1 Department of Medicine and Medical Science (Medicine 1), Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2 Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
Correspondence
Kenji Yokota
yokochan{at}md.okayama-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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The 60 kDa heat-shock protein (HSP60), an immune-potent antigen of H. pylori (Sharma et al., 1997), induces IL-8 secretion from human gastric epithelial cells (Yamaguchi et al., 1999
). We have reported that humoral immune responses to HSP60 are closely associated with gastric inflammation and play a role in the pathogenesis of mucosa-associated lymphoid tissue lymphoma (Hayashi et al., 1998
; Kawahara et al., 1999
). With regard to host-cell receptors for HSP60, sulfated glycolipids are reportedly involved (Huesca et al., 1996
), but the host-cell receptor(s) involved in HSP60-induced IL-8 production have not been fully elucidated. Some studies have shown that Toll-like receptors (TLRs) act as receptors for bacterial structures such as LPS and HSP60 (Kawahara et al., 2001
; Ohashi et al., 2000
; Sasu et al., 2001
; Vabulas et al., 2001
) in the development of an innate immune response against bacterial infection. Among the TLR family, TLR2 and/or TLR4 (Ohashi et al., 2000
, Sasu et al., 2001
; Vabulas et al., 2001
) are reportedly important in the recognition of HSP60 in human cells and chlamydia. TLR2 and TLR5 are reported to be required for H. pylori-induced NF-
B activation and chemokine expression by epithelial cells (Smith et al., 2003
).
In the present study, to investigate the involvement of the interaction between bacterial HSP60 and host TLRs in inflammatory responses against H. pylori infection, we reacted cultured KATO III human gastric epithelial cells with H. pylori HSP60 or recombinant H. pylori HSP60 (rHpHSP60) in the presence or absence of anti-TLR antibodies. The effects on IL-8 production and NF-B activation were measured.
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METHODS |
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KATO III human gastric cell lines (obtained from the Japanese Collection at Research Bioresources, Tokyo, Japan) were grown in RPMI 1640 supplemented with 10 % FCS, 50 U penicillin G sodium ml1 and 50 µg streptomycin sulfate ml1 at 37 °C in an atmosphere of 5 % CO2 and 99 % humidity. KATO III cells were used at a final concentration of 5x105 cells ml1.
Preparation of rHpHSP60.
rHpHSP60 was prepared as described by Ishii et al. (2001). In brief, the ORF of HSP60 taken from H. pylori ATCC 43504 genomic DNA was amplified and cloned into the vector pGEX-5X3 (Amersham Biosciences). The resultant plasmid was transformed into Escherichia coli DH5
. Cells were cultured in LuriaBertani broth containing 2 % glucose and ampicillin (100 µg ml1), harvested by centrifugation and disrupted by sonication on ice for 5 min with a probe sonicator (Astron). Soluble rHpHSP60 was purified by glutathione-Sepharose 4B affinity chromatography (Amersham Biosciences) according to the manufacturer's instructions.
Protein production in KATO III cells (ELISA).
KATO III cells were co-cultured with various amounts of intact H. pylori or various concentrations of rHpHSP60. Cell-culture supernatants were collected and IL-8 was measured by ELISA (IL-8 ELISA Development kit; Techne). To evaluate the interaction between rHpHSP60 and TLRs in IL-8 production, cells were preincubated with 10 µg TL2.1 ml1 (anti-human TLR2 mouse mAb; Sanbio), 10 µg HTA125 ml1 (anti-human TLR4 mouse mAb; Sanbio) or 10 µg control irrelevant mouse mAb ml1 (anti-botulinum toxin type C), which we had prepared previously (Mahmut et al., 2002), for 30 min before stimulation under FCS-free conditions. In addition, KATO III cells were pretreated with ammonium pyrrolidinedithiocarbamate (PDTC) (SigmaAldrich), an inhibitor of NF-
B-mediated transcriptional activation (Kawai et al., 1995
; Ziegler-Heitbrock et al., 1993
), for 1 h before being reacted with intact H. pylori (1x107 c.f.u. ml1) or rHpHSP60 (50 µg ml1). Cell-culture supernatants were collected and IL-8 was measured as described above.
