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Endocytosed HSP60s Use Toll-like Receptor 2 (TLR2) and TLR4 to Activate the Toll/Interleukin-1 Receptor Signaling Pathway in Innate Immune Cells

2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês

10.1074/jbc.m103217200

ISSN

1083-351X

Autores

R. Martin Vabulas, Parviz Ahmad‐Nejad, Clarissa da Costa, Thomas Miethke, Carsten J. Kirschning, Hans Häcker, Hermann Wagner,

Tópico(s)

Yersinia bacterium, plague, ectoparasites research

Resumo

Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic "danger" signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-κB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands. Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic "danger" signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-κB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands. heat shock protein antigen-presenting cells interleukin lipopolysaccharide Toll-like receptor Toll/IL-1 receptor myeloid differentiation marker 88 TNF receptor-associated factor c-Jun N-terminal kinase extracellular signal-regulated kinase I-κB kinase hemagglutinin polymyxin B monodansylcadaverine signal transducers and activators of transcription interferon fetal calf serum bone marrow derived dendritic cells tumor necrosis factor Heat shock proteins (HSPs)1 represent a collective of evolutionary conserved proteins, molecular chaperones, that bind nonnative states of other proteins and assist them to reach functional conformation (1Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3116) Google Scholar, 2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2425) Google Scholar). In addition, HSPs act as molecular shuttles for antigens (3Srivastava P.K. Menoret A. Basu S. Binder R.J. McQuade K.L. Immunity. 1998; 8: 657-665Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). For example, certain HSPs isolated from (tumor) cells are associated with a large repertoire of proteins and peptides, including antigenic peptides (4Udono H. Srivastava P.K. J. Exp. Med. 1993; 178: 1391-1396Crossref PubMed Scopus (565) Google Scholar, 5Arnold D. Faath S. Rammensee H. Schild H. J. Exp. Med. 1995; 182: 885-889Crossref PubMed Scopus (293) Google Scholar, 6Nieland T.J. Tan M.C. Monne-van Muijen M. Koning F. Kruisbeek A.M. van Bleek G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6135-6139Crossref PubMed Scopus (192) Google Scholar, 7Blachere N.E. Li Z. Chandawarkar R.Y. Suto R. Jaikaria N.S. Basu S. Udono H. Srivastava P.K. J. Exp. Med. 1997; 186: 1315-1322Crossref PubMed Scopus (491) Google Scholar). Upon internalization by immature dendritic cells, HSP-peptide complexes efficiently trigger cytotoxic T cell responses (8Arnold-Schild D. Hanau D. Spehner D. Schmid C. Rammensee H.G. de la S.H. Schild H. J. Immunol. 1999; 162: 3757-3760Crossref PubMed Google Scholar, 9Castellino F. Boucher P.E. Eichelberg K. Mayhew M. Rothman J.E. Houghton A.N. Germain R.N. J. Exp. Med. 2000; 191: 1957-1964Crossref PubMed Scopus (359) Google Scholar, 10Binder R.J. Harris M.L. Menoret A. Srivastava P.K. J. Immunol. 2000; 165: 2582-2587Crossref PubMed Scopus (112) Google Scholar, 11Binder R.J. Han D.K. Srivastava P.K. Nat. Immunol. 2000; 1: 151-155Crossref PubMed Scopus (595) Google Scholar). This raises the question why HSP-peptide complexes require no adjuvant to confer immunogenicity to bound peptides. Since initiation of antigen-specific responses depends on activation of immature dendritic cells into professional antigen-presenting cells (APCs) expressing costimulatory molecules and producing cytokines such as interleukin (IL)-12, HSP may directly activate immature APC (12Singh-Jasuja H. Scherer H.U. Hilf N. Arnold-Schild D. Rammensee H.G. Toes R.E. Schild H. Eur. J. Immunol. 2000; 30: 2211-2215Crossref PubMed Scopus (327) Google Scholar, 13Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1076) Google Scholar, 14Binder R.J. Anderson K.M. Basu S. Srivastava P.K. J. Immunol. 2000; 165: 6029-6035Crossref PubMed Scopus (185) Google Scholar). Activation of APC can be induced by either T helper cells or pathogen-derived ligands (15Banchereau J. Steinman R.M. Nature. 