Induction of Bacterial Lipoprotein Tolerance Is Associated with Suppression of Toll-like Receptor 2 Expression

Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-α and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-κB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-α and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2 expression. Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-α and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-κB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-α and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2 expression. lipopolysaccharide bacterial lipoprotein mitogen-activated protein kinases extracellular signal-regulated kinases c-Jun NH2-terminal kinases nuclear factor-κB toll-like receptor membrane-bound CD14 soluble CD14 systemic inflammatory response syndrome acute respiratory distress syndrome multisystem organ dysfunction syndrome tumor necrosis factor-α interleukin-6 monoclonal antibody polyclonal antibody fluorescence-activated cell sorter phosphate-buffered saline electrophoretic mobility shift assays fluorescein isothiocyanate enzyme-linked immunosorbent assay S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R) Lipopolysaccharide (LPS),1 a predominant glycolipid in the outer membrane of Gram-negative bacteria, activates monocytes and macrophages to produce several pro-inflammatory cytokines such as TNF-α and IL-6. Excessive activation of monocytes and macrophages by LPS with overwhelming production of pro-inflammatory cytokines is thought to be responsible for the clinical manifestation of septic shock (1Manthey C.L. Vogel S.N. Rev. Med. Microbiol. 1992; 3: 72-81Google Scholar, 2Sessler C.N. Bloomfield G.L. Fowler A.A. Clin. Chest Med. 1996; 17: 213-235Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Bacterial lipoprotein (BLP) is the most abundant protein in the outer membrane of both Gram-positive and Gram-negative bacteria (3DiRienzo J.M. Nakamura K. Inouye M. Annu. Rev. Biochem. 1978; 47: 481-532Crossref PubMed Scopus (250) Google Scholar, 4Henderson B. Poole S. Wilson M. Microbiol. Rev. 1996; 60: 316-341Crossref PubMed Google Scholar). Recently, BLP has also been shown to activate a variety of host inflammatory cells to produce pro-inflammatory cytokines (5Brightbill H.D. Libraty D.H. Krutzik S.R. Yang R.B. Belisle J.T. Bleharski J.R. Maitland M. Norgard M.V. Plevy S.E. 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Reduced nuclear factor-κB (NF-κB) activation and NF-κB-DNA binding activity accompanied by a decreased degradation of both IκB-α and IκB-β are also found in LPS-tolerized cells in response to a second LPS stimulation (13Tominaga K. Saito S. Matsuura M. Nakano M. Biochim. Biophys. Acta. 1999; 1450: 130-144Crossref PubMed Scopus (61) Google Scholar, 14Medvedev A.E. Kopydlowski K.M. Vogel S.N. J. Immunol. 2000; 164: 5564-5574Crossref PubMed Scopus (450) Google Scholar, 15Kohler N.G. Joly A. Biochem. Biophys. Res. Commun. 1997; 232: 602-607Crossref PubMed Scopus (48) Google Scholar, 16Sato S. Nomura F. Kawai T. Takeuchi O. Muhlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (350) Google Scholar). On the other hand, BLP as well as several other non-LPS bacterial cell wall components have been shown to not only activate host inflammatory cells but also induce tolerance in these cells to the subsequent stimulation (16Sato S. Nomura F. Kawai T. Takeuchi O. Muhlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (350) Google Scholar, 17Biberstine K.J. Darr D.S. Rosenthal R.S. Infect. Immun. 1996; 64: 3641-3645Crossref PubMed Google Scholar, 18Kreutz M. Ackermann U. Hauschildt S. Krause S.W. Riedel D. Bessler W. Andreesen R. Immunology. 1997; 92: 396-401Crossref PubMed Scopus (78) Google Scholar). We have shown previously (19Doyle M. Wang J.H. Redmond H.P. Surg. Forum. 2000; 51: 191-193Google Scholar) that pre-exposure of MF-1 mice to a sub-lethal dose of BLP induces BLP tolerance that protects against a subsequent lethal BLP challenge. More interestingly, induction of BLP tolerance also protects against the lethality induced by LPS challenge, indicating a cross-tolerance to LPS (19Doyle M. Wang J.H. Redmond H.P. Surg. Forum. 2000; 51: 191-193Google Scholar). 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Miller D.S. Jahr T.G. Sundan A. Bazil V. Espevik T. Finlay B.B. Wright S.D. J. Exp. Med. 1992; 176: 1665-1671Crossref PubMed Scopus (609) Google Scholar, 22Haziot A. Rong G.W. Silver J. Goyert S.M. J. Immunol. 