To exclude the possible effects of trace amounts of LPS on IL-8 secretion induced by rHpHSP60 preparations, 20 µg polymyxin B ml1 (PMB) (Wako Pure Chemical Industries), a potent LPS antagonist (Stokes et al., 1989), was added when KATO III cells were reacted with rHpHSP60, and E. coli LPS (SigmaAldrich) was used as control. In addition, a PMB-agarose gel column (Pierce) was used to remove LPS and LPS-associated molecules in rHpHSP60 preparations. The endotoxin activity of rHpHSP60 and PMB-agarose column fractions was determined by using the LAL assay kit (BioWhittaker) according to the manufacturer's instructions. IL-8 secretion from KATO III cells that had been reacted with the original rHpHSP60 preparation was compared with that from cells reacted with the PMB-agarose column pass-through fractions.
NF-B activation in KATO III cells that had been reacted with rHpHSP60 was detected and quantified by the use of Trans AM NF-
B (Active Motif) according to the manufacturer's instructions. Briefly, nuclear extracts were prepared and incubated for 1 h on a 96-well plate with immobilized oligonucleotide containing the NF-
B consensus site (5'-GGGACTTTCC-3'). After incubation with antibody to p65 (RelA), a subunit of the activated NF-
B dimer, horseradish peroxidase-conjugated secondary antibody was added, followed by developing solution included in Trans AM NF-
B. The optical density was read at 450 nm with a reference wavelength of 655 nm on a spectrophotometer. Recombinant tumour necrosis factor
(TNF-
) (SigmaAldrich) was used as a positive control.
Detection of rHpHSP60 in KATO III cells with bacterial or rHSP stimulation.
To measure the rHpHSP60 concentration when live or heat-killed bacteria contacted and stimulated KATO III cells, the culture supernatants and cells were analysed by Western blotting. KATO III cells (5x106 cells ml1) were reacted with several concentrations of rHpHSP60 (1001 µg ml1), or intact or heat-killed H. pylori [from OD600=0·5 (approx. 5x107 c.f.u. or cells ml1) to OD600=0·01 (approx. 1x105 c.f.u. or cells ml1)]. After incubation for 8 h, supernatants were gently collected. Cells were resuspended in 1 ml PBS. Aliquots of 10 µl of supernatants and cell suspensions were taken for SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were reacted with 1 : 3000-diluted anti-H. pylori HSP60 polyclonal antibody prepared as described previously (Yunoki et al., 2000). After washing with PBS, the membranes were reacted with peroxidase-conjugated anti-rabbit immunoglobulins (Dako). The reactions were detected by ECL Western blotting detection reagents (Amersham Pharmacia). The luminol reaction was visualized by a LAS-1000mini Bio-Imaging Analyser System (Fuji).
mRNA expression in KATO III cells (RT-PCR and Northern blotting).
Using Northern blotting analysis, we analysed IL-8 mRNA expression in KATO III cells treated with H. pylori and rHpHSP60. Total RNA was isolated from KATO III cells using RNA zol-B (Tel-Test Inc.), and IL-8 cDNA was prepared by RT-PCR using ReverTra Dash (Toyobo) with the following oligonucleotide primer set for IL-8 (5'-AAGGAACCATCTCACTG-3' and 5'-GATTCTTGGATACCACAGAG-3'; Crabtree et al., 1994) and labelled with horseradish peroxidase by an ECL direct nucleic acid labelling and detection system (Amersham Biosciences). Human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was also prepared, as a control, with a primer set in an RT-PCR kit (ReverTra Dash) according to the manufacturer's protocol. After treatment with rHpHSP60 (50 µg ml1), total RNA was isolated from KATO III cells and separated by electrophoresis (15 µg per lane) through a 1 % agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond-N+; Amersham Biosciences) in 20x SSC (1x SSC is 0·15 M sodium chloride/15 mM sodium citrate). The membrane was baked for 30 min at 80 °C and cross-linked by a UV cross-linker (Stratagene). After pre-hybridization, the membrane was hybridized with labelled IL-8 or G3PDH cDNA probes for 12 h at 42 °C. The membrane was washed twice with 0·5x SSC containing 0·4 % SDS for 10 min at 55 °C and then washed twice more with 2x SSC for 5 min at room temperature. RNA in the blots was detected by using the ECL direct nucleic acid labelling and detection system and analysed with the LAS-1000mini Bio-Imaging Analyser System.
