# Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism

Alain Gobert, Jean-Christophe Bambou, Catherine Werts, Viviane Balloy, Michel Chignard, Anthony Moran, Richard Ferrero

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## To cite this version:

Alain Gobert, Jean-Christophe Bambou, Catherine Werts, Viviane Balloy, Michel Chignard, et al. Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism. Journal of Biological Chemistry, 2004, 279 (1), pp.245-250. 10.1074/jbc.M307858200. hal-05495925

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# *Helicobacter pylori* Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism*

Received for publication, July 21, 2003, and in revised form, October 15, 2003
Published, JBC Papers in Press, October 22, 2003, DOI 10.1074/jbc.M307858200

Alain P. Gobert‡§¶, Jean-Christophe Bambou‡‖, Catherine Werts**, Viviane Balloy‡‡,
Michel Chignard‡‡, Anthony P. Moran§§¶¶, and Richard L. Ferrero‡‖‖

*From the ‡Unité de Pathogénie Bactérienne des Muqueuses (UPBM), **Unité de Bactériologie Moléculaire et Médicale, and ‡‡Unité Défense Innée et Inflammation, INSERM E336, Institut Pasteur, Paris Cedex15, France and the §§Department of Microbiology, National University of Ireland, IRL Galway, Ireland*

*Helicobacter pylori* has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which *H. pylori* induces IL-6 production in macrophages, however, has not been investigated. To identify the *H. pylori* factor responsible for this activity, we fractionated soluble proteins from *H. pylori* strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as *H. pylori* heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of *H. pylori* HSP60, soluble protein extracts of *H. pylori* 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with *H. pylori* HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of *H. pylori* HSP60 responses from these signaling molecules. *H. pylori* HSP60-induced IL-6 mRNA expression, and NF-κB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by *H. pylori* HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.

The Gram-negative microaerophilic bacterium *Helicobacter pylori* colonizes mammalian stomachs, and its persistence in human tissues is linked to the development of chronic inflammation. Chronic gastritis is such a common feature of *H. pylori* infection that histopathologists commonly use this as a criterion for diagnosing the presence of *H. pylori* bacteria in gastric biopsies. In contrast, the development of peptic ulceration and gastric cancer in response to *H. pylori* infection is much less frequent, occurring in ~10 and 1% of infections, respectively (1). Although there has been a concerted effort in the last decade to identify the bacterial factors that are associated with these severe forms of *H. pylori* related disease, little is currently known regarding those factors that induce chronic gastritis. One of the major *H. pylori* virulence factors is the *cag* pathogenicity island (*cag*PAI),<fnref n="1" /> which has been correlated with the more severe *H. pylori* associated pathologies and mediates its effects through gastric epithelial cells (2). Although only a proportion of *H. pylori* strains harbor a *cag*PAI, all of the cases of *H. pylori* infection are associated with the development of chronic gastritis. Therefore, it is likely that non-*cag*PAI factors promote chronic inflammation (3). One mechanism by which inflammatory processes might occur is via the activation of antigen-presenting cells, such as macrophages, within the gastric mucosa. Indeed, professional phagocytes appear to play a role in the chronic inflammatory lesions associated with *H. pylori* infection (4, 5). Nevertheless, as *H. pylori* LPS has a 100-fold lower biological activity to that of other Gram-negative bacteria (6–8), it is not likely to play a major role in macrophage activation *in vivo*. Conversely, certain soluble proteins in *H. pylori* extracts have been reported to activate mononuclear cells *in vitro* (9–11), but the identities of these proteins and of their receptors, for the most part, remain unknown.

Macrophage-derived cytokine production is strongly up-regulated during *H. pylori* infection (11). These innate responses are principally characterized by increases in the expression levels of IL-1β, tumor necrosis factor-α, and IL-6 in infected gastric tissues (11–14). Strikingly, a relationship exists be-

* This work was funded by the Institut Pasteur (PTR 94) (to C. W., M. C., and R. L. F.) and ARC (project number 4428) (to R. L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver-tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Bourse Roux postdoctoral fellowship from the Institut Pasteur.

¶ Present address: Laboratoire de Microbiologie, Inra de Clermont-Ferrand-Thiel, F-63122 Saint-Genes-Champanelle, France.