1993; 151: 1500-1507PubMed Google Scholar). The presence of CD14 has been also found to facilitate BLP signaling (6Wooten R.M. Morrison T.B. Weis J.H. Wright S.D. Thieringer R. Weis J.J. J. Immunol. 1998; 160: 5485-5492PubMed Google Scholar, 23Sellati T.J. Bouis D.A. Kitchens R.L. Darveau R.P. Pugin J. Ulevitch R.J. Gangloff S.C. Goyert S.M. Norgard M.V. Radolf J.D. J. Immunol. 1998; 160: 5455-5464PubMed Google Scholar, 24Hirschfeld M. Kirschning C.J. Schwandner R. Wesche H. Weis J.H. Wooten R.M. Weis J.J. J. Immunol. 1999; 163: 2382-2386Crossref PubMed Google Scholar). However, a CD14-dependent pathway used by BLP is distinct from that used by LPS (23Sellati T.J. Bouis D.A. Kitchens R.L. Darveau R.P. Pugin J. Ulevitch R.J. Gangloff S.C. Goyert S.M. Norgard M.V. Radolf J.D. J. 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Immunol. 2000; 164: 3476-3479Crossref PubMed Scopus (651) Google Scholar) that the expression of CD14 is not altered in LPS-tolerized monocytes and macrophages, whereas dysregulation of tlr2andtlr4 gene expression and a reduced surface expression of TLR4 are implicated in LPS tolerance (14Medvedev A.E. Kopydlowski K.M. Vogel S.N. J. Immunol. 2000; 164: 5564-5574Crossref PubMed Scopus (450) Google Scholar, 37Nomura F. Akashi S. Sakao Y. Sato S. Kawai T. Matsumoto M. Nakanishi K. Kimoto M. Miyake K. Takeda K. Akira S. J. Immunol. 2000; 164: 3476-3479Crossref PubMed Scopus (651) Google Scholar). More interestingly, when LPS tolerance in mouse peritoneal macrophages occurs via suppressed TLR4-MD2 surface expression (37Nomura F. Akashi S. Sakao Y. Sato S. Kawai T. Matsumoto M. Nakanishi K. Kimoto M. Miyake K. Takeda K. Akira S. J. Immunol. 2000; 164: 3476-3479Crossref PubMed Scopus (651) Google Scholar), tolerance to LPS induced by mycoplasmal lipopeptides, the 2-kDa macrophage-activating lipopeptides (MALP-2), is not through the down-regulation of TLR4-MD2 expression (16Sato S. Nomura F. Kawai T. Takeuchi O. Muhlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (350) Google Scholar). However, it is unknown whether TLR2, particularly TLR2 protein expression, is associated with the development of BLP tolerance as well as LPS tolerance. In the present study, we report that a synthetic bacterial lipopeptide induces tolerance in human THP-1 monocytic cells not only to subsequent BLP stimulation but also to LPS, indicating a cross-tolerance. Furthermore, induction of BLP tolerance is CD14-independent. When similar levels of suppressed MAP kinase phosphorylation and NF-κB activation are present in both BLP-tolerized and LPS-tolerized cells, BLP tolerance, but not LPS tolerance, prevents BLP-induced TLR2 overexpression. These results indicate the involvement of TLR2 in the development of BLP tolerance. RPMI 1640 medium, PBS, fetal calf serum, penicillin, streptomycin sulfate, and glutamine were purchased from Invitrogen. LPS from Escherichia coli serotype O55B5 was purchased from Sigma. BLP, a synthetic bacterial lipopeptide (Pam3-Cys-Ser-Lys4-OH) derived from the immunologically active NH2 terminus of bacterial lipoproteins, was purchased from Roche Molecular Biochemicals, which was LPS-free as confirmed by the Limulus amebocyte lysate assay (Charles River Endosafe, Charleston, SC). [γ-32P]ATP (3000 Ci/mmol) and poly(dI-dC) were obtained from Amersham Biosciences. FITC-conjugated mouse anti-human CD14 mAb (anti-Leu-M3) was obtained from BD Biosciences. Rabbit pAbs against active (phosphorylated) ERK1 and -2, active JNK1 and -2, and active p38 were obtained from Promega (Madison, WI) and New England Biolabs (Beverly, MA), respectively. Goat pAb against human TLR2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A specific mouse anti-human CD14 blocking mAb (MEM-18) was obtained from HBT (Uden, Netherlands), and a specific mouse anti-human TLR2 blocking mAb (2392) was a gift from Genentech, Inc. (San Francisco, CA). THP-1 cells (a human monocytic cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin sulfate (100 μg/ml), and glutamine (2 mm) at 37 °C in a humidified 5% CO2atmosphere. Tolerance in THP-1 cells was induced as described previously (14Medvedev A.E. Kopydlowski K.M. Vogel S.N. J. Immunol. 