1998; 392: 245-252Crossref PubMed Scopus (12240) Google Scholar). Recognition of pathogen-derived ligands such as lipopolysaccharide (LPS), peptidoglycans, and bacterial CpG-DNA by Toll-like receptor 4 (TLR4), TLR2, and TLR9, respectively, has recently been discovered (16Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2617) Google Scholar, 17Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5359) Google Scholar). Subsequent cell activation is brought about by recruitment through TLR's Toll/IL-1 receptor (TIR) domain of the adapter molecule myeloid differentiation marker 88 (MyD88) followed by activation of the IL-1 receptor-associated kinase and recruitment of the adapter molecule tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (18Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar). This leads ultimately to activation of kinases controlling activity of transcription factors responsible for induction of proinflammatory cytokines and costimulatory molecules. The ability of HSP to activate innate immune cells has been best documented for HSP60 (19Kol A. Sukhova G.K. Lichtman A.H. Libby P. Circulation. 1998; 98: 300-307Crossref PubMed Scopus (502) Google Scholar, 20Kol A. Bourcier T. Lichtman A.H. Libby P. J. Clin. Invest. 1999; 103: 571-577Crossref PubMed Scopus (464) Google Scholar, 21Chen W. Syldath U. Bellmann K. Burkart V. Kolb H. J. Immunol. 1999; 162: 3212-3219PubMed Google Scholar). Interestingly, chlamydial HSP60 and its human homologue accumulate in atherosclerotic lesions and stimulate macrophages and endothelial cells as well (19Kol A. Sukhova G.K. Lichtman A.H. Libby P. Circulation. 1998; 98: 300-307Crossref PubMed Scopus (502) Google Scholar). Although the signaling pathways involved are yet poorly understood, we were intrigued by similarities of CpG-DNA-driven and HSP60-driven activation of immune cells. Therefore, we analyzed whether HSP60s activate innate immunity cells via the TLR signaling pathway. Here we report that both chlamydial and human HSP60 induce in murine macrophages the stress-activated protein kinases c-Jun N-terminal kinase 1/2 (JNK1/2) and p38, the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2), and I-κB kinase (IKK). Activation, at least of JNK and IKK, depends on MyD88 and TRAF6. Finally, we demonstrate that chlamydial ("exogenous") and human ("endogenous") HSP60 are recognized by TLR2 and TLR4 but not by TLR9 and that endocytosis of HSP60 precedes initiation of signaling. A 5′-FLAG epitope-tagged C terminus of murine MyD88 (MyD-C) was amplified by reverse transcription polymerase chain reaction from murine spleen RNA and cloned into a modified pcDNA3 vector (Invitrogen), containing an untranslated intervening sequence from the mouse IgG heavy chain gene for improved expression in eukaryotic cells (pCX). The expression vectors for the C terminus of human TRAF6 (TRAF-C) and human FLAG-tagged TLR2 and TLR4 were gifts from Tularik, Inc. The cDNA for human TLR9, a gift from B. Beutler (University of Texas Southwestern Medical Center, Dallas, TX) was expressed in a modified pcDNA3 vector, containing the EF-1α promoter. The human MD-2 expression vector was kindly provided by K. Miyake (Saga Medical School, Nabeshima, Japan). The cDNA for hemagglutinin epitope (HA)-tagged IKKα, a gift from R. Schmid (University of Ulm, Ulm, Germany), was expressed in pCX. HA-JNK1 was a gift from M. Karin (University of California at San Diego, La Jolla, CA). The luciferase reporter driven by synthetic enhancer harboring 6 NF-κB binding consensus sites was a gift from P. Baeuerle (Munich, Germany). Recombinant human HSP60 was purchased from StressGen Biotechnologies (Victoria, Canada). TheEscherichia coli expression vector for His epitope-tagged chlamydial HSP60 was kindly provided by B. Kaltenboeck (Auburn University, Auburn, AL). Recombinant chlamydial HSP60 was produced by standard protein purification procedures using a Ni2+-nitrilotriacetic acid column (Qiagen, Hilden, Germany) followed by size exclusion chromatography (Superdex 200; Amersham Pharmacia Biotech). The purity of produced protein was determined by SDS-polyacrylamide gel electrophoresis and silver staining. Endotoxin contamination of recombinant chlamydial HSP60 was less than 0.1 pg/µg protein as determined by LAL-Test (Acila GMN, Weiterstadt, Germany). Phosphothioate-stabilized CpG oligonucleotides 1668 (TCC ATG ACG TTC CTG ATG CT) and 2006 (TCG TCG TTT TGT CGT TTT GTC GTT) were purchased from TIB MOLBIOL (Berlin, Germany). LPS from Salmonella minnesota Re 595, polymyxin B (PMB), monodansylcadaverine (MDC), and anisomycin were purchased from Sigma, poly(dI·dC) was fromAmersham Pharmacia Biotech, and murine recombinant interferon-γ (IFN-γ) was from PeproTech (Rocky Hill, NJ). Antibodies to ERK1/2 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and antibodies from New England Biolabs included anti-phospho-JNK1/2 (Thr183/Tyr185), anti-JNK1/2, anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-IκB-α, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-STAT1 (Tyr701), and anti-STAT1. The murine macrophage cell line RAW264.7 was grown in VLE-RPMI medium (Biochrom KG, Berlin, Germany) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin G, and 100 IU/ml streptomycin sulfate (all from Biochrom KG). 