‖ Present address: EMI 0212, Faculte Necker Enfants-malades, 75743 Paris, France.

¶¶ Supported by the Health Research Board (Ireland).

‖‖ To whom correspondence should be addressed: UPBM, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France. Tel.: 33-1-40613324; Fax: 33-1-40613640; E-mail: rferrero@pasteur.fr.

<footnote n="1">¹ The abbreviations used are: *cag*PAI, *cag* pathogenicity island; LPS, lipopolysaccharide; IL, interleukin; PKA and PKC, protein kinases A and C, respectively; HSP, heat shock protein; NF-κB, nuclear factor κB; TLR, toll-like receptor; MyD88, myeloid differentiation factor 88; MG-132, Z-Leu-Leu-Leu-CHO; H89, *N*-[2-(*p*-bromocinnamyl)amino]ethyl]-5-isoquinoline-sulfonamide; Gö 6983, 2-[1-(3-dimethylaminopropyl)]5-me-thoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide; MAPK kinase, mitogen-ac-tivated protein kinase kinase; WT, wild-type; PBS, phosphate-buffered saline; Pam₃Cys, *S*-[2,3-bis(palmitoyloxy)-(*2-RS*)-propyl]-*N*-palmitoyl-(*R*)-Cys-(*S*)-Ser-Lys4-OH, trihydrochloride.</footnote>
tween *H. pylori* and IL-6 since IL-6 mRNA levels in the gastric mucosa have been correlated with the level of inflammation (11, 15) and serum IL-6 concentrations have been linked to the status of *H. pylori* induced gastric cancer (16). Normally, IL-6 plays an important role in host defense mechanisms as a messenger between innate and adaptive systems by stimulating interferon-γ production in T-cells, by promoting immunoglobulin secretion in activated B-cells, and via polymorphonuclearphil activation (17). Hence, IL-6 has been implicated in the control of virus or bacteria *in vivo* (18).

The findings above suggest that IL-6 could have critical functions for the pathophysiology of *H. pylori* infection. Thus, the aim of the current study was to identify novel *H. pylori* proteins capable of inducing IL-6 in macrophages. To this end, we fractionated a soluble cell extract of the genome-sequenced *H. pylori* 26695 strain by anion exchange and size exclusion chromatography and tested the fractions for IL-6-inducing activity on RAW 264.7 cells. We have identified HSP60 as the main *H. pylori* factor inducing IL-6 release from murine macrophages through a signaling pathway involving NF-κB activation, but that is independent of TLR-2, TLR-4, or MyD88 signaling pathways.

## EXPERIMENTAL PROCEDURES

*Materials*—Pharmacological inhibitors of NF-κB (MG-132), PKA (H89), PKC (Gö 6983), MAPK kinase (PD98 059), and phosphoinositide 3-kinase (wortmannin) were purchased from Calbiochem. Materials for chromatography were from Amersham Biosciences. Highly purified *H. pylori* LPS was extracted by the phenol-water procedure after pretreatment of bacteria with Pronase E and subsequent purification by enzymatic treatments with RNase A, DNase II, and proteinase K and ultracentrifugation as described elsewhere (6). *Escherichia coli* LPS (19) was prepared by Dr. U. Zähringer (Center for Medicine and Biosciences, Borstel, Germany) and kindly provided by Dr. D. J. Philpott (Institut Pasteur, Paris, France). All of the other chemicals were from Sigma.

*Bacteria*—*H. pylori* SS1 (20) and 26695 (21) strains were used throughout. Bacteria were maintained on blood agar base number 2 (Oxoid) plates containing 10% horse blood, 10 µg/ml vancomycin, 2.5 IU/liter polymyxin B, 5 µg/ml trimethoprim, and 2.5 µg/ml fungizone under microaerobic conditions at 37 °C. Prior to each experiment, *H. pylori* was grown overnight in the same conditions by shaking at 140 rpm in Brain-Heart Infusion (Oxoid) liquid medium containing 0.2% β-cyclodextrin. The bacteria were washed twice and suspended in PBS. Bacterial density was estimated by spectrophotometry (A<sub>600</sub>) and by microscopic observation. *H. pylori* soluble proteins were obtained by vortexing 10<sup>9</sup> bacteria in 1 ml of water followed by three freeze-thaw cycles and by centrifugation at 12,000 × g for 10 min. Protein concentrations of the supernatants were determined using the Bio-Rad Bradford Assay. *H. pylori* urease activity was determined at A<sub>540</sub> using urea-indole reagent (22).