2000; 164: 5564-5574Crossref PubMed Scopus (450) Google Scholar). Briefly, cells cultured in a serum-present or serum-free medium were preincubated with culture medium (non-tolerant), 10 ng/ml BLP (BLP tolerant), or 10 ng/ml LPS (LPS tolerant) for 24 h, washed twice with PBS, and incubated in a fresh culture medium for 2 h. Then cells were stimulated with 100 and 1,000 ng/ml BLP or LPS for different time points. For blocking experiments, the cells were incubated with CD14 blocking mAb (MEM-18, 10 μg/ml) (38Plotz S.G. Lentschat A. Behrendt H. Plotz W. Hamann L. Ring J. Rietschel E.T. Flad H.D. Ulmer A.J. Blood. 2001; 97: 235-241Crossref PubMed Scopus (57) Google Scholar) or TLR2 blocking mAb (2392, 25 μg/ml) (39Aliprantis A.O. Yang R.B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1278) Google Scholar) for 30 min to totally block CD14 or TLR2 before they were exposed to BLP or LPS. THP-1 cells (1 × 105 cells/100 μl) were stained with 20 μl of FITC-conjugated mouse anti-human CD14 mAb at 4 °C for 30 min. FITC-conjugated isotype IgG2b mAb was used as a negative control. mCD14 expression on THP-1 cells was analyzed on a FACscan flow cytometer (BD Biosciences) for detecting the log of the mean channel fluorescence intensity with an acquisition of FL1. A minimum of 10,000 events were collected and analyzed using the CellQuest software (BD Biosciences). THP-1 cells (2 × 105 cells/well) incubated in 24-well plates (Falcon, Lincoln Park, NJ) were subjected to different challenges as described above. Cells were then stimulated with 100 and 1,000 ng/ml BLP or LPS for 6 h. Cell-free supernatants were collected by centrifugation, transferred to new tubes, and stored at −70 °C until analysis. The levels of TNF-α and IL-6 in cell supernatants were assessed using commercially available ELISA kits (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. Non-tolerized and BLP- or LPS-tolerized THP-1 cells were further stimulated with 100 ng/ml BLP or LPS for different time points. After being extensively washed with cold PBS, cells were lysed in ice with lysis buffer (1% Triton X-100, 20 mm Tris, 137 mm NaCl, 1 mmphenylmethylsulfonyl fluoride, 2 mmNa3VO4, 10 μg/ml leupeptin, 2 μg/ml aprotinin). Protein concentrations were determined using a micro BCA protein assay reagent kit (Pierce). The proteins were denatured at 95 °C for 10 min in loading buffer (60 mm Tris, 2.5% SDS, 10% glycerol, 5% mercaptoethanol, 0.01% bromphenol blue). Aliquots containing equal amount of total proteins from each sample were separated in SDS-polyacrylamide gels and transblotted onto nitrocellulose membranes (Schleicher & Schuell). After blocking for 2 h with TBS containing 0.1% Tween 20 and 6% nonfat milk, membranes were probed overnight at 4 °C with anti-MAP kinase pAbs or anti-TLR pAb. Blots were further incubated for 1 h with secondary horseradish peroxidase-conjugated anti-rabbit IgG (Promega) or anti-goat IgG (Santa Cruz Biotechnology) and developed using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology) according to the manufacturer's instructions. Dual transfections of THP-1 cells were accomplished using 12 μl/ml Dmrie-C reagent (Invitrogen) and 4.0 μg/ml pNF-κB-Luc vector DNA (CLONTECH, Palo Alto, CA) that contains the firefly luciferase gene as the reporter. The pRL-CMV vector (Promega) containing the Renilla luciferase gene was used as internal control. Briefly, THP-1 cells were plated in 24-well plates (2 × 105 cells/well) and preincubated for 24 h with culture medium, 10 ng/ml BLP, or 10 ng/ml LPS. After being washed with PBS and incubated in fresh culture medium for 2 h, non-tolerized and BLP- or LPS-tolerized cells were transfected with Dmrie-C-DNA complexes that were formed by mixture of pNF-κB-Luc vector DNA (0.8 μg/well) and pRL-CMV vector DNA (0.08 μg/well) with Dmrie-C reagent (2.4 μl/well) in 200 μl of serum-free Opti-MEM I (Invitrogen) for 30 min. Cells were transfected with the complexes for 4 h and cultured for a further 14-h period after replacement of the medium. Each transfection was performed in duplicate. After transfection, cells were stimulated with BLP (100 ng/ml) or LPS (100 ng/ml) for 6 h. Cell extracts were prepared using the Passive lysis buffer (Promega), and protein content in each sample was determined using a micro BCA protein assay reagent kit (Pierce). Firefly and Renillaluciferase activities were measured using the Dual luciferase reporter assay system (Promega) to assess promoter activity and transfection efficiency. Non-tolerized