TLR mRNA and protein expression in KATO III cells was examined by RT-PCR. Total RNA was isolated from KATO III cells by using RNA zol-B. After DNase I (Invitrogen) treatment, RT-PCR was performed using ReverTra Dash. PCR amplification was performed for 35 cycles at 95 °C for 10 s, 53 °C for 2 s and 74 °C for 30 s using the following oligonucleotide primer sets: for TLR2, 5'-CTTATCCAGCACACGAATACACAG-3' (sense 14551478) and 5'-TGGAGAGTCACACAGGTAATTTGC-3' (antisense 18071830); and for TLR4, 5'-CTCCAGGTTCTTGATTACAGTCTC-3' (sense 18001823) and 5'-TAGCTCATTCCTTACCCAGTCCT-3' (antisense 22182240). These primer sets were designed by using GENETYX-MAC (Genetyx Corporation) based on the DNA sequences of TLR2 and TLR4 from the GenBank Database. As a housekeeping gene for the control reaction, PCR was performed simultaneously with G3PDH primers (ReverTra Dash). Appropriate negative controls, including amplification of a non-specific RT-PCR reaction product, were included in each run. PCR products were resolved on 2 % agarose gels and DNA fragments were visualized under UV light by staining with ethidium bromide.
Real-time PCR for mRNA of TLR2 and 4.
Total RNA was isolated from KATO III cells using RNA zol-B (Tel-Test Inc.). After DNase I (Invitrogen) treatment, cDNA was prepared using ReverTra Dash. Quantitative PCR analysis was performed on a LightCycler using the LightCycler Primer set of human TLR2, TLR4 and G6PDH. Real-time PCR was done in a 24 µl volume consisting of 10 µl cDNA sample, 10 µl H2O, 2 µl LightCycler primer set and 2 µl LightCycler FastStart DNA master SybrGreen I. The reaction mixtures were loaded into capillary tubes and thermal cycling was carried out as follows: activation of hotstart Taq DNA polymerase, 95 °C for 10 min; cycling, 95 °C for 10 s, 68 °C for 10 s, 72 °C for 16 s for 40 cycles. The DNA concentration was calculated by LightCycler software and the ratio of TLR2 or TLR4/GAPDH was also calculated.
Immunocytochemistry of TLR and NF-B in KATO III cells.
KATO III cells were seeded on chamber slides and fixed in 70 % methanol. After incubation with TL2.1 (anti-human TLR2 antibody) or HTA125 (anti-human TLR4 antibody) as primary antibodies, the cells were incubated with tetraethylrhodamine isothiocyanate-conjugated goat anti-mouse immunoglobulins (SigmaAldrich). As negative controls, irrelevant mouse mAb (anti-botulinum toxin type C) and PBS were used instead of the primary antibodies. The stained cells were observed under a confocal laser-scanning microscope (LSM510; Carl Zeiss).
For NF-B activation analysis, KATO III cells were seeded in poly-L-lysine (Wako Pure Chemicals Industries)-treated chamber slides 3 days before the experiment. After pretreatment with TL2.1 (anti-human TLR2 antibody), HTA125 (anti-human TLR4 antibody) or medium control for 30 min, cells were stimulated with rHpHSP60 or intact H. pylori for 1 h and fixed in methanol (70 %) containing glycerol (30 %) at 20 °C for 30 min. After washing, cells were treated with 0·1 % Triton X100 for 4 min, incubated with rabbit anti-p65 antibody (Santa Cruz Biotechnology) and then reacted with fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-rabbit immunoglobulins (SigmaAldrich). The stained cells were observed under a confocal laser-scanning microscope.