*Mice, Macrophages, and Culture Conditions*—The murine macrophage cell line RAW 264.7 was maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 10 mM HEPES at 37 °C in a humidified 5% CO<sub>2</sub> atmosphere. Wild-type (WT) C57BL/6 mice were purchased from Charles River. TLR-2, TLR-4, and MyD88 knock-out C57BL/6 mice were obtained from Professor S. Akira (Osaka University, Osaka, Japan) and bred in the animal facilities of the Institut Pasteur. Double TLR-2/TLR-4 knock-out mice were generated by V. Balloy Resident peritoneal macrophages from these animals were purified as described previously (23). For the experiments, RAW 264.7 cells or peritoneal macrophages were plated in 24-well plates (5 × 10<sup>5</sup> cells/well) or in 6-well plates (2 × 10<sup>6</sup> cells/well) in the same medium for 2 h, washed, and stimulated with *H. pylori* with a multiplicity of infection of 10, with purified HSP60, with *H. pylori* or *E. coli* LPS, or with Pam<sub>3</sub>Cys. Cells were stimulated for the appropriate times (24 h for IL-6 assays, 6 h for mRNA analysis, and 5 h for NF-κB analysis).

*Column Chromatography of H. pylori Soluble Extracts*—Soluble proteins of *H. pylori* 26695 (6.7 mg) were separated by ion exchange chromatography on a Q-Sepharose High Performance column (80-ml bed volume) at pH 8.0. Proteins were eluted using a linear gradient of NaCl from 25 to 500 mM at a flow rate of 1 ml/min. Fractions (4 ml) were collected from the column. Aliquots (20 µl) from fractions were then

added to RAW 264.7 cells for 24 h, and IL-6 concentrations were determined in each cell-free supernatant. The fractions that induced IL-6 production were pooled, desalted, and concentrated with Centriprep YM-50 and Centricon YM-50 columns (Millipore). The resulting concentrate was subjected to further purification by size exclusion chromatography on a Sephadryl S-100 column (2000-ml bed volume). Fractions (4 ml) were collected after elution with 400 ml of PBS, pH 7.4, and assayed on macrophages for IL-6-inducing activity. The corresponding fractions were pooled and concentrated. An aliquot was separated by SDS-PAGE using a 10% gel, and the gel was stained with Coomassie Blue or with silver nitrate. A protein band was extracted from the gel and analyzed by mass spectrometry at the Plate-Form Technique 3 (Proteomic Genopole, Institut Pasteur).

*Immunopurification of H. pylori HSP60*—An AminoLink Plus immobilization kit (Pierce) was used to purify HSP60. 600 µg of *H. pylori* total soluble protein extracts were applied to an agarose column that had been coupled with 6 mg of a polyclonal antibody to *H. pylori* HSP60 (24) according to the manufacturer's instructions. The column was washed with PBS, and the flow-through was collected. HSP60 was eluted from the column using 0.1 M glycine, pH 2.5. The protein concentrations of the fractions were determined prior to concentration using Centricon YM-10 ultrafiltration units (Millipore).

*Determination of IL-6 Concentration*—IL-6 levels were determined in macrophage supernatants using the Duo Set enzyme-linked immunosorbent assay development kit (R&D Systems) according to the manufacturer's protocol.

*Measure of Endotoxin Contamination*—LPS concentration was determined using the Limulus amebocyte lysate colorimetric assay QCL-1000 from BioWhittaker.

*Western Blotting*—*H. pylori* proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes by electroblotting. Membranes were blocked for 2 h at 4 °C using PBS containing 5% nonfat dry milk and 0.1% Tween 20. Polyclonal antibody to HSP60 (1:50000) (24) or to UreB (1:20000) (25) and a goat anti-rabbit antibody conjugated to horseradish peroxidase (1:20000, Bio-Rad) were used each for 2 h at room temperature, respectively. Chemiluminescent detection was performed using Western blotting detection reagents ECL (Amersham Biosciences) and exposure to Hyperfilm MP (Amersham Biosciences).

