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Induction of Tolerance to Lipopolysaccharide and Mycobacterial Components in Chinese Hamster Ovary/CD14 Cells Is Not Affected by Overexpression of Toll-Like Receptors 2 or 4^{1 ,2}

Andrei E. Medvedev^*, Philipp Henneke, Andra Schromm, Egil Lien, Robin Ingalls, Matthew J. Fenton, Douglas T. Golenbock and Stefanie N. Vogel^3,*

^* Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; Maxwell Finland Laboratory for Infectious Diseases, Boston Medical Center, Boston, MA 02118; Norwegian University of Science and Technology, Trondheim, Norway; and Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA 02118

	Abstract

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Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-B-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-B activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-B translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-B responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.

	Introduction

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Microbial infection activates cells of the host innate immune system that recognize conserved molecular structures common to a large group of pathogens (pathogen-associated molecular patterns, PAMPs)⁴ by a limited number of germline-encoded receptors (1). LPS, the major constituent of the outer membrane of Gram-negative bacteria, is an example of one such PAMP. LPS activates neutrophils, monocytes, macrophages, and other cell types to up-regulate expression of adhesion molecules, produce a number of pro- and anti-inflammatory cytokines, and secrete low m.w. proinflammatory mediators (2). Mycobacterium tuberculosis (Mtb) is the causative agent of pulmonary tuberculosis and represents an intracellular pathogen that is internalized primarily by macrophages. Several PAMPs have been identified in Mtb, including mycobacterial glycolipid lipoarabinomannan (LAM), soluble tuberculosis factor (STF), and lipopeptides (3, 4). Mtb is internalized primarily by macrophages, and this process is accompanied by secretion of a number of cytokines and chemokines, production of the reactive nitrogen intermediate, NO, and activation of the transcription factor NF-B (5). Recognition of LPS and several other bacterial products, including peptidoglycan (PGN), lipoteichoic acid (LTA), and LAM is mediated by CD14 (6, 7, 8). However, CD14 lacks transmembrane and intracellular domains necessary for signal transduction and is now generally believed to bind various PAMPs and present them to signal-transducing molecules that have been identified as belonging to the Toll-like receptor (TLR) family. Indeed, using fluorescence resonance energy transfer techniques, LPS was recently shown to trigger a physical association between CD14 and TLR4 (9).

TLR family members are transmembrane proteins consisting of an extracellular region that contains leucine-rich repeats flanked by cysteine-rich clusters, a transmembrane portion, and a cytoplasmic domain that is homologous to the signaling domain of the IL-1R type I (10). Toll was initially shown to control dorsoventral patterning during embryogenesis of Drosophila melanogaster (11) and to mediate antifungal immune response in the adult fly (12). Transfection of constitutively active Toll leads to NF-B activation, cytokine production, and expression of costimulatory molecules involved in the activation of adaptive immune response through a pathway homologous to the NF-B signaling cascade initiated by ligand engagement of the IL-1R type I (13). Ten mammalian TLRs have been cloned to date, of which only three (TLR2, TLR4, and TLR9) have known ligands (10, 13, 14, 15, 16, 17). A point mutation within the tlr4 gene in the C3H/HeJ mouse strain results in profound hyporesponsiveness to LPS (18, 19). Another LPS-refractory mouse strain, C57BL10/ScCr, was reported to have a deletion of the tlr4 gene (19, 20). These studies, combined with results demonstrating acquisition of LPS sensitivity upon TLR4 transfection of otherwise LPS-unresponsive cell lines (21, 22, 23), and data on LPS hyporesponsiveness of TLR4 knockout mice (24), strongly support the hypothesis that TLR4 is the primary signal-transducing receptor for enterobacterial LPS. LPS-mediated signaling through TLR4 requires concomitant expression of an accessory protein, myeloid differentiation protein-2 (MD-2), which physically associates with TLR4 (23, 25, 26, 27). TLR2 has also been reported to confer LPS responsiveness upon its transfection into LPS-unresponsive HEK 293 cells (28, 29). However, this effect was likely to be due to the presence of contaminating TLR2 ligands in commercial LPS preparations as repurification of LPS completely abolished its ability to activate TLR2-transfected cells, leaving TLR4-mediated responses intact (22). In addition, LPS evokes biological responses in Chinese hamster ovary (CHO)/CD14 cells that express endogenous Chinese hamster TLR4 but not TLR2 (30). In contrast, Dziarski et al. (31) have recently reported that overexpression of TLR2, MD-2, and CD14 conferred upon HEK 293 cells responsiveness to purified LPS or lipid A from Gram-negative bacteria and showed a physical association of MD-2 with either TLR2 or TLR4. Furthermore, recent data from our laboratory (32) and that of Ulevitch and colleagues (33) have also demonstrated that, in contrast to enterobacterial LPS, LPS purified from Porphyromonas gingivalis and Leptospira interrogans, respectively, signal via a TLR2-dependent mechanism. Together, these results indicate that some endotoxins can activate TLR proteins other than TLR4, although TLR4 appears to be the preeminent receptor for enterobacterial LPS in primary cells. Live Mtb have been demonstrated to signal via both TLR2 and TLR4, whereas their structural components, LAM and STF, activate cells through TLR2 only (3, 4). Using transfection approaches and knockout techniques, TLR2 was shown to function as a pattern recognition receptor for diverse bacteria and their products, including Gram-positive bacteria, PGN, spirochetal pathogens Borrelia burgdorferi and Treponema pallidum, as well as mycoplasma and their lipopeptides (3, 4, 34, 35, 36, 37, 38, 39). Signaling initiated by TLR2 and TLR4 involves several common intracellular signaling intermediates, including MyD88, IL-1R-associated kinases (IRAKs), and TNFR-associated factor (TRAF)-6 that are also engaged in IL-1R type I-initiated NF-B activation (40). However, LPS is capable of inducing delayed NF-B and mitogen-activated protein (MAP) kinase activation in macrophages derived from MyD88 knockout mice, indicating that additional MyD88-independent signal transduction pathways also exist (41).