Statistical analysis.
Results were expressed as means±SD. Data were compared using Scheffe's multiple comparison test and differences were considered significant at P values <0·05.
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RESULTS |
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Expression of IL-8 mRNA in KATO III cells stimulated with H. pylori (Fig. 1c) or rHpHSP60 (Fig. 1d
) was examined by Northern blotting analysis. IL-8 mRNA expression increased and reached a maximum level at 3 h after rHpHSP60 stimulation.
Involvement of TLRs in rHpHSP60-induced IL-8 release from KATO III cells
We next examined whether interaction of rHpHSP60 with TLRs was involved in IL-8 secretion induced by rHpHSP60. First, we confirmed the expression of TLR2 and TLR4 in KATO III cells. TLR2 and TLR4 mRNAs were constitutively expressed in the cells, irrespective of the presence of rHpHSP60 stimulation, as demonstrated by RT-PCR (Fig. 3a). Also, TLR2 and TLR4 proteins were identified immunocytochemically along the cell surface of KATO III cells (Figs 4
b and 3c). Real-time PCR was performed to confirm the expression of TLR2 or TLR4 mRNA. The number of PCR product (copies) are shown in Fig. 3(d)
and the TLR2 or TLR4/G6PDH ratio is shown in Fig. 3(e)
. rHSP60 did not enhance TLR mRNA expression.
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NF-B activation in KATO III cells reacted with rHpHSP60
Next, we investigated NF-B activation in KATO III cells reacted with rHpHSP60. An increase of intranuclear NF-
B was observed 10 min after rHpHSP60 stimulation and reached a maximum level 1 h after treatment (Fig. 5
a). This reaction was significantly inhibited by anti-TLR2 antibody (TL2.1) treatment (Fig. 5b
). Immunocytochemically, p65 (RelA), a subunit of the activated NF-
B dimmer, was localized exclusively in the cytoplasm before reaction with intact H. pylori or rHpHSP60 (Fig. 6
a). After reaction with intact H. pylori (Fig. 6b
) or rHpHSP60 (Fig. 6c
), p65 was translocated into the nucleus. The HSP60-induced translocation of p65 was inhibited by pretreatment with TL2.1 anti-TLR2 antibody (Fig. 6d
).
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DISCUSSION |
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HSPs are a family of proteins induced in prokaryotic and eukaryotic cells by environmental stress. These proteins function as chaperones to facilitate folding, unfolding and translocation of intracellular polypeptides (Ellis, 1990; Young, 1990
). HSP60 has been shown to play a role in the adherence and attachment of H. pylori to gastric epithelium (Huesca et al., 1996
; Yamaguchi et al., 1997
) and to induce IL-8 secretion from KATO III human gastric epithelial cells (Yamaguchi et al., 1999
). The results of the present study, which confirmed that rHpHSP60 can induce IL-8 release from KATO III cells and further found that IL-8 secretion was accompanied by an increase of IL-8 mRNA, indicate that H. pylori HSP60 is a bacterial virulence factor that can induce host inflammatory responses.
It has been reported that commercially available recombinant human HSP60 induced TNF- release from murine macrophages in a manner similar to that of LPS, but Gao & Tsan (2003)
reported that the TNF-
-inducing activity of this HSP60 was due to the presence of contaminating LPS and LPS-associated molecules. In the present study, three experiments excluded the possibility that LPS contamination of our rHpHSP60 preparation was responsible for the release of IL-8: (1) the addition of PMB did not inhibit rHpHSP60-induced IL-8 release; (2) endotoxin activities in the rHpHSP60 preparation were negligible; and (3) fractions of rHpHSP60 further purified by passage through a PMB-agarose column induced as much IL-8 release from KATO III as did the original rHpHSP60 preparation.
