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Involvement of Toll-like Receptors 2 and 4 in Cellular Activation by High Mobility Group Box 1 Protein

2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês

10.1074/jbc.m306793200

ISSN

1083-351X

Autores

Jong Sung Park, Daiva Svetkauskaite, Qianbin He, Jae‐Yeol Kim, Derek Strassheim, Akitoshi Ishizaka, Edward Abraham,

Tópico(s)

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Resumo

High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-κB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produced activation of NF-κB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increased the activity of IKKβ, HMGB1 exposure resulted in activation of both IKKα and IKKβ. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-κB-dependent transcription by HMGB1. Transfections with dominant negative constructs demonstrated that TLR 2 and TLR 4 were both involved in HMGB1-induced activation of NF-κB. In contrast, RAGE played only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS. High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-κB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, we explored the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produced activation of NF-κB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increased the activity of IKKβ, HMGB1 exposure resulted in activation of both IKKα and IKKβ. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-κB-dependent transcription by HMGB1. Transfections with dominant negative constructs demonstrated that TLR 2 and TLR 4 were both involved in HMGB1-induced activation of NF-κB. In contrast, RAGE played only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS. HMGB1 1The abbreviations used are: HMGB1, high mobility group box 1; IRAK, IL-1R-associated kinase family; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL-1, interleukin-1; ERK, extracellular signal-regulated kinase; JNK, c-Jun amino-terminal kinase; TLR, Tell-like receptor; IKK, IκB kinase; RAGE, receptor for advanced glycation end products; SAPK, stress-activated protein kinase; PGN, peptidoglycan; PAM, tripamitoyl lipopeptide PAM3-Cys-Ser-Lys4; E2, ubiquitin carrier protein; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; NIK, NF-κB-inducing kinase; MKK, mitogen-activated protein kinase kinase. (formerly HMG1) was originally described as a non-histone, chromatin-associated nuclear protein (1Agresti A. Bianchi M.E. Curr. Opin. Genet. Dev. 2003; 13: 170-178Crossref PubMed Scopus (329) Google Scholar, 2Andersson U. Erlandsson-Harris H. Yang H. Tracey K.J. J. Leukoc. Biol. 2002; 72: 1084-1091PubMed Google Scholar, 3Czura C.J. Wang H. Tracey K.J. J. 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However, such RAGE-independent HMGB1 receptors have not yet been identified. Additionally, no studies have examined the relative importance of RAGE versus other putative HMGB1 receptors. In the present experiments, we explored the role of RAGE, TLR 2, and TLR 4, as well as associated kinases, in HMGB1-induced cellular activation. Our studies demonstrate that RAGE plays only a minor role in macrophage activation by HMGB1, whereas signaling through TLR 2 and TLR 4 appears to be of much greater importance. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation of the ability of HMGB1 to potentiate many inflammatory responses initiated by LPS. Reagents—LipofectAMINE 2000, RPMI 1640, Dulbecco's modified Eagle's medium, and penicillin/streptomycin were obtained from Invitrogen Corp. (Carlsbad, CA). Defined fetal bovine serum was purchased from HyClone (Logan, Utah). LPS (from Escherichia coli O111: B4) was obtained from Sigma Chemical Co. (St. Louis, MO). The LPS was re-extracted twice with phenol then precipitated from the aqueous phase to ensure that signaling only occurred through TLR 4 (35Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (985) Google Scholar). The TLR 2 ligands peptidoglycan (PGN) and tripamitoyl lipopeptide PAM3-Cys-Ser-Lys4 (PAM) were purchased from InvivoGen (San Diego, CA). Poly(dI-dC)·poly(dI-dC) was purchased from Amersham Biosciences (Piscataway, NJ). The Coomassie-Plus protein assay reagent and BCA protein assay reagent were obtained from Pierce (Rockford, IL). Antibodies for IKKα and IKKβ were purchased from Upstate Inc. (Lake Placid, NY). IκBα protein was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HMGB1 was purified from pig thymus by the method of Sanders (36Sanders C. Biochem. Biophys. Res. Commun. 1977; 78: 1034-1042Crossref PubMed Scopus (90) Google Scholar) and contained less than 10 pg/ml LPS by chromogenic assay (37Schwartz M.D. Repine J.E. Abraham E. Am. J. Respir. Cell Mol. Biol. 1995; 12: 434-440Crossref PubMed Scopus (85) Google Scholar). Mice—Male C3H/HeN (Harlan Sprague-Dawley, Indianapolis, IN) and C3H/HeJ (Jackson Laboratories, Bar Harbor, ME) mice were purchased at 6 weeks of age and were maintained in the animal colony at the University of Colorado Health Sciences Center (Denver, CO). Transgenic mice lacking TLR 2 (38Takeuchi O. Hoshino K. Akira S. J. Immunol. 