Prior exposure to LPS significantly inhibits the ability of various cell types to respond to subsequent LPS challenge. This phenomenon, known as endotoxin tolerance, may protect the host from developing a shock syndrome caused by excessive production of inflammatory cytokines by host monocytes and macrophages hyperactivated by persisting bacteria. LPS tolerance results in decreased activation of MAP (42, 43, 44) and IB kinases (45), diminished expression and activation of IRAK-1 (46, 47), inhibited induction of transcription factors NF-B and AP-1 (44, 48), and suppressed gene expression and production of many cytokines, including TNF-, IL-1, IL-6, and IL-12 (reviewed in Ref. 49). However, expression of other mediators (e.g., IL-10, IL-1R antagonist, TNFRII, and NO) in LPS-tolerant cells is either unaffected or may even be enhanced (49), suggesting that endotoxin tolerance does not inhibit cellular functions globally, but rather, represents a "reprogramming" of cells, possibly as a means of adaptation to microbial infection (50). Although molecular mechanisms of tolerance to microbial products have been studied extensively, they still remain largely unknown. Inhibition of the LPS signaling cascade involves upstream signaling intermediates, e.g., decreased membrane GTP binding capacity and G protein content (51), and altered expression of phospholipase C-1 and phosphatidylinositol-3'-kinase (52). Whereas endotoxin tolerance does not affect cell surface expression of CD14 (53), LPS hyporesponsiveness induced by pretreatment of mouse peritoneal macrophages with LPS was recently reported to decrease cell surface expression of the TLR4-MD-2 receptor complex (54). In contrast, other results suggest that modulation of the downstream signal-transducing molecules of the TLR4 signaling pathway is responsible for the development of an LPS-tolerant state (44, 46, 47).

In the present work, we report that tolerance to LPS and mycobacterial products can be induced in CHO/CD14 cells stably transfected with human TLR2 or TLR4 under conditions where cell surface expression of these TLRs is not influenced by exposure to microbial stimuli. Similarly, prior exposure to endotoxin was found to inhibit LPS-mediated NF-B translocation significantly in CHO cells that concomitantly overexpressed TLR4 and MD-2, under conditions where cell surface expression of both TLR4 and MD-2 was not down-regulated. Furthermore, TLR4- (LPS) and TLR2- (LAM and STF) specific bacterial ligands were found to induce a state of cross-tolerance analogous to that induced by LPS and IL-1 in mouse macrophages (44). These data indicate that down-regulation of TLR4/MD-2 cell surface expression alone cannot account for the induction of tolerance to microbial pathogens, and suggest involvement of additional ancillary and/or downstream signaling intermediates.