*Reverse Transcriptase-PCR*—Macrophage total RNA was isolated using TRIzol reagent (Invitrogen). Subsequently, RNA (1 µg) from each sample was reverse-transcribed using 25 units of Superscript II reverse transcriptase (Invitrogen) and PCR was conducted using 2 µl of cDNA and 0.25 units of TaqDNA polymerase (Amersham Biosciences). For IL-6, 15 pmol each of 5' and 3' primers were used with 3 pmol each of β-actin primers in a multiplex reaction. One PCR cycle consisted of the following: 94 °C for 45 s, 58 °C for 1 min, and 72 °C for 1 min. The total cycle numbers were 30. A final elongation step of 7 min at 72 °C was then used. Primer sequences were designed with Primer3 software (www.genome.wi.mit.edu/cgi-bin/primer/primer3 www.cgi) and were as follows: murine IL-6, 5'-AACGATGATGCACTTGCAGA-3' and 5'-GAGCATTGGAAATTGGGGTA-3'; and murine β-actin, 5'-CCAGAGCAAGAGGTATCC-3' and 5'-CTGTGGTGGTGAAAGCTGTAG-3'. PCR products (283 bp for IL-6 and 436 bp for β-actin) were run on 1.5% agarose gels with 0.4 µg/ml ethidium bromide. Stained bands were visualized under UV light and photographed with Image Master VDS (Amersham Biosciences). Band intensity was evaluated using NIH Image 1.63 program.

*Electrophoretic Mobility Shift Assay*—Macrophages were lysed with a buffer containing 10 mM HEPES, pH 8, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40. Suspensions were centrifuged at 1200 × g for 5 min at 4 °C. Nuclear pellets were resuspended in 100 µl of a solution containing 25 mM Tris-HCl, pH 8.0, 400 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 20% w/v glycerol and were rapidly frozen and thawed three times. After centrifugation at 4000 × g at 4 °C for 12 min, supernatants containing nuclear proteins were removed and aliquot were used for protein determination. Nuclear extracts (1 µg of protein) were incubated for 30 min with a DNA probe corresponding to the κB site of the H-2KB promoter (31), which had previously been biotin-labeled (Pierce). NF-κB activation was determined using a chemiluminescent electrophoretic mobility shift assay technique (Pierce) according to the manufacturer's instructions.

*Statistical Analyses*—The Student Newman-Keul's test was used for comparisons between multiple groups. The Student's t test was used for single comparisons between two groups.
<figure><img src="image_3.png" /><figcaption>FIG. 1. Chromatographic elution profiles of *H. pylori* HSP60. A, ion exchange chromatography. *H. pylori* 26695-soluble proteins were separated on a Q-Sepharose High Performance column. Fractions were added to RAW 264.7 macrophages for 24 h, and IL-6 concentration was determined in culture supernatants (●). In the same eluates, urease was evaluated by urea-indole assay (□). B, size exclusion chromatography. Fractions with an IL-6-inducing activity obtained after ion exchange chromatography were pooled, concentrated, and separated on a Sephacryl S-100 column. Fractions were added to RAW 264.7 macrophages for 24 h, and IL-6 concentration was determined in culture supernatants. C, SDS-PAGE analysis of the purified protein. The 40- and 80-kDa bands correspond to molecular mass markers.</figcaption></figure>

RESULTS

**H. pylori HSP60 Is the Activator of IL-6 Production by Macrophages**—To purify the *H. pylori* factor responsible for IL-6 synthesis by macrophages, soluble bacterial proteins were subjected to anion exchange chromatography. Fractions with IL-6-inducing activity on RAW 264.7 macrophages were identified (Fig. 1A), corresponding to fractions 88–124. Because urease is another major protein of *H. pylori* known to induce cytokine production by macrophages (26), we determined the urease activity of the eluted fractions. Urease activity was detected in fractions 60–96 (Fig. 1A). To exclude the possible

involvement of urease on IL-6 activation, only fractions (numbers 96–120) eluted after the urease peak were concentrated. The pooled fractions were subjected to size exclusion chromatography. The fractions tested for IL-6-inducing activity (Fig. 1B) (22–31) were concentrated and separated by electrophoresis. A single 55–60-kDa protein band was revealed by Coomassie Blue staining (Fig. 1C). This protein was identified by mass spectrometry as being a homolog of *E. coli* GroEL, designated here as *H. pylori* HSP60 (also referred to as HspB) (24). Urease activity was not detected in this purified extract, and no other bands were observed on the polyacrylamide gel after silver staining (data not shown).

