFN-ß is reported to be mediated by TLR4 signaling, not by TLR2 signaling (31). To investigate the involvement of TLR in LSO-induced cytokine production, we first compared the gene expression and production of cytokines induced by LSO between PECs from C3H/HeN and C3H/HeJ mice that are known to be functionally defective in TLR4-dependent signaling (37). Cells were stimulated by rLSO530, rLSO483, PGN and LPS for 4 h, thereafter gene expressions for IL-6 and iNOS were examined by RT–PCR. While all the ligands induced the expression of these genes in C3H/HeN macrophages, a similar level of expression in C3H/HeJ mice was induced only by PGN (Fig 6A). The amount of IL-6 and NO produced in the culture supernatant was measured by ELISA and the Griess reagent assay, respectively. Consistent with the gene expression, production of IL-6 and NO by rLSO530, rLSO483 and LPS was not observed in C3H/HeJ macrophages (Fig 6B). The result clearly showed that rLSO530 and rLSO483 utilized at least TLR4 to mediate the signaling for cytokine expression.



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Fig. 6. (A) Expression of mRNA for IL-6 and iNOS in adherent PECs of C3H/HeN and C3H/HeJ mice. Adherent PECs were stimulated with rLSO530 (200 nM), rLSO483 (200 nM), PGN (5 µg ml–1) and LPS (1 µg ml–1) for 4 h. Total RNA was extracted and subjected to RT–PCR for detection of cytokine mRNA for IL-6, iNOS and ß-actin. PCR products were run on 2% agarose gel and visualized using ethidium bromide. Lanes: 1, PBS; 2, LSO530; 3, LSO483; 4, PGN; 5, LPS. (B) Adherent PECs of C3H/HeN and C3H/HeJ mice were stimulated for 24 h with the bacterial products. The level of IL-6 and NO in culture supernatant was measured. A representative result was shown. Similar results were obtained in two independent experiments. Data are means ± SE for three determinations. Asterisks indicate that the value is significantly different from the culture containing PBS as determined by one-way analysis of variance and the Scheffe test (P < 0.05).

 
Next, we evaluated TLR2 signaling in LSO-induced cytokine production. After stimulation of adherent PECs from TLR2 KO mice, TLR4 KO mice and wild-type mice with rLSO530, rLSO483, PGN and LPS for 24 h, TNF{alpha}, IL-12 p40 and IL-6 in the supernatant were measured by ELISA. LSO-induced TNF{alpha}, IL-12 p40 and IL-6 production was suppressed not only in TLR4 KO mice but also in TLR2 KO mice (Fig. 7).



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Fig. 7. PECs of C57/B6 (wild type), TLR2 KO and TLR4 KO mice were stimulated for 24 h with the bacterial products. The level of TNF{alpha}, IL-6 and IL-12p40 in the culture supernatant was measured. A representative result was shown. Similar results were obtained in two independent experiments. Data are means ± SE for three determinations. Asterisks indicate that the value is significantly different from the culture of wild-type cells as determined by one-way analysis of variance and the Scheffe test (P < 0.05).

 
When PECs were stimulated with PGN and LPS at the same time, the production of TNF{alpha} and NO was stronger than that after stimulation with rLSO530 or rLSO483. However, IL-12 p40 and IL-12 p70 production in PGN plus LPS at the same dose did not overcome those in rLSO530 or rLSO483 (data not shown). Considering that LSO-induced cytokine production is depending on TLR2 and TLR4 (Figs 6 and 7), LSO-induced signaling seemed to be transduced simultaneously, but not independently, via TLR2 and TLR4.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In our previous report, it was shown that LSO from L. seeligeri was highly potent in the induction of IFN-{gamma} from the spleen cells of mice and that cytokine-inducing activity was not dependent on the cholesterol-binding domain of LSO by using full-length rLSO530 and C-terminus-truncated rLSO483 (17). In the present study using peritoneal macrophages of mice, we have looked at the ability of rLSO530 and rLSO483 to induce cytokines and NO production that are important for the host defense against bacterial infection (38). We have also attempted to compare the profile of cytokine and NO response among various bacterial ligands known to stimulate NF-{kappa}B activation in macrophages.