	Materials and Methods

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Cells and reagents

CHO fibroblast cell line stably transfected with a NF-B-dependent reporter construct endothelial leukocyte adhesion molecule CD25, the hygromycin vector pCEP4 (EL-1 cells), and the EL-1/CD14 cell line (clone 3E10) were described previously (30, 34, 38, 55). Upon stimulation with LPS, TNF-, or IL-1, a NF-B-dependent reporter plasmid drives the expression of surface CD25 as a result of NF-B translocation. The 3E10/TLR2 cell line that expresses Flag-tagged TLR2 was engineered by stable transfection of the 3E10 reporter cell line with the cDNA for human TLR2 in the pFLAG-CMV-1 vector as detailed previously (34, 38). The EL-1/TLR4 and 3E10/TLR4 cell lines were obtained by stable transfection of cells with native human TLR4 cDNA inserted into the pcDNA3 expression vector (provided by Dr. R. Medzhitov, Yale University, New Haven, CT) by the calcium phosphate precipitation method (34, 38). Transfectants were selected in G418, and a clonal cell line was established subsequently by combining FACS cell sorting with the anti-TLR4 mAb HTA125 (a gift from Dr. K. Miyake, Saga Medical School, Saga, Japan) with limiting dilution cloning as described previously (34, 38). The EL-1/TLR4/MD-2 cell line, which concomitantly overexpresses native human TLR4 and Flag-tagged human MD-2, was generated by stable transfection of EL-1/TLR4 cells with Flag-tagged MD-2 cDNA cloned into the pEFBOS expression vector using XhoI/BamHI sites (provided by Dr. K. Miyake). Following cell sorting with M2 anti-Flag Ab, cell clones were obtained by a standard limiting dilution cloning technique and maintained in the presence of G418. 3E10 and EL-1 cells were cultured in Ham’s F12 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 400 U/ml hygromycin B (Calbiochem, San Diego, CA). EL-1/TLR4, EL-1/TLR4/MD-2, 3E10/TLR4, and 3E10/TLR2 cell clones were maintained in the same medium supplemented with 1 mg/ml G418 (Life Technologies, Gaithersburg, MD). Table I summarizes TLR expression patterns of the cell lines used in this study. Protein-free, phenol/water-extracted Escherichia coli LPS K235 was prepared according to the method of McIntire et al. (56). Arabinose-capped mycobacterial LAM was provided by Dr. J. Belisle (Colorado State University, Fort Collins, CO) under the provisions of National Institutes of Health Contract NO1 AI25147. Soluble H37Ra-conditioned culture supernatant, termed STF, was prepared as described (3). Levels of contaminating LPS in LAM and STF preparations were determined using a Limulus lysate assay (BioWhittaker) and were <1 pg/ml final concentration in all experiments. Anti-Flag M2 mAb, mouse IgG1, mouse IgG2a, and anti-mouse IgG (Fab-specific) FITC conjugate were purchased from Sigma (St. Louis, MO), recombinant murine TNF- was from R&D Systems (Minneapolis, MN), and anti-CD25 (anti-IL-2R) FITC conjugate was obtained from BD Biosciences (San Jose, CA). Anti-ACTIVE MAP kinase and c-Jun NH₂-terminal kinase (JNK) polyclonal Abs directed to phosphorylated forms of extracellular signal-regulated kinase (ERK) 1/2 and JNK 1/2, and donkey-anti-rabbit IgG (H + L) HRP conjugate were obtained from Promega (Madison, WI); phospho-p38 MAP kinase Ab and p38 MAP kinase Ab were purchased from New England Biolabs (Beverly, MA). Rabbit polyclonal IgG against IB- (C21) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cells were plated in 12-well tissue culture plates at a density of 3 x 10⁵ cells/well in respective selective medium and incubated overnight at 37°C in a 5% CO₂ atmosphere. Thereafter, cells were washed three times with PBS, resuspended in medium, and pretreated for 5 h with various bacterial components. Following three washes with PBS, cells were restimulated for 18 h with medium, LPS, or mycobacterial components, detached from the plastic with trypsin/EDTA, and examined by FACS analysis for the presence of surface CD25 as described (3, 4, 34, 38, 55). Flag-tagged proteins were detected by FACS analysis with anti-Flag M2 mAb as described (3, 4, 34, 38). Native human TLR4 expression was measured by staining cells for 45 min with anti-TLR4 mAb HTA125 or mouse IgG2a isotype control Ab (5 µg/ml each) on ice, washing twice with PBS/2% FCS, and incubating for 30 min with secondary FITC-labeled anti-mouse IgG (5 µg/ml). Thereafter, cells were subjected to flow cytometry analysis on an Epics-Coulter cell analyzer (Coulter, Miami, FL).

Preparation of nuclear extracts and EMSA

Cells were plated into six-well plates (Costar) at a density of 2 x 10⁶ cells/well, grown overnight, washed with PBS, and treated as described in the figure legends. Nuclear extracts were prepared as described previously (44). Protein concentration was determined using the Bio-Rad assay kit (Bio-Rad, Hercules, CA). The NF-B-specific oligonucleotide probe 5'-AGTTGAGGGGACTTTCCCAGGC-3' from the murine Ig-B light chain gene enhancer was synthesized by the BIC Synthesis and Sequencing Facility (Uniformed Services University of the Health Sciences, Bethesda, MD) and ³²P-end-labeled with T4 polynucleotide kinase (Promega) as recommended by the manufacturer. Nuclear extracts (4 µg) were incubated with 0.2 ng radiolabeled DNA probe in a binding buffer (final volume 20 µl) containing 2 µg poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ), 10 mM Tris-HCl (pH 7.9), 50 mM KCl, 4% glycerol, 1 mM DTT, and 0.25 mg/ml BSA for 30 min at room temperature. After incubation, a portion of each reaction (18 µl) was loaded onto a 5% nondenaturing polyacrylamide gel, and the DNA-protein complexes were resolved from free oligonucleotide by electrophoresis (0.25 x Tris borate/EDTA, 150 V, 2 h). The gels were dried (80°C, 2 h) and exposed to x-ray films (X-OMAT AR; Eastman Kodak, Rochester, NY).