5–10·106 RAW264.7 cells were transfected by electroporation in a 400-µl final volume (RPMI, 25% FCS) at 280 V (for JNK kinase assay) or 300 V (for IKK kinase assay) and 960 microfarads in a Gene Pulser (Bio-Rad). 5 µg of HA-JNK1 or 9 µg of HA-IKKα were used for transfection together with different amounts of specific expression vectors as indicated in the figure legends. The overall amount of plasmid DNA was held constant at 30 µg/electroporation by the addition of the appropriate empty expression vector. After electroporation, cells were washed and split into six-well plates for subsequent stimulation and lysis. The human embryonic kidney fibroblasts 293T were cultured in Dulbecco's modified Eagle's medium (Biochrom KG) with the same supplements as for RAW264.7 macrophage cell cultures. For luciferase reporter assays, the 293T cells were transfected by the calcium phosphate method as described (22Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4820) Google Scholar). Briefly, 24 h before transfection, 293T cells were plated at 105 cells/well in 12-well plates in 10% FCS, Dulbecco's modified Eagle's medium. 1 ng of 6× NF-κB luciferase reporter together with 0.1 µg of respective expression vector or empty control vector (as indicated in legend of Fig. 5 A) per transfection were used. The overall DNA amount was held constant at 6 µg by the addition of pcDNA3. After the addition of DNA mix, the cells were held in 3% CO2 incubator for 15–20 h, subsequently washed, and held in 7% CO2 incubator for an additional 5–10 h before stimulation and lysis. Mice deficient in TLR2 generated by gene targeting were obtained from Tularic Inc. (South San Francisco, CA). Briefly, a portion of the TLR2 gene sequence encoding a portion of the extracellular and of the transmembrane domain was replaced by gene targeting in ES cells from 129/SvJ mice with a neomycin cassette oriented in the opposite direction of the gene (Deltagen Inc., Menlo Park, CA). TLR2-deficient mice were generated by aggregation in C57BL/6 mice and intercrossing of resulting heterozygous mice. Inactivation of the TLR2 gene was confirmed by Western blotting of extracts from thioglycolate-elicited peritoneal macrophages using rabbit polyclonal serum raised against TLR2. 2C. J. Kirschning, manuscript in preparation. Groups of corresponding wild-type and TLR2-deficient mice were applied. All experiments used homozygous F1 generation mice. TLR4-mutated C3H/HeJ and wild-type C3H/HeN mice were purchased from Charles River (Sulzfeld, Germany). Age-matched groups of wild-type and TLR-deficient mice were used for the experiments. Bone marrow-derived dendritic cells (BMDDC) were prepared as described (23Lutz M.B. Kukutsch N. Ogilvie A.L. Rossner S. Koch F. Romani N. Schuler G. J. Immunol. Methods. 1999; 223: 77-92Crossref PubMed Scopus (2502) Google Scholar). For stimulation, nonadherent BMDDC at days 7–9 were washed and plated at 7.5·105 in medium without FCS, and after a 4-h rest cells were stimulated in duplicates for 18 h. Unless otherwise stated, stimulations were performed in the presence of 20 µg/ml PMB, sufficient to block at least 50 ng/ml LPS. TNF-α levels in culture supernatants were determined by a commercially available enzyme-linked immunosorbent assay kit (R&D Systems) according to the instructions of the manufacturer. For Western blotting, cells were grown and stimulated as indicated in the figure legends. After stimulation, cells were lysed in Triton lysis buffer containing 25 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 10 mm sodium pyrophosphate, 20 mm β-glycerophosphate, 2 mm sodium orthovanadate, 10 mm sodium fluoride, 10 µg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride. Lysates were boiled in SDS sample buffer, sonicated, centrifuged at 10,000 ×g for 10 min, resolved on a 10% SDS-polyacrylamide gel electrophoresis, and blotted onto Protran nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked in 5% skim milk solution, probed with the indicated antibodies, and visualized using the chemiluminescence kit Renaissance (PerkinElmer Life Sciences). For