and BLP- or LPS-tolerized THP-1 cells in 6-well plates (1 × 106cells/well) were further treated with 100 ng/ml BLP or LPS for 60 min. Nuclear and cytoplasmic extracts were prepared as described previously (40Bowie A.G. Moynagh P.N. O'Neill L.A. J. Biol. Chem. 1997; 272: 25941-25950Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Briefly, cells were lysed in a hypotonic solution (10 mm Hepes, 1.5 mm MgCl2, 10 mm KCl, 0.1% Nonidet P-40, pH 7.9) on ice for 10 min and centrifuged at 13,000 rpm to pellet nuclei. Cytoplasmic supernatants were removed, and nuclei were resuspended in nuclear extract buffer (20 mm Hepes, 25% glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, pH 8.0) on ice for 15 min. The lysates were centrifuged at 13,000 rpm, and supernatants containing the nuclear proteins were collected. All buffers contained freshly added 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche Molecular Biochemicals). Protein concentrations were determined using a micro BCA protein assay reagent kit (Pierce). All nuclear extracts were stored at −70 °C until analysis. EMSA were performed as described previously (41Bourke E. Kennedy E.J. Moynagh P.N. J. Biol. Chem. 2000; 275: 39996-40002Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Briefly, 2.0–4.0 μg of nuclear extracts were incubated with 30,000 cpm of double-stranded oligonucleotide, 5′-AGT TGA GGGGACTTTCCC AGG C-3′, containing the NF-κB consensus sequence (underlined) (Promega) that had been labeled previously with [γ-32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase (Promega). The DNA-binding reactions were performed in the presence of 2.0 μg of poly(dI-dC) as nonspecific competitor in binding buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1.0 mm EDTA, 5.0 mmdithiothreitol, 4% glycerol, 100 μg/ml nuclease-free bovine serum albumin) at room temperature for 30 min. For competition experiments, unlabeled double-stranded oligonucleotide, 5′-AGT TGA GGCGACTTTCCC AGG C-3′, containing the mutated NF-κB consensus sequence (underlined) (Promega) was added to the nuclear extracts 30 min before the addition of the radiolabeled probe. All reaction mixtures were subjected to electrophoresis on native 5% (w/v) polyacrylamide gels, which were subsequently dried and autoradiographed. All data are presented as the mean ± S.D. Statistical analysis was performed using analysis of variance. Differences were judged statistically significant when thep value was less than 0.05. LPS activates monocytes and macrophages to produce pro-inflammatory cytokines such as TNF-α and IL-6. Fig.1, A and B, shows that BLP is a more potent stimulus than LPS for human THP-1 monocytic cell activation, as naive (non-tolerant) THP-1 cells exposed to BLP at 100 and 1,000 ng/ml for 6 h produced much higher levels of TNF-α and IL-6 (p < 0.05, versus LPS stimulation). A synergistic effect of mycoplasmal lipopeptides (MALP-2) and LPS on TNF-α production in murine peritoneal macrophages has been reported previously (16Sato S. Nomura F. Kawai T. Takeuchi O. Muhlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (350) Google Scholar). However, there were no further increases in TNF-α and IL-6 production found in THP-1 cells stimulated with combinations of BLP and LPS, when compared with cells stimulated with BLP alone (Fig. 1, A and B). When THP-1 cells were preincubated with 10 ng/ml BLP for 24 h and then subjected to a second stimulation with high doses of BLP (100 and 1,000 ng/ml), TNF-α and IL-6 production was significantly attenuated (Fig. 1, C and D), indicating induction of BLP tolerance. Most interestingly, BLP-tolerized THP-1 cells had an impaired ability to produce TNF-α and IL-6 in response to LPS stimulation or LPS plus BLP stimulation (Fig. 1, C andD), indicating a novel cross-tolerance to LPS induced by BLP pretreatment. Preincubation of THP-1 cells with LPS induced LPS tolerance, as demonstrated by attenuated TNF-α and IL-6 production in response to a second LPS stimulation (Fig. 1, E andF). Notably, LPS-tolerized THP-1 cells exhibited markedly reduced production of TNF-α and IL-6 in response to subsequent BLP or BLP plus LPS stimulation (Fig. 1, E and F). However, unlike BLP-tolerized THP-1 cells, which were effectively unresponsive when challenged with LPS or LPS plus BLP, LPS-tolerized cells still produced significant levels of TNF-α and IL-6 when challenged with BLP or BLP plus