Western blot analysis

Cellular extracts were prepared as described (57). Twenty micrograms of total protein was added in Laemmli buffer, boiled for 5 min, resolved by SDS-12% PAGE in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), and transferred onto Immobilon P transfer membranes (Millipore, Bedford, MA) (100 V, 1.5 h, 4°C). After blocking for 2 h in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk, membranes were washed three times in TBS-T and probed for 20 h at 4°C with the respective Abs (dilution 1/1000 in TBS-T/0.5% nonfat milk). Following washing in TBS-T, membranes were incubated with secondary HRP-conjugated, donkey anti-rabbit IgG (1/10,000 dilution) and washed five times in TBS-T, and bands were detected using ECL reagents (Amersham Pharmacia Biotech) according to manufacturer’s directions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell surface expression of transfected TLR4 and TLR2 is not affected by LPS

Induction of LPS tolerance has been reported to be accompanied by diminished staining of mouse macrophages with a mAb that detects an epitope defined by the interaction of TLR4 with MD-2 (54). Hence, we first sought to examine whether overexpression of TLR4 could compensate for such an LPS-mediated decrease in TLR4-MD-2 expression, resulting in a reduced ability of LPS to cause endotoxin tolerance. As TLR2 has been reported to confer responsiveness to a large variety of Gram-positive, fungal, and mycobacterial structures (3, 4, 34, 35, 36, 37, 38, 39), the effect of TLR2 overexpression on the induction of tolerance to LPS and mycobacterial components was also studied. As a model system, we used the CHO/CD14 fibroblast cell line that expresses a reporter plasmid comprised of the human CD25 reporter gene under the control of a NF-B-dependent endothelial leukocyte adhesion molecule-1 promoter (the 3E10 cell clone). This reporter cell line was further stably transfected with the plasmids containing either native human TLR4 cDNA or Flag-tagged human TLR2 cDNA under control of a strong, constitutively active, CMV promoter. Clonal 3E10/TLR4 and 3E10/TLR2 cell lines were obtained by combining cell sorting and limiting dilution cloning techniques. FACS analysis with anti-Flag M2 mAb or anti-TLR4 HTA125 mAb confirmed high levels of cell surface expression of the transfected TLRs, whereas the control cell line 3E10 showed no staining with either anti-Flag M2 (Fig. 1) or HTA125 mAb (data not shown). The expression of transfected TLRs in 3E10/TLR4 and 3E10/TLR2 cells was also confirmed by RT-PCR with primers specific for human TLR4 and TLR2 (data not shown). As measured by FACS analysis, treatment of 3E10/TLR4 or 3E10/TLR2 cells with LPS for 5 h did not alter cell surface levels of transfected TLRs when compared with those detected after treatment with medium alone (Fig. 1). Likewise, no change in the expression of transfected TLRs was seen after restimulation with LPS for 20 h or after pretreatment or restimulation with LAM or STF (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Cell surface expression of TLR4 and TLR2 in 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines following treatment with medium or LPS. 3E10, 3E10/TLR2, and 3E10/TLR4 cell clones were treated for 5 h with medium or 10 ng/ml LPS. Thereafter, cells were detached, stained with anti-TLR4 mAb HTA125, anti-FLAG mAb M2, or isotype-matched control Abs, and analyzed for cell surface expression of TLR4 and TLR2 by FACS. Thin dotted lanes show staining with isotype-matched control Abs; bold dotted and solid lanes represent TLR-specific staining of cells pretreated with medium or LPS, respectively. Shown are data of a representative experiment (n = 3).

MAP kinases play an important role in mediating many cell functions, including activation of various transcription factors and production of pro- and anti-inflammatory cytokines (58). Therefore, we sought to examine whether overexpression of TLR2 or TLR4 would affect the capacity of LPS to induce an endotoxin-tolerant state as reflected by decreased MAP kinase activation. Because phosphorylation of the MAP kinases closely correlates with their activation (59), MAP kinase phosphorylation was measured in cellular extracts by Western analysis with Abs specific for phosphorylated forms of the respective MAP kinases. Similar to our previous findings in mouse macrophages (44), stimulation of medium-pretreated 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines with 10 ng/ml LPS resulted in phosphorylation of the JNK 1/2 and p38 MAP kinases (Fig. 2). However, in contrast to macrophages, no ERK 1/2 phosphorylation was seen after treatment of any of these three cell lines with LPS (data not shown). These results are consistent with our previously published observation that LPS fails to induce tyrosine phosphorylation of proteins with molecular mass corresponding to ERK 1/2 MAP kinases in CHO/CD14 cells, whereas PMA induced a strong response (60). Overexpression of human TLR4 in 3E10 cells led to enhanced phosphorylation of JNK 1/2 and p38 MAP kinases in response to stimulation with 10 ng/ml LPS compared with the responses detected in the parental 3E10 cell line and in 3E10/TLR2 cells (Fig. 2). Pretreatment of 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines for 5 h with 10 ng/ml LPS led to a complete abrogation of the ability of these cells to respond to subsequent LPS challenge with phosphorylation of JNK 1/2 and p38 MAP kinases (Fig. 2). Comparable levels of total p38 MAP kinase expression were detected in all three cells lines that were not modulated by LPS (Fig. 2), indicating that the observed changes in MAP kinase phosphorylation were not due to unequal protein loading. Thus, overexpression of either TLR2 or TLR4 in 3E10 cells does not affect the capacity of LPS to induce an endotoxin-tolerant state at the level of JNK and p38 activation.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Comparison of LPS-mediated activation of the JNK 1/2 and p38 MAP kinases in the 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines pretreated with medium or endotoxin. Cells were pretreated for 5 h with either medium or 10 ng/ml LPS followed by washing with PBS and restimulation for 30 min with either medium or 10 ng/ml LPS. Cellular extracts were prepared, and MAP kinase phosphorylation was examined by Western analysis with Ab specific for either the phosphorylated (p) forms of the respective MAP kinases or with anti-total p38 Ab. The results of a representative experiment are presented (n = 6).