Sulfated glycolipid is reportedly a host-cell receptor for H. pylori HSP60 (Huesca et al., 1996), and HSP60s of human cells and chlamydia bind to TLR2 or TLR4 (Ohashi et al., 2000
; Sasu et al., 2001
; Vabulas et al., 2001
). In H. pylori infection, TLR4 involvement is reported in the activation of mitogen oxidase 1 by LPS of H. pylori in gastric pit cells (Kawahara et al., 2001
). Other investigators reported that human gastric epithelial cells recognize and respond to H. pylori at least in part via TLR2 and TLR5 (Smith et al., 2003
). Recently, H. pylori HSP60 was reported to mediate IL-6 production by macrophage via TLRs independent mechanism (Gobert et al., 2004
). However, the interaction of the TLRs with H. pylori HSP60 on IL-8 production by human gastric epithelial cells had not been studied. In the present study, we demonstrated that treatment of KATO III cells with anti-TLR antibodies, especially anti-TLR2, inhibited HSP60-induced IL-8 secretion. This result is persuasive evidence that TLRs are host-cell receptors for H. pylori HSP60 in the induction of initial inflammatory responses.
In vertebrates, TLRs play a key role in the recognition of infectious pathogens and initiate innate immunity as a first line of defence (Aderem & Ulevitch, 2000). For this purpose, it is reasonable that TLRs should be directed to specific motifs on pathogens. Because HSPs are highly conserved proteins with significant molecular mimicry among prokaryotic and eukaryotic cells, the recognition of H. pylori HSP by TLRs is a likely mechanism for the initiation of innate immunity and the concurrent host inflammatory response against this bacterium. In support of this possibility, we found that anti-TLR2 antibody significantly inhibited IL-8 secretion. However, the inhibition was incomplete, even when anti-TLR4 antibody was added. Thus, receptors other than TLR2 and TLR4 may also recognize H. pylori HSP60. At present it remains unclear whether the observed induction of IL-8 is specific to HSP60 from H. pylori. However, preliminary data suggest that production of this cytokine from monocytes involves the less conserved regions of the protein.
Activation of the TLR signalling pathway leads to NF-B translocation to the nucleus and transactivation (Aderem & Ulevitch, 2000
; Akira & Hemmi, 2003
). In the present study, we demonstrated that nuclear translocation of RelA (p65) was induced by the addition of rHpHSP60 and inhibited by pretreatment with the anti-TLR antibody. Involvement of NF-
B activation was further confirmed by the observation that IL-8 secretion was inhibited after treatment with PDTC, an inhibitor of NF-
B-mediated transcriptional activation. However, the possible involvement of other signalling pathways, such as the mitogen-activated protein kinase pathway, which is also triggered after TLR ligation (Aderem & Ulevitch, 2000
; Akira & Hemmi, 2003
), deserves consideration.
Adherence and attachment of H. pylori to gastric epithelial cells cause host responses, including the synthesis of various inflammatory mediators and cytokines that lead to the development of gastric injury. The cag pathogenicity island of H. pylori is reportedly important in the production of these cytokines through NF-B activation (Covacci et al., 1999
; Crabtree et al., 1995b
; Sharma et al., 1998
). Other reports have indicated that not only cag pathogenicity island, but also flagella, LPS, outer-membrane protein and HSP60 of H. pylori are associated with IL-8 induction from epithelial cells or monocytes (Bhattacharyya et al., 2002
; Cunningham et al., 2000
; Lee et al., 2003
; Yamaguchi et al., 1999
; Yamaoka et al., 2002
). These non-cag antigens are located on the bacterial surface and make direct contact with host cells. The cag-independent pathway of cytokine induction may also involve induction of mucosal inflammation. The results of the present study, which indicate that H. pylori HSP60 acts as a bacterial virulence factor for the induction of host inflammatory responses, acting through binding to TLRs, further characterize the mechanisms by which H. pylori can cause disease.
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ACKNOWLEDGEMENTS |
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Received 29 July 2004;
accepted 2 September 2004.
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