**Immunopurification of H. pylori HSP60**—Because chaperone proteins of the HSP60 family have an essential function in bacteria, we did not attempt to inactivate the *hsp60* gene. Instead, the involvement of *H. pylori* HSP60 on IL-6 stimulation was confirmed by an immunopurification approach. *H. pylori* HSP60 protein was detected in soluble protein extracts of *H. pylori* strains 26695 and SS1 (Fig. 2A). The concentration of HSP60, however, was dramatically decreased after immunopurification on a column coated with an anti-*H. pylori* HSP60 antibody (Fig. 2A). In addition, the HSP60 protein was detected in the concentrated eluates by Western blotting (data not shown). Conversely, the concentration of UreB in immunopurified extracts was not affected by chromatography (Fig. 2A), suggesting that the column specifically retained HSP60. IL-6 synthesis by RAW 264.7 macrophages was significantly increased following stimulation with soluble protein extracts of the two *H. pylori* strains when compared with unstimulated cells (data not shown). Nonetheless, IL-6 production was decreased by 88.8 ± 3.1 and 94.5 ± 1.9% when macrophages were activated with 2.5 µg/ml of the concentrated flow-through when compared with the stimulation with the same concentration of total *H. pylori* soluble proteins for 26695 and SS1 strains, respectively (Fig. 2B). A concentration-dependent increase in macrophage IL-6 production was observed in RAW 264.7 cells that had been stimulated with the immunopurified HSP60 (Fig. 3).

We determined that immunopurified *H. pylori* HSP60 contained 15 enzyme units/ml, equivalent to 75 enzyme units of contaminating LPS for 400 ng of HSP60. When *H. pylori* and *E. coli* LPS, corresponding to the amounts detected in 400 ng of HSP60 preparation, were added to RAW 264.7 cells for 24 h, IL-6 concentrations were 0 and ~200 pg/ml, respectively. IL-6 production by HSP60-stimulated RAW 264.7 macrophages was not altered by the use of 10 µg/ml polymyxin B, whereas IL-6 synthesis by macrophages activated with 1 µg/ml *H. pylori* LPS or with 10 ng/ml *E. coli* LPS was inhibited by ~93 and ~95% when polymyxin B was added to the cultures, respectively (data not shown). In addition, IL-6 production was inhibited by 93 ± 2,5% when RAW 264.7 cells were stimulated with 400 ng/ml boiled HSP60 for 24 h compared with the stimulation with the same concentration of native *H. pylori* HSP60 (data not shown; *p* < 0.05, *n* = 2). Together, these results suggest that contaminating LPS is not responsible for IL-6 induction observed with the *H. pylori* HSP60.

**H. pylori and HSP60-induced Macrophage IL-6 Production Does Not Require TLR-2, TLR-4, or MyD88**—Previous reports have demonstrated that TLRs-MyD88 complexes may mediate the signaling of HSPs (27–29). To determine whether whole *H. pylori* and purified *H. pylori* HSP60 induce IL-6 production in macrophages through these signaling pathways, we stimulated peritoneal macrophages from WT, TLR-2⁻/⁻, TLR-4⁻/⁻, TLR-2⁻/⁻/TLR-4⁻/⁻, or MyD88⁻/⁻ mice. As shown in Fig. 4A, IL-6 synthesis was induced by purified *H. pylori* HSP60 or by intact bacteria in primary cultures of WT mouse macrophages,
FIG. 2. Effect of *H. pylori* soluble proteins on IL-6 production. Total soluble proteins (SP) of *H. pylori* 26695 (left panels) and SS1 (right panels) strains were extracted and passaged on an immunopurification column to which was coupled a polyclonal antibody to *H. pylori* HSP60. The column was washed, and the flow-through (FT) was collected and concentrated. A, immunodetection of HSP60 and UreB. Western blot analysis of HSP60 (~55-kDa band) and UreB (~60-kDa band) performed on SP and FT. Equal amounts of proteins (1 µg/lane) were used. B, IL-6 synthesis by macrophages. RAW 264.7 cells were stimulated for 24 h with 2.5 µg/ml SP or FT, and the IL-6 concentration was measured in supernatants. Data are expressed as the mean ± S.E. of two experiments. *, p < 0.05; **, p < 0.01 versus SP-stimulated macrophage.