in vitro kinase assays, 18 h after transfection cell cultures were deprived of FCS for 2 h and then stimulated as indicated in legend of Fig. 4. Afterward, medium was aspirated, cells were lysed in Triton lysis buffer, and lysates were precleared by centrifugation at 10,000 × g for 10 min. To precipitate HA-tagged kinases, antibodies to the HA tag (clone 12CA5, Roche Molecular Biochemicals) together with protein A-Sepharose (Amersham Pharmacia Biotech) were incubated with the lysates overnight. Precipitates were washed 3 times with HNTG buffer (20 mmHEPES, pH 7.5, 150 mm NaCl, 0.1% Triton X-100, 10% glycerol) and equilibrated in kinase buffer (25 mm HEPES, pH 7.5, 10 mm MgCl2, 10 mmβ-glycerophosphate, 0.5 mm EGTA, 0.5 mmsodium fluoride, 0.5 µm sodium orthovanadate, 20 µm ATP). Kinase assays were initiated by adding [γ-32P]ATP (Hartmann Analytic, Braunschweig, Germany) and the number of amino acids from C-JUN (GST-c-Jun (79)) for JNK or the number of amino acids from IKB (GST-IκBα (54)) for IKK. Reactions were performed at 30 °C for 30 min for JNK or 40 min for IKK and stopped by boiling in SDS sample buffer. Probes were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, and the radioactive intensity was measured using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). For loading control, membranes were probed with antibodies to HA-tag (clone 3F10; Roche Molecular Biochemicals). For luciferase assays, transfected cells were stimulated as described in Fig. 5 A and lysed, and luciferase activity in extracts was measured with the Luciferase Assay System kit from Promega(Mannheim, Germany) according to manufacturer's instructions. Chlamydial HSP60 was labeled using the Alexa FluorTM546 Protein Labeling Kit (Molecular Probes, Leiden, The Netherlands) following the manufacturer's instructions. Concentration of protein after labeling was controlled by the BCA assay kit from Pierce. To study the cellular uptake of chlamydial HSP60, 7×105 RAW264.7 macrophages were plated 1 day before image collection in Lab-Tek II two-chambered cover glasses (Nunc International, Wiesbaden-Biebrich, Germany) in 10% FCS, RPMI medium. Two h prior to stimulation, medium was exchanged as detailed in Fig. 2. The specimens were mounted in a Zeiss Tempcontrol 37-2 digital and held at 37 °C during the recordings. The uptake was viewed with a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) using a helium/neon 543-nm laser, a Plan-Neofluar 40 1.3 oil lens, and LSM 510 version 2.02 software. 1-µm thick slices were collected. Initial attempts to correlate HSP60-induced TNFα production with activation of IKK or JNK consistently resulted in weak signals or no signals. However, if macrophage cell line RAW264.7 cultures were withdrawn of FCS for several hours and then stimulated with HSP60, an unequivocal activation of, for example, stress-activated protein kinase JNK1/2 ensued (Fig.1 A). This was paralleled with production of higher amounts of TNFα at each concentration of HSP60 tested (Fig. 1 B). The potential of contamination with LPS could be excluded, since all experiments were performed in the presence of PMB at concentrations able to neutralize 50 ng/ml LPS (Figs.1 B and 3 B). We speculated that FCS might block the contact of HSP60 with macrophages. To analyze this possibility, we compared stimulation of cells with fluorescently labeled HSP60 in the presence or absence of serum. Labeling of HSP60 did not affect its stimulatory capacity as measured by TNFα production (data not shown). Confocal laser-scanning microscopy revealed that in the absence of FCS fluorescently labeled HSP60 became rapidly endocytosed by macrophages (Fig. 2). On the other hand, FCS (10% in culture medium) effectively blocked the internalization of HSP60 (Fig.2). Overall, these data implied that component(s) within FCS interfered with HSP60 endocytosis by macrophages and perhaps with subsequent triggering of intracellular signaling cascades.Figure 3Signaling cascades triggered by HSP60. A, 1 day before stimulation, 2.5·105 RAW264.7 macrophages/well in 0.5 ml of 10% FCS/RPMI medium were plated in 24-well plates. 