Endotoxin pretreatment inhibits LPS-mediated NF-B activation in 3E10, 3E10/TLR2, and 3E10/TLR4 cell clones

The next experiments were conducted to examine whether overexpression of TLR2 or TLR4 affects the induction of endotoxin tolerance, as measured by suppressed NF-B activation in response to LPS challenge. We first assessed the effect of LPS pretreatment on LPS-mediated degradation of IB- in 3E10, 3E10/TLR2, and 3E10/TLR4 cell clones. Stimulation of medium-pretreated 3E10, 3E10/TLR2, and 3E10/TLR4 cells for 30 min with 1 ng/ml LPS resulted in a partial degradation of IB-, whereas a complete degradation was reached when LPS was used at a concentration of 10 ng/ml (Fig. 3). In contrast, LPS pretreatment of all three cell lines almost completely abolished LPS-induced IB- degradation (Fig. 3). To confirm and extend these results, we examined LPS-induced NF-B activation in medium- or LPS-pretreated 3E10, 3E10/TLR2, and 3E10/TLR4 cells based on NF-B DNA binding activity and on the ability to transactivate NF-B-dependent expression of the CD25 reporter gene. As depicted in Fig. 4, EMSA analyses of 3E10 and 3E10/TLR2 cells showed a suboptimal NF-B response following their stimulation with 0.1 ng/ml LPS, which reached a plateau at 10 and 100 ng/ml LPS. Overexpression of TLR4 enhanced LPS-mediated NF-B translocation 10-fold, because as little as 1 ng/ml LPS was required to induce maximal levels of NF-B translocation in the 3E10/TLR4 cell line (Fig. 4). Pretreatment with LPS led to a >100-fold shift of the LPS dose-response curve to the right in all three cell lines (Fig. 4), indicating a significant decrease in LPS-induced NF-B translocation. In 3E10, 3E10/TLR4 (data not shown), and 3E10/TLR2 (Fig. 8) cells, TNF--mediated NF-B translocation was not influenced by prior exposure to LPS, and, conversely, pretreatment of these cell lines with TNF- did not affect LPS-induced NF-B translocation (data not shown). To measure the transactivating potential of NF-B, CD25 cell surface expression was subsequently measured in either the parental 3E10 cell line or in 3E10/TLR2 and 3E10/TLR4 clones. LPS stimulation of medium-pretreated 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines resulted in a potent induction of CD25 cell surface expression (Fig. 5, A–C, top). In all three cell lines, prior exposure to LPS led to significantly diminished expression of membrane CD25 in response to LPS restimulation (Fig. 5, A–C, bottom), consistent with suppressed LPS-mediated IB- degradation and NF-B translocation observed in LPS-tolerant cells. Notably, TNF--induced NF-B-dependent CD25 cell surface expression was similar in these cell lines and was not affected by LPS pretreatment (data not shown), indicating specific down-regulation of the LPS signaling pathways in LPS-tolerant cells. Consistent with suppressed NF-B activation observed in 3E10, 3E10/TLR2, and 3E10/TLR4 cells, LPS pretreatment also led to a marked inhibition of LPS-inducible endogenous IL-6 gene expression in all three cell lines (data not shown). Thus, overexpression of TLR2 or TLR4 in 3E10 cells does not affect the capacity of LPS to induce a state of endotoxin tolerance.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Effect of LPS pretreatment on LPS-mediated IB- degradation in 3E10, 3E10/TLR2, and 3E10/TLR4 cell clones. Cells were pretreated for 5 h with medium or 10 ng/ml LPS, washed with PBS, and restimulated for 30 min with either medium or LPS. Thereafter, cellular extracts were prepared and assayed for the expression of IB- by Western analysis. The results of a representative experiment are shown (n = 5).

View larger version (64K): [in this window] [in a new window]   FIGURE 4. LPS-mediated NF-B translocation in 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines pretreated with medium or LPS. Following pretreatment for 5 h with medium or 10 ng/ml LPS, 3E10, 3E10/TLR2, and 3E10/TLR4 cells were washed with PBS and restimulated with medium or indicated concentration of LPS for 45 min. Cells were then harvested, and nuclear extracts were prepared as described in Materials and Methods. NF-B DNA-binding activity was analyzed by EMSA with a 32P-labeled, NF-B-specific oligonucleotide probe. The data from a representative experiment are shown (n = 8).