<figure><img src="image_4.png" /></figure>

FIG. 3. Immunopurified HSP60 induces IL-6 production by RAW 264.7 cells. Macrophages were stimulated for 24 h with various concentrations of *H. pylori* HSP60. IL-6 concentration was determined by enzyme-linked immunosorbent assay in the supernatants. Data represent the mean ± S.E. of one experiment performed in duplicate.

demonstrating that IL-6 synthesis is not a property restricted to cell lines. In addition, IL-6 production in WT peritoneal macrophages was not significantly different from that observed in macrophages recovered from TLR-2⁻¹⁻, TLR-4⁻¹⁻, or MyD88⁻¹⁻ mice following stimulation with any of these factors (Fig. 4A).

Conversely, a significant increase of IL-6 production was observed when macrophages from WT mice were stimulated with the TLR-2 ligand Pam₃Cys (30), whereas IL-6 was not synthesized by macrophages from TLR-2⁻¹⁻ mice activated with this compound (Fig. 4B). Additionally, macrophages from TLR-4⁻¹⁻ mice, which had been stimulated with *E. coli* LPS or *H. pylori* LPS, produced 93.3 ± 1.5 and 69.4 ± 18.5% lower amounts of IL-6 when compared with macrophages from WT animals, respectively (Fig. 4C).

*H. pylori*- and HSP60-induced Macrophage IL-6 Expression Is NF-κB-dependent—The signaling mechanism responsible for IL-6 expression in macrophages was investigated. IL-6 mRNA levels in RAW 264.7 cells were up-regulated in response to purified HSP60 or to intact 26695 *H. pylori* (Fig. 5A) when compared with control macrophages. This increase was inhibited by 100 and ~ 85% when the proteasome inhibitor MG-132 was used on macrophages activated by HSP60 or by *H. pylori*,

respectively. In contrast, *H. pylori* or HSP60-induced IL-6 mRNA expression was not affected by the use of H89, Gö 6983, PD98 059, and wortmannin, the specific inhibitors of PKA, PKC, MAPK kinase, and phosphoinositide 3-kinase, respectively. By electrophoretic mobility shift assay, MG-132 treatment was also shown to abrogate the induction of NF-κB activation in HSP60-stimulated macrophages, thus confirming the role of this transcription factor in IL-6 production (Fig. 5B).

DISCUSSION

Interleukin-6 production in the gastric mucosa has been shown to be consistently induced by *H. pylori* infection and to correlate with the development of chronic gastritis (12, 13). Although endothelial (31) and epithelial cells (32) can produce minor quantities of IL-6 upon *H. pylori* stimulation, activated macrophages are the main source of IL-6. Thus, our goal was to describe how *H. pylori* might activate IL-6 in macrophages. For this reason, we fractionated total soluble *H. pylori* proteins by ion exchange and size exclusion chromatography and tested the eluates for IL-6 production in macrophages. This resulted in the identification of HSP60 as the main *H. pylori* factor responsible for the induction of IL-6 production in macrophages. Bacterial proteins belonging to the *E. coli* GroEL family are highly immunogenic and have been reported to confer protective immunity against pathogens (33), including *H. pylori* (24). In addition, it has been previously shown that the HSPs from eukaryotic organisms and from other pathogenic bacteria can stimulate pro-inflammatory cytokine production in macrophages (34, 35). However, to our knowledge, this is the first report showing the direct activation of macrophages by a *Helicobacter* HSP.