2 h before stimulation, cell cultures were deprived of FCS. Cells were stimulated with 1 µg/ml of human or chlamydial HSP60 for 60 and 120 min and with 2 µm CpG oligonucleotide 1668 for 30 min. 20 µg/ml PMB was used throughout the stimulations to exclude the effects of trace amounts of LPS in protein preparations. Cells were lysed, and Western blotting for the activated form of p38 (p-p38), for I-κBα, and for activated forms of ERK1/2 (p-ERK1/2) was performed. Additionally, the total amount of p38 and ERK1/2 in lysates was determined. One representative experiment out of three is shown. B, RAW264.7 cells were pretreated as in A and then stimulated in the absence of FCS for 30 min with 2 µm 1668 or 50 ng/ml LPS. In parallel, the same stimulations were performed in medium containing 10% FCS. To probe the efficiency of PMB, part of the cells were in addition stimulated with 50 ng/ml LPS with and without PMB. Cells were lysed, and Western blotting for the activated form of p38 was performed. Additionally, the total amount of p38 in lysates was determined. One representative experiment out of three is shown.View Large Image Figure ViewerDownload (PPT) Having established that serum-free conditions allow efficient HSP60-mediated activation of macrophages, we compared the signaling pathways induced with those triggered by bacterial CpG-DNA or LPS. In addition to JNK1/2 (Fig. 1 A) HSP60 also activated stress-activated protein kinase p38, although slightly delayed when compared with CpG-DNA (oligonucleotide 1668) or LPS (Fig.3 A and data not shown). Furthermore, chlamydial HSP60 and its human homologue activated IKK (see below), caused degradation of IκB-α (Fig. 3 A) and induced in macrophages NF-κB luciferase reporter activity (data not shown). Finally, both HSP60s activated the classic mitogen-activated protein kinase ERK1/2 (Fig. 3 A), as shown for LPS (24Weinstein S.L. Sanghera J.S. Lemke K. DeFranco A.L. Pelech S.L. J. Biol. Chem. 1992; 267: 14955-14962Abstract Full Text PDF PubMed Google Scholar) or CpG-DNA (25Hacker H. Mischak H. Hacker G. Eser S. Prenzel N. Ullrich A. Wagner H. EMBO J. 1999; 18: 6973-6982Crossref PubMed Scopus (106) Google Scholar). Pathogen-derived (exogenous) ligands such as LPS or bacterial CpG-DNA activate innate immune cells via the evolutionary ancient TIR signaling pathway (26Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar, 27Hacker H. Vabulas R.M. Takeuchi O. Hoshino K. Akira S. Wagner H. J. Exp. Med. 2000; 192: 595-600Crossref PubMed Scopus (418) Google Scholar). Signaling through TIR is believed to occur via sequential recruitment of the adapter molecule MyD88, the IL-1 receptor-associated kinase, and the adapter molecule TRAF6 (18Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 28Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). To address the question of whether HSP60s share intracellular signaling cascades with classic bacterial ligands, we employed transient transfections of RAW264.7 with dominant negative forms of signaling intermediates, concentrating our analysis on downstream activation of JNK and IKK. Having established that the peak of JNK and IKK activity in RAW264.7 macrophages stimulated with 5 µg/ml of HSP60 was reached at 60 and 30 min, respectively (data not shown), these time points were used to "read out" the effects of dominant-negative constructs. First, we analyzed whether HSP60-mediated activation of the intracellular signaling cascades is controlled by MyD88. To this, RAW264.7 cells were cotransfected with either HA-tagged JNK1 or IKKα together with a C terminus of MyD88 (MyD-C) known to act as a dominant negative version of MyD88 (28Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). As shown in Fig.4 A, increasing amounts of MyD-C inhibited dose-dependently both JNK and IKK activation after stimulation with human or with chlamydial HSP60. Dose-dependent expression of the MyD-C construct was confirmed by Western blotting (data not shown). To control for unspecific effects of MyD-C overexpression, we used poly(dI·dC) as an MyD88-independent stimulus (27Hacker H. Vabulas R.M. Takeuchi O. Hoshino K. Akira S. Wagner H. J. Exp. Med. 2000; 192: 595-600Crossref PubMed Scopus (418) Google Scholar). Fig.4 A shows that induction of JNK and IKK activity by poly(dI·dC) was not affected by transfection of dominant negative MyD-C. TRAF6, originally cloned as a CD40-interacting molecule, is composed of a highly conserved C-terminal TRAF domain and an N-terminal RING finger domain (29Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (516) Google Scholar). The overexpressed C-terminal TRAF domain (TRAF-C) acts as a dominant-negative molecule in TIR- and CD40-dependent signaling (18Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 30Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1119) Google Scholar, 31Kashiwada M. Shirakata Y. Inoue J.I. Nakano H. Okazaki K. Okumura