View larger version (38K): [in this window] [in a new window]   FIGURE 8. Pretreatment of the 3E10/TLR2 cell line with LPS, LAM, or STF down-regulates NF-B translocation in response to these bacterial compounds. A, 3E10 and 3E10/TLR2 cell lines were pretreated for 5 h with medium, 10 ng/ml LPS, or 1 µg/ml LAM, washed with PBS, and incubated for 45 min with medium, 10 ng/ml LPS, or 1 µg/ml LAM. B, Following incubation for 5 h with medium, 10 ng/ml LPS, or 10 µl/ml STF, 3E10/TLR2 and 3E10 cells were washed with PBS and restimulated for 45 min with medium, 10 ng/ml LPS, 50 ng/ml TNF- or 10 µl/ml STF. Thereafter, nuclear extracts were prepared, and NF-B translocation was assessed by EMSA. The results of a representative experiment are shown (n = 3).

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Analysis of LPS-induced NF-B-dependent CD25 cell surface expression in 3E10, 3E10/TLR2, and 3E10/TLR4 cell lines pretreated with medium or LPS. 3E10 (A), 3E10/TLR2 (B), and 3E10/TLR4 (C) cell lines were pretreated for 5 h with medium or 1 ng/ml LPS, washed with PBS, and restimulated for 20 h with either medium (dotted lines) or 10 ng/ml LPS (solid lines). Thereafter, the cells were detached, stained with FITC-labeled anti-CD25 mAb, and subjected to flow cytometry analysis to measure CD25 expression. The results of a representative experiment are depicted (n = 6).

Thus far, our data suggest that down-regulation of TLR4 expression is not the mechanism underlying early endotoxin tolerance. Because the previously published results implying this mechanism were obtained with an Ab that detected a TLR4/MD-2-shared epitope (54), we next sought to determine whether overexpression of MD-2 would diminish the ability of TLR4-expressing cells to become tolerant to LPS. To this end, CD14-negative CHO cells were stably transfected with native human TLR4 and Flag-tagged human MD-2 and their LPS responses were compared with those seen in the parental EL-1 cell line or in EL-1 cells engineered to overexpress human TLR4 only. Cell surface expression of TLR4 was clearly detectable in both EL-1/TLR4 and EL-1/TLR4/MD2 cells by FACS analysis with anti-TLR4 mAb HTA125, whereas staining with anti-Flag Ab M2 verified that MD-2 was expressed only on the cell surface of the EL-1/TLR4/MD-2 cell line (Fig. 6, A and B, top). Interestingly, concomitant expression of TLR4 and MD-2 significantly enhanced the fluorescence intensity of TLR4 compared with that measured in EL-1/TLR4 cells. Functional analyses demonstrated that in the absence of CD14, LPS failed to induce NF-B translocation in the parental EL-1 cell line even when used at a concentration as high as 1 µg/ml (data not shown). Overexpression of TLR4 in CD14-negative CHO cells enabled NF-B activation in response to 1000 ng/ml LPS, whereas cotransfection of TLR4 and MD-2 conferred upon CHO cells the ability to respond to as little as 1 ng/ml LPS (Fig. 6, A and B, bottom). Fig. 6, A and B, shows that pretreatment of both EL-1/TLR4 and EL-1/TLR4/MD2 cells with 10 ng/ml LPS for 5 h did not significantly modulate expression of TLR4 or MD-2, whereas LPS-mediated NF-B activation was dramatically inhibited in both cell lines, indicating the induction of endotoxin tolerance. In contrast, stimulation with TNF- resulted in comparable NF-B translocation in both cell lines regardless of whether they were pretreated with LPS or medium (Fig. 6, A and B, bottom), demonstrating specific inhibition of the LPS signaling pathway in endotoxin-tolerant cells. These data indicate that concomitant expression of TLR4 and MD-2 in CHO cells failed to prevent the induction of endotoxin tolerance.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Pretreatment of EL-1/TLR4 and EL-1/TLR4/MD-2 cell lines with LPS does not modulate the expression of TLR4 or MD-2, whereas it significantly inhibits NF-B activation in response to LPS, but not to TNF-. EL-1/TLR4 (A) and EL-1/TLR4/MD-2 (B) cells were pretreated for 5 h with either medium or 10 ng/ml LPS, washed three times with PBS, and resuspended in medium. Thereafter, cells were analyzed for cell surface expression of TLR4 and MD-2 by FACS with anti-TLR4 mAb HTA125 or anti-Flag mAb M2 followed by staining with FITC-conjugated anti-mouse IgG (top), or for NF-B activation in response to LPS or 50 ng/ml TNF- by EMSA (bottom). The results of a representative experiment (n = 3) are shown.