Macrophages play an important role in innate immune responses to microbial pathogens. Indeed, IL-6 release by macrophages activated with various intact Gram-positive or Gram-negative bacteria or with bacterial-derived products has been fully documented. Interestingly, it was shown previously that HSPs from bacterial species of the *Mycobacterium*, *Legionella*, or *Escherichia* genera are potent IL-6 activators in mouse macrophages (34). It was also reported that *H. pylori* urease is a potent activator of human mucosal macrophages for IL-6 generation (26); nonetheless, the amount of recombinant urease used in that study was 100-times greater than the quantity of *H. pylori* HSP60 necessary to obtain similar levels of IL-6 by

<figure><img src="image_5.png" /></figure>
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FIG. 4. HSP60 and *H. pylori* induce macrophage IL-6 production independently of TLR-2, TLR-4, and MyD88. A, effect of *H. pylori* HSP60 and whole bacteria. Peritoneal macrophages from WT (n = 8 mice), TLR-2⁻/⁻ (n = 3 mice), TLR-4⁻/⁻ (n = 3 mice), TLR-2⁻/⁺/⁺ TLR-4⁻/⁻ (n = 6 mice), or MyD88⁻/⁻ (n = 3 mice) were activated with 400 ng/ml *H. pylori* HSP60 (black bars) or with intact *H. pylori* 26695 with a multiplicity of infection of 10 (hatched bars). Open bars represent unstimulated macrophages. IL-6 concentrations were measured in culture supernatants after 24 h. Data are expressed as the mean ± S.E. **, p < 0.01 versus macrophages stimulated with HSP60 or *H. pylori*. ND, not detectable. B, effect of Pam₃Cys on TLR-2. Macrophages from WT (n = 3 mice) and TLR-2⁻/⁻ mice (n = 3 mice) were stimulated with 1 µg/ml Pam₃Cys (black bars). Open bars represent control macrophages. Data are the mean ± S.E. of IL-6 concentration measured in culture supernatants after 24 h. ***, p < 0.001 versus unstimulated macrophages. C, effect of LPS on TLR-4. Macrophages from WT and TLR-4⁻/⁻ mice were stimulated with 10 ng/ml *E. coli* LPS (open bars, n = 3 mice) or with 1 µg/ml *H. pylori* LPS (black bars, n = 2 mice), and IL-6 concentrations were determined in culture supernatants after 24 h. Data represent the mean ± S.E. *, p < 0.05; ***, p < 0.001 versus macrophages from WT mice.

murine macrophages. Therefore, HSP60 appears to be the main IL-6 inducer, even if other factors may be capable of inducing IL-6 when present in high concentrations. Therefore, we can hypothesize that, in vivo, it is possible that IL-6 expression in macrophages is essentially HSP60-dependent. In support of this hypothesis, cytoplasmic HSP60 has been found in the extracellular medium of *H. pylori* cultures (36, 37) and has been detected in the lamina propria of the gastric antrum from patients with *H. pylori* gastritis (38). Thus, *H. pylori* HSP60 release is likely to occur in vivo and may be capable of reaching mucosal macrophages within the gastric epithelium as a consequence of the disruption of epithelial tight junctions during *H. pylori* infection.

Recently, it was shown that tumor necrosis factor-α production by murine macrophages in response to stimulation by recombinant human HSP60 or HSP70 was due to contamination by bacterial LPS (39, 40). For this reason, we chose not to

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<figure><img src="image_9.png" /></figure>

FIG. 5. Identification of signal transduction pathways required for IL-6 expression. A, pharmacological inhibition of signal transduction proteins. MG-132 (5 µM), H89 (10 µM), Gö 6983 (10 µM), PD98 059 (50 µM), or wortmannin (0.1 µM) was added to RAW 264.7 macrophages 30 min before stimulation with 400 ng of HSP60 or infection with *H. pylori*. IL-6 and β-actin mRNA expression was then examined by reverse transcriptase-PCR after 6 h. The levels of mRNA were compared with those in unstimulated macrophages (Ctrl). B, analysis of NF-κB activation by electrophoretic mobility shift assay. Nuclear proteins from unstimulated (Ctrl) RAW 264.7 macrophages and in those that had been stimulated for 5 h with 400 ng of *H. pylori* HSP60, in the presence or absence of MG-132, were extracted and analyzed. Equal amounts of proteins (1 µg/lane) were loaded on the gel.