2000; 165: 5392-5396Crossref PubMed Scopus (915) Google Scholar, 39Takeuchi 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 (2830) Google Scholar) (TLR 2–/–) were a gift from Dr. Shizuo Akira. All mice were 7–10 weeks of age when experiments were initiated. Experimental procedures were approved by the University of Colorado Health Sciences Center Institutional Animal Care and Use Committee. Isolation and Culture of Mouse Neutrophils—Peripheral neutrophils were purified from bone marrow cell suspensions as previously described (40Arcaroli J. Yang K.Y. Yum H.K. Kupfner J. Pitts T.M. Park J.S. Strassheim D. Abraham E. J. Leukoc. Biol. 2002; 72: 571-579PubMed Google Scholar, 41Yang K.Y. Arcaroli J. Kupfner J. Pitts T.M. Park J.S. Strasshiem D. Perng R.P. Abraham E. Cell. Signal. 2003; 15: 225-233Crossref PubMed Scopus (59) Google Scholar, 42Abraham E. Gyetko M.R. Kuhn K. Arcaroli J. Strassheim D. Park J.S. Shetty S. Idell S. J. Immunol. 2003; 170: 5644-5651Crossref PubMed Scopus (92) Google Scholar). To obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with 5 ml of RPMI 1640/penicillin/streptomycin, and the cells were passed through a glass wool column. The cell pellets from the bone marrow were resuspended in RPMI 1640/5%-defined fetal calf serum and then incubated with 10 μl of primary antibodies specific for the cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4 °C. This custom mixture (Stem Cell Technologies) is specific for T and B cells, red blood cells, monocytes, and macrophages. After 15-min incubation with the antibody mixture, 100 μl of antibiotin tetrameric antibody complexes was added for an additional 15 min at 4 °C. Following this, 60 μl of colloidal magnetic dextran iron particles were added to the suspension and incubated for 15 min at 4 °C. The entire cell suspension was then placed into a column surrounded by a magnet. The T cells, B cells, red blood cells, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative selection methods. Viability, as determined by trypan blue exclusion, was consistently greater than 98%. Neutrophil purity, as determined by Cytospin preparations stained with Wright's stain, was greater than 98%. Neutrophils were resuspended in RPMI 1640/10%-defined fetal calf serum at a final concentration of 5 × 106 cells/ml and stimulated with 1000 ng/ml HMGB1 or 100 ng/ml LPS. Control cultures or those containing HMGB1 were supplemented with polymyxin B (10 μg/ml) to block any effects of contaminating endotoxin. Transient Transfection and Luciferase Reporter Assay—The mouse macrophage cell line, RAW 264.7, was plated on 6-well plates at 5 × 105 cells/ml on the day before transfection. Combinations of expression plasmid DNAs (1 μg/ml) were transfected by using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Duplicate wells were set up for each group. The reporter plasmid used was pNF-κBluc (Clontech, Palo Alto, CA). The plasmids expressing dominant negative mutant proteins were gifts: MyD88-(152–296), ΔIRAK-1, and IRAK-2-(97–590) from Dr. Alberto Mantovani, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; IRAK-4 [KK213AA], TRAF6-(289–522), NIK[KK429,430AA], IKKα[S176A], and IKKβ[S177A] from Tularik, South San Francisco, CA; TAK-1[K63W] and TAB2[1281–2551] from Dr. Kuni Matsumoto, Nagoya University, Nagoya, Japan; p38α[TGY180–182AGF] from Dr. Philips E. Scherer, Albert Einstein College of Medicine, New York, NY; TIRAP [PH] from Dr. Ruslan Medzhitov, Yale University, New Haven, CT; RAGE[Δcyto] from Dr. Henri Huttunen, University of Helsinki, Helsinki, Finland; TRAF2-(87–501) from Dr. David Sassoon, Mount Sinai School of Medicine, New York, NY; TLR2[P681H] and TLR4[P721H] from Dr. David M. Under-hill, Institute for Systems Biology, Seattle, WA; and MKK3[A] and MKK6[A] from Dr. Jiahuai Han, Scripps Research Institute, La Jolla, CA. At 48 h after transfection, cells were stimulated with either LPS or HMGB1 for 1 h. The cells were then lysed, and luciferase activity was measured using a luciferase reporter assay kit (Promega) according to the manufacturer's instructions. All of the luciferase assays were repeated at least three times. Representative results are shown for each experiment. Immune Complex Kinase Assay—Cells were lysed in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) at 5 × 106 cells/ml. The lysates (5 × 106 cells/sample) were incubated with 1 μg of polyclonal antibodies (either anti-IKKα or anti-IKKβ) for 3 h at 4 °C, and then protein G-Sepharose beads (Invitrogen) were added for an additional 2 h. The beads were washed three times in lysis buffer and then once in kinase buffer (20 mm Tris-HCl, pH 7.4; 20 mm MgCl2; 2 mm EGTA; 0.5 mm sodium vanadate; 10 mm β-glycerophosphate; 1 mm dithiothreitol). The kinase reaction was initiated by the addition of 30 μl of kinase buffer containing 10 μm ATP, 5 μCi of [32P]ATP, and 1 μg of IκBα (Santa Cruz Biotechnology Inc.) as a substrate; this step was allowed to proceed for 30 min at 30 °C. The reaction was terminated by the addition of 5× SDS sample buffer. Samples were boiled and resolved by 10% SDS-polyacrylamide gel electrophoresis, and the fixed gel was then exposed to an x-ray