Next, we examined whether the mycobacterial TLR2 ligands, LAM and STF, can elicit tolerance against themselves in 3E10 cells that overexpress human TLR2. In addition, the abilities of TLR4- (LPS) and TLR2- (LAM, STF) specific bacterial products to induce cross-tolerance were studied. Neither LAM (Fig. 7A) nor STF (data not shown) induced NF-B-dependent CD25 expression in the 3E10/TLR4 cell line that expresses functional TLR4, but not TLR2 (30), whereas strong NF-B activation was seen in response to LPS. Similarly, no NF-B translocation was elicited by LAM or STF in the parental 3E10 cell line, which lacks functional endogenous TLR2, whereas marked NF-B activation was induced by both LPS and TNF- (Fig. 8, A and B). Confirming previously published observations (3, 4, 34), overexpression of TLR2 rendered 3E10 cells sensitive to both LAM and STF, as evidenced by NF-B-dependent CD25 expression (Fig. 7B and data not shown) and NF-B translocation (Fig. 8, A and B). Pretreatment of 3E10/TLR2 cells with LPS, LAM, or STF for 5 h reduced their capacity to exhibit NF-B responses upon restimulation with any of these bacterial structures (Figs. 7B and 8, A and B). Of note, the induction of cross-tolerance was observed despite the failure of these bacterial products to affect cell surface expression of TLR2 (Fig. 1 and data not shown). In contrast, prior exposure of the 3E10/TLR4 cell line to LAM or STF did not change LPS-mediated CD25 cell surface expression (Fig. 7A and data not shown) or NF-B translocation (Fig. 8, A and B), whereas LPS pretreatment markedly decreased NF-B activation induced by subsequent LPS challenge. LPS pretreatment of 3E10/TLR2 cells significantly inhibited NF-B activation in response to subsequent stimulation with LPS, LAM, or STF, whereas pretreatment with LAM or STF suppressed LPS-mediated NF-B activation to a lesser extent than that induced by restimulation with LAM or STF (Figs. 7B and 8, A and B). Thus, pretreatment of 3E10/TLR2 cells with either a TLR4-specific bacterial ligand (LPS) or TLR2-specific mycobacterial components, LAM and STF, results in the development of cross-tolerance.

View larger version (36K): [in this window] [in a new window]   FIGURE 7. LPS and LAM cross-tolerize for NF-B transactivation in 3E10/TLR2 cells. The 3E10/TLR4 (A) and 3E10/TLR2 (B) cells were pretreated for 5 h with medium, 1 ng/ml LPS, or 1 µg/ml LAM. Thereafter, cells were washed with PBS and restimulated for 20 h with medium, 10 ng/ml LPS, and 1 µg/ml LAM followed by FACS analysis of CD25 cell surface expression. The results of a representative experiment are given (n = 3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the current study, we reasoned that if tolerance to microbial pathogens occurs mainly due to down-regulation of their signaling receptors, then overexpression of TLR2 or TLR4 should reduce the ability of TLR2- and TLR4-specific bacterial products to induce tolerance. To address this question, clonal 3E10/TLR2 and 3E10/TLR4 cell lines were obtained following stable transfection of the parental 3E10 cell clone with expression plasmids that contain human TLR2 or TLR4 cDNA under the control of a strong constitutively active CMV promoter. In line with previously published observations (3, 4), transfection of TLR2 into 3E10 cells, which express functional endogenous TLR4 but not TLR2 (30), imparted responsiveness to TLR2-specific mycobacterial components LAM and STF. Proper functional activities of transfected TLR4 were evidenced by increased LPS sensitivity of 3E10/TLR4 transfectants, confirming earlier reports on augmentation of cell responsiveness to LPS upon an increase in expression levels of cell surface TLR4 (61, 62). Importantly, FACS analyses showed that LPS, LAM, or STF did not influence cell surface expression of transfected TLR2 or TLR4. Thus, even if LPS might transiently decrease expression of endogenous TLR4, the preponderance of heterologously expressed human TLR4 molecules, whose expression is not affected by LPS, would ensure proportionately lower down-regulation of total TLR4 in 3E10/TLR4 cells compared with the 3E10 cell line. In turn, this should result in a marked difference in the capacities of 3E10/TLR4 and 3E10 cell lines to become tolerant to LPS. In contrast, the absence of functional, endogenous TLR2 in 3E10 cells creates a situation where induction of tolerance to TLR2-specific mycobacterial products would occur despite their failure to affect cell surface expression of transfected human TLR2.