use recombinant *H. pylori* HSP60 to stimulate our cells. Three findings support the contention that the *H. pylori* HSP60-induced IL-6 production reported here was LPS-independent. (i) *E. coli* or *H. pylori* LPS added to macrophages at comparable amounts to that detected in the HSP60 preparation failed to stimulate IL-6 production. (ii) IL-6 synthesis was not significantly activated by heat-inactivated HSP60. (iii) IL-6 was produced by HSP60-stimulated peritoneal macrophages from TLR-4⁻/⁻ mice at levels similar to those from WT animals with TLR-4 being recognized as the receptor for bacterial LPS. Possible protein contamination of HSP60 preparations was also envisaged. However, we found a single protein band after SDS-PAGE of pooled IL-6-inducing fractions and only one *H. pylori* protein species was detected in this band by mass spectrometry. One of the major protein constituents of *H. pylori* is urease, which was not detected in our purified extract. Therefore, even if HSP60 was associated with other minor proteins, these are highly unlikely to have an effect on IL-6 activation when compared with the high specific activity of *H. pylori* HSP60.

Although eukaryotic and prokaryotic HSPs are known to stimulate macrophages (35), there is no consensus regarding the nature of the receptor of HSPs. Several studies have demonstrated that TLR-2 (27), TLR-4 (28), or both (41) are implicated in the recognition of HSPs. In contrast, it has been reported that the TLR-4 receptor complex is not involved in the binding of human HSP60 by murine macrophages and that the receptor of human HSP60 is different from that of HSP70 and Gp96 (42), which signal through TLR-2 and TLR-4 (29, 41). Therefore, we determined the potential involvement of TLRs on IL-6-inducing activity by *H. pylori* HSP60 or by intact *H. pylori* and showed that IL-6 expression did not require either TLR-2 or TLR-4. This result was confirmed by the fact that macrophages from mice deficient for MyD88, an adapter molecule recruited by TLR-2 and TLR-4 (43), responded similarly to WT macrophages upon both types of stimulation. Accordingly, it has been shown that cytokine synthesis by peritoneal macro-
phages stimulated with intact *Staphylococcus* bacteria was TLR-4-independent and partially TLR-2-independent (44). As proposed by Habich *et al.* (45), it is possible that HSPs from different organisms may bind to different receptor complexes. Together, these observations and the present results raise the question regarding the nature of the macrophage receptor for *H. pylori* HSP60. Alternative receptors for human HSP60 (46) and for the HSP60 of the fungus *Histoplasma capsulatum* (47) have already been described. We also demonstrated that a complete inhibition of IL-6 expression induced by HSP60 occurred with the specific proteasome inhibitor, MG-132, whereas inhibitors of PKA, PKC, MAPK kinase, and phosphoinositide 3-kinase had no effect, suggesting that macrophage IL-6 expression in response to *H. pylori* involves NF-κB activation. This result was confirmed by showing that NF-κB is induced in RAW 264.7 cells stimulated with HSP60. Moreover, this activity could be abrogated by treatment of the cells with MG-132 (Fig. 5B). Consistent with this finding, *H. pylori* has been shown to induce NF-κB activation in mice peritoneal macrophages and in the human monocytic cell line, THP-1 (48). Thus, further studies are warranted to identify the receptor(s) for *H. pylori* HSP60 on macrophages that leads to NF-κB activation but that signal(s) through a pathway independent of TLR-2, TLR-4, and MyD88.

In conclusion, we have described a new role for *H. pylori* HSP60, implicating it in the pathophysiology of *H. pylori* gastritis, through the release of IL-6 by macrophages. This finding supports the hypothesis that the host is able to recognize and respond to a *H. pylori* protein that is constitutively synthesized and/or essential for bacterial survival and is present in all of the *H. pylori* strains. Indeed, the recognition of *H. pylori* HSP60 by innate immune system cells may represent a mechanism by which *H. pylori* induces chronic gastritis, which is a common feature of all infections attributed to this Gram-negative pathogen.

*Acknowledgment*—We thank Professor S. Akira (Osaka University, Osaka, Japan) for the generous gift of TLR knock-out breeder mice.

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