film. Electrophoretic Mobility Shift Assays—To obtain nuclear extracts, the neutrophils were suspended in lysis buffer containing 10 mm Tris·HCl (pH 7.5), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 0.1 mm Na3VO4, and 0.1% Triton X-100, and the samples were incubated on ice for 20 min. After cytoplasm was removed from the nuclei by 15 passages through a 25-gauge needle, the nuclei were collected by centrifugation at 5,000 × g for 10 min at 4 °C. The pellets were suspended in extraction buffer containing 20 mm Tris·HCl (pH 7.5), 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 0.1% Triton X-100, 25% glycerol, 0.5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, and 0.1 mm Na3VO4. After a 30-min incubation on ice, the suspension was centrifuged at 14,000 × g for 20 min at 4 °C, and the supernatants were collected. The protein concentration in the supernatants was determined using Coomassie Plus protein assay reagent (Pierce). Nuclear extracts (5 μg) were incubated at room temperature for 15 min in 20 μl of reaction buffer containing 10 mm Tris·HCl (pH 7.5), 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 50 mm NaCl, and 4% glycerol with 32P-end-labeled, double-stranded oligonucleotide probe specific for the κB site, 5′-GCCATGGGGGGATCCCCGAAGTCC-3′ (Geneka Biotechnology) and 1 μg of poly(dI-dC)·poly(dI-dC). The complexes were resolved on 5% polyacrylamide gels in Tris·HCl (pH 8.0)-borate-EDTA buffer at 10 V/cm. Dried gels were exposed with Kodak Biomax MS film (Rochester, NY) for 1–24 h at 70 °C. Quantification was performed by image analysis using densitometry (ChemiDoc system; Bio-Rad, Hercules, CA). Statistical Analysis—Data are presented as mean ± S.E. for each experimental group. Student's t test (for comparisons between LPS- and HMGB1-stimulated cells) was used. p < 0.05 was considered significant. HMGB1 Enhances Nuclear Translocation of NF-κB through Mechanisms Independent of TLR 4 —In a previous study (13Park J.S. Arcaroli J. Yum H.K. Yang H. Wang H. Yang K.Y. Choe K.H. Strassheim D. Pitts T.M. Tracey K.J. Abraham E. Am. J. Physiol. Cell Physiol. 2003; 284: C870-C879Crossref PubMed Scopus (390) Google Scholar), we found that exposure of neutrophils to HMGB1 increased nuclear accumulation of NF-κB to approximately the same level as that seen after stimulation with LPS. Although interactions with TLR 4 are primarily responsible for LPS-induced cellular activation, the involvement of TLR 4 in HMGB1 signaling had not previously been determined. To examine the importance of TLR 4 and TLR 2 in HMGB1-associated NF-κB activation, we utilized neutrophils from transgenic mice lacking TLR 2 (TLR 2–/–), C3H/HeJ, and control C3H/HeN mice. C3H/HeJ mice lack a functional TLR 4 receptor due to a missense point mutation that results in the substitution of histidine for proline within the cytoplasmic portion of TLR 4 (43Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. 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 (6578) Google Scholar, 44Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1365) Google Scholar). After exposure to HMGB1, nuclear translocation of NF-κB was increased to approximately the same degree in neutrophils from C3H/HeN and C3H/HeJ mice (Fig. 1A). In both mouse strains, maximal nuclear accumulation of NF-κB was present 15 min after stimulation with HMGB1. In contrast, after culture with LPS, the nuclear content of NF-κB was not altered in C3H/HeJ neutrophils, but was increased in neutrophils from C3H/HeN mice (Fig. 1B). Such results demonstrate that receptors other than TLR 4 participate in HMGB1-induced cellular activation. HMGB1 and LPS both increased nuclear translocation of NF-κB in neutrophils from TLR 2 –/– mice (Fig. 1C). As expected, exposure of TLR 2 –/– neutrophils to the TLR 2-specific ligands PGN and PAM did not produce any alterations in the nuclear concentrations of NF-κB. Such results indicate that receptors other than TLR 2 are involved in cellular activation after exposure to HMGB1. To delineate mechanisms by which HMGB1 leads to enhanced nuclear translocation of NF-κB, we examined activation of IKKα and IKKβ in HMGB1- and LPS-stimulated neutrophils. IKKα and IKKβ are catalytically active components of the IκB kinase (IKK) complex that, when activated after cellular exposure LPS or other stimuli, can phosphorylate members of the IκB family, freeing cytoplasmic NF-κB to translocate into the nucleus and initiate transcriptional activity (45O'Connell M.A. Bennett B.L. Mercurio F. Manning A.M. Mackman N. J. Biol. Chem. 1998; 273: 30410-30414Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 46Fischer C. Page S. Weber M. Eisele T. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: 24625-24632Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 47Dunne, A., and O'Neill, L. A. (2003) Science's STKE http://stke.sciencemag.org/cgi/reprint/sigtrans;2003/171/re3Google Scholar). Although IKKα is dispensable for NF-κB activation induced by mediators such as LPS and IL-1, cells from mice deficient in IKKβ show impaired NF-κB activation and IL-6 produ

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