A number of observations presented herein suggest that tolerance to LPS and mycobacterial products can be induced independently of down-regulation of cell surface expression of TLR2 and TLR4. First, both the parental 3E10 cell line and 3E10 cell clones that overexpress TLR2 or TLR4 showed comparable inhibition of various LPS-induced responses following LPS pretreatment, including MAP kinase phosphorylation, NF-B activation, and IL-6 gene expression. Second, not only LPS, but also TLR2-specific LAM and STF, rendered 3E10/TLR2 cells tolerant to these microbial products. These data extend a previously reported observation on the induction of LAM tolerance in human monocytes by LAM (63) and demonstrate that in 3E10/TLR2 cells, tolerance to LAM or STF can be induced despite the failure of mycobacterial products to affect TLR2 expression. Third, LPS, LAM, and STF were found to induce cross-tolerance in the 3E10/TLR2 cell line. Similar findings have been reported in other models of cross-tolerance using distinct TLR2 bacterial ligands, including muramyl dipeptide and mycoplasma-derived macrophage-activating lipopeptide, 2 kDa (MALP-2) (64, 65). Furthermore, Lehner et al. (66) have recently demonstrated the induction of cross-tolerance in mouse macrophages by LPS and highly purified LTA that have been shown in this report to act via TLR4 and TLR2, respectively. Our results extend these observations and indicate that engagement of the TLR2 signaling pathway by prior exposure to mycobacterial products that "bypass" the TLR4 receptor stage inhibits TLR4-mediated signal transduction events initiated by LPS. Fourth, this study shows that prior exposure of 3E10/TLR2 cells to LPS down-regulated NF-B activation induced not only by LPS, but also by the TLR2 ligands LAM and STF. Of note, LPS pretreatment caused comparable decreases in NF-B activation in response to LPS, STF, or LAM, whereas the TLR2-specific mycobacterial products potently induced tolerance against themselves and only partially inhibited LPS-mediated NF-B translocation. Similar to our data, prior exposure of mouse macrophages to LPS has been shown to suppress significantly both LPS- and LTA-induced TNF- release, whereas LTA pretreatment strongly inhibited TNF- production in response to LTA but was a weaker inducer of LPS tolerance (66). Sato et al. (65) also reported that LPS pretreatment rendered mouse macrophages tolerant to LPS and to MALP-2, although in their system, LPS-mediated induction of tolerance to MALP-2 was less effective compared with significant suppression of LPS responses observed in endotoxin-tolerant cells. The reason for these quantitative differences in the induction of cross-tolerance among LPS and various TLR2 ligands (LAM, STF, LTA, and MALP-2) is unclear but might be related to their differential use of other TLRs that could associate with TLR2 or TLR4. Indeed, a dominant-negative mutant of TLR6 was reported to inhibit TNF- production in response to TLR2-specific PGN, but not to another TLR2 ligand, the synthetic tripalmitoylated lipopeptide Pam₃CSK₄ (67).

MD-2 is required for enabling LPS-mediated signal transduction via TLR4 (23, 25, 26, 27). Hence, LPS tolerance may occur due to down-regulation of endogenous MD-2, leading to suppression of TLR4 signal transduction. However, if this were the case, then overexpression of MD-2, together with TLR4, would be predicted to inhibit the induction of LPS tolerance. To examine this possibility, CD14-negative CHO cells (the EL-1 cell clone) were stably transfected with either native human TLR4 alone or concomitantly with TLR4 and MD-2, and the effect of LPS pretreatment on the expression of TLR4 and MD-2, as well as on functional responses of transfected cell lines, was evaluated. FACS analyses of the EL-1/TLR4/MD-2 cell line revealed significantly higher fluorescence intensity of TLR4 compared with that detected in the EL-1/TLR4 cells, extending similar results obtained from a comparative analysis of human HEK 293/TLR4/MD-2 vs 293/TLR4 cells (31, 68). The mechanism by which MD-2 expression facilitates the detection of TLR4 is unknown. It is interesting that a MD-2 related protein, MD-1, binds to RP105, a member of the TLR family expressed on B lymphocytes (69), and is thought to regulate RP105 cell surface expression (70). Analogous to MD-1, MD-2 may bind to TLR4, stabilizing its expression on the cell surface, which could facilitate TLR4 detection and signal transduction. To study consequences of overexpression of TLR4 and MD-2 on cell responsiveness to LPS, we undertook EMSA analysis of NF-B translocation. In the absence of CD14, LPS failed to induce NF-B translocation in EL-1 cells despite the presence of endogenous TLR4 (data not shown), consistent with an important role for membrane CD14 in mediating LPS responsiveness (7). Even though cells were stimulated in the presence of FCS that contains soluble CD14, soluble CD14 does not confer LPS sensitivity upon CHO cells (60). In agreement with previously published data in HEK 293 cells (21), overexpression of TLR4 alone in CHO cells induced NF-B translocation only in response to high concentrations of LPS, whereas cotransfection of TLR4 together with MD-2 significantly enhanced LPS-mediated NF-B activation. Of importance, LPS pretreatment markedly suppressed LPS-mediated NF-B translocation in both EL-1/TLR4 and EL-1/TLR4/MD-2 cell lines despite the failure of LPS to decrease cell surface expression of either TLR4 or MD-2. It could be argued that stable transfection of CHO cells with TLR4 and MD-2 may produce a large number of these molecules that are not down-regulated by LPS and are expressed on the cell surface without being associated in a functional signal-transducing complex. However, MD-2 is a secreted protein that has neither a transmembrane domain nor GPI linkage capable of anchoring this protein to the cell surface (25, 27). Therefore, MD-2 would be predicted to be present on the cell surface only if associated with TLR4, assuming that TLR4 is its only binding partner. Indeed, when MD-2 was expressed alone, its cell surface expression was undetectable by FACS, whereas cotransfection of MD-2 along with TLR4 resulted in MD-2 cell surface expression (25). Furthermore, several reports have demonstrated coimmunoprecipitation of MD-2 with TLR4 (27, 31, 68). Using coimmunoprecipitation, we have also been able to show physical interaction of TLR4 with MD-2 in EL-1/TLR4/MD-2 cells (data not shown). Altogether, our results indicate that endotoxin tolerance can be induced in CHO cells that overexpress TLR2, TLR4, or TLR4 plus MD-2, whose expression levels are not subject to LPS regulation. Thus, it seems unlikely that down-regulation of TLR4/MD-2 complexes only underlies the phenomenon of endotoxin tolerance. Indeed, we demonstrated