K. Yamamoto T. Nagaoka H. Takemori T. J. Exp. Med. 1998; 187: 237-244Crossref PubMed Scopus (113) Google Scholar). To investigate whether TRAF6 is involved in HSP60-induced signaling, we cotransfected TRAF-C together with HA-tagged JNK1 or IKKα into RAW 264.7 cells, activated them with human or chlamydial HSP60, and measured induced kinase activities. As shown in Fig. 4 B, TRAF-C suppressed dose-dependently JNK and IKK activity induced by HSP60 yet did not affect JNK and IKK activity triggered by poly(dI·dC). Dose-dependent expression of TRAF-C construct was confirmed by Western blotting (data not shown). These results implied that HSP60s signal via MyD88 and TRAF6, leading to downstream activation of JNK and IKK. Activation of the TIR pathway by bacterial ligands LPS, peptidoglycans, or CpG-DNA is brought about via TLR4, TLR2, or TLR9, respectively (17Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5359) Google Scholar, 32Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6421) Google Scholar, 33Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 34Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2779) Google Scholar). Participation of MyD88 and TRAF6 in HSP60-triggered signaling events prompted us to consider the involvement of Toll-like receptors. To test whether one of these TLR acts as a HSP60 receptor component we resorted to the system of transient reconstitution of unresponsive human embryonic kidney fibroblasts 293T. These cells were transiently transfected with luciferase reporter driven by synthetic enhancer harboring NF-κB binding consensus sites. Upon cotransfection of human TLR2 expression vector but not of the empty control vector, both human and chlamydial HSP60 activated the NF-κB reporter in these cells (Fig.5 A). Furthermore, transient transfection with human TLR4 was insufficient to make 293T responsive to HSP60, yet cotransfection of TLR4 plus human MD-2 conferred responsiveness (Fig. 5 A). Cotransfection of TLR2 and TLR4 plus MD-2 did not yield in synergistic responsiveness (data not shown), indicating that TLR2 and TLR4 appear not to cooperate in the response to HSP60, at least in regard to NF-κB activation. Finally, transfection of a human TLR9 construct selectively conferred responsiveness to CpG-DNA but not to HSP60 (Fig. 5 A), implying specificity in "gain of function." These data suggested that TLR2 or a complex of TLR4 and MD-2 represent essential receptor components for endogenous (human) and exogenous (chlamydial) HSP60. To verify the gain of function experiments, we performed "loss of function" assays by using BMDDC from TLR2-k.o. mice and from C3H/HeJ mice known to carry the codominant point mutationLps d that compromises the function of TLR4 (32Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6421) Google Scholar). Compared with wild type BMDDC, TLR2-deficient BMDDC displayed a strongly reduced capacity to respond to HSP60 as measured by TNFα production (Fig. 5 B). The reactivity to LPS was, however, only slightly compromised due to contaminating lipoproteins in the commercially available LPS preparation (35Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (969) Google Scholar). Similarly, TLR4-defective BMDDC from C3H/HeJ mice responded under serum-free conditions to HSP60, but the response was curtailed compared with control cells from C3H/HeN mice (Fig. 5 B). To control for equal stimulatory conditions for both wild-type and mutant cells, we included stimulation with CpG-DNA (Fig. 5 B) known to be TLR9-dependent but not TLR2- or TLR4-dependent. Based on the TLR2- and TLR4-associated gain of function and loss of function data, we concluded that both TLR2 and TLR4 confer responsiveness to HSP60. Since FCS effectively impaired HSP60 endocytosis by macrophages and HSP60 triggered signaling as well (Fig. 1 and 2), we analyzed whether endocytosis of HSP60 is a condition for signal initiation or whether it reflects only scavenging of the ligand. There are at least five independent pathways known for endocytic internalization: the clathrin-dependent pathway, the caveolar pathway, a clathrin- and caveolin-independent pathway, macropinocytosis, and phagocytosis (36Riezman H. Woodman P.G. van Meer G. Marsh M. Cell. 1997; 91: 731-738Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In view of the known receptor-dependent uptake of certain HSP, that is HSC70, HSP70, gp96 and HSP90 (8Arnold-Schild D. Hanau D. Spehner D. Schmid C. Rammensee H.G.

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