As NF-{kappa}B is an essential DNA-binding protein that promotes the gene expression of several cytokines including TNF{alpha} (36), macrophages were stimulated with rLSO530, rLSO483, LPS and PGN at the doses that induced a similar level of NF-{kappa}B activation. Under this condition, a different profile was observed in the induction of IL-12 p40, IL-12 p70 and NO production among bacterial ligands tested. PGN induced IL-6 production stronger than rLSOs and LPS. IL-12 p40 and p70 were strongly induced by rLSOs but not by LPS, in contrast, NO production was induced only by LPS. It has been shown that autocrine/paracrine IFN-{alpha} mediates LPS-induced iNOS gene expression (24). In addition, LPS, a TLR4 ligand, is reported to induce IFN-ß gene expression more strongly than PGN, a TLR2 ligand (31), and IFN-ß is known to be produced from macrophages infected with L. monocytogenes expressing LLO while LLO itself did not induce IFN-ß (22). Based on these reports, we have analyzed the involvement of IFN-ß in the different profile with special reference to the induction of NO. As expected, LPS strongly induced the expression of the IFN-ß gene whereas PGN, rLSO530 and rLSO483 did not (Fig. 3). LPS-induced NO production was suppressed significantly in the presence of IFN-ß-specific neutralizing antibody (Fig. 4B) and the addition of rIFN-ß up-regulated NO production induced by PGN, LSO530 and LSO483. These data suggested that the different level of NO production was highly dependent on IFN-ß production.

The profile of IL-12 p40 or IL-12 p70 production among the bacterial ligands tested in this study was different from that of NO. Both rLSO530 and rLSO483 were able to induce the production of higher levels of IL-12 than LPS or PGN (Fig. 2). Furthermore, the effect of IFN-ß on IL-12 production was different among the ligands whereas IFN-ß was essential for NO production. Additional rIFN-ß enhanced the PGN-induced IL-12 production, whereas rIFN-ß suppressed the LSO-induced IL-12 production. A recent report demonstrated that IFN-{alpha} receptor KO mice were more resistant to L. monocytogenes infection and that the resistance was correlated with elevated levels of IL-12 p70 (39). Our results concur with this finding in the point that type I IFNs can inhibit IL-12 production. There are several controversial reports about the effect of additional IFN-ß on IL-12 production. SAC enhanced IL-12 p70 production in the presence of IFN-ß (28), whereas SAC plus IFN-{gamma}-induced IL-12 p40 and IL-12 p70 or Chlamydia pneumoniae antigen-induced IL-12 p40 were suppressed by additional IFN-ß (28, 40). In contrast, a recent study showed that exogeneous IFN-ß enhanced LPS-induced IL-12 p70 secretion in dendritic cells (41).

Among several TLR ligands known to date, it is shown that the representative TLR4 ligand, LPS, induced less IL-12 p40 mRNA compared with a TLR9 ligand, CpG DNA (42). In a recent article, pneumolysin, which is produced by Streptococcus pneumoniae and a member of CDCs like LSO, was reported to be a TLR4 ligand (43). From our present result using macrophages from C3H/HeJ, TLR2 KO and TLR4 KO mice, LSO was suggested to be a TLR2/TLR4 ligand (Fig. 6). LPS-induced TLR4 signaling has two pathways, MyD88-dependent and MyD88-independent pathways (44). IFN-ß production was reported to be associated with the MyD88-independent pathway (32). Although both LSO and LPS appear to share the TLR4-mediated signaling cascade, LSO did not induce IFN-ß production like LPS. The result suggested that LSO was different from LPS in that LSO required TLR2 in addition to TLR4 for signaling. Even after stimulation with PGN plus LPS, PGN-induced IL-12 production did not overcome LSO-induced IL-12 production. Therefore, LSO signaling may be different from PGN signaling. It remains to be determined whether LSO recognizes TLR2 and TLR4 simultaneously as a heterodimeric receptor or independent receptor at the same time.

In conclusion, we showed in this study that LSO induced the production of several cytokines from macrophages and was a strong inducer of IL-12 but not IFN-ß, and the lack of NO induction was ascribed to the absence of IFN-ß induction. Although LSO signaling was suggested to require both TLR2 and TLR4, the cytokine production profile was different from that of PGN or LPS. The results suggested that LSO stimulates a signaling pathway different from that for PGN or LPS.


    Acknowledgements
 
This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) from The Ministry of Education, Science and Culture of Japan and a Grant-in-Aid for Scientific Research (B) and (C) from The Japan Society for the Promotion of Science.


    Abbreviations
 
CDC   cholesterol-dependent cytolysin
iNOS   inducible nitric oxide synthase
KO   knockout
LLO   listeriolysin O
LSO   seeligeriolysin O
NF-{kappa}B   nuclear factor {kappa}-B
NO   nitric oxide
PEC   peritoneal exudates cell
PGN   peptidoglycan
PMB   polymyxin B
SAC   Staphylococcus aureus Cowan
TLR   Toll-like receptor
TNF{alpha}   tumor necrosis factor alpha
rIFN-ß   recombinant IFN-ß
rLSO   recombinant LSO
RT   reverse transcription

    Notes
 
Transmitting editor: S. Koyasu

Received 9 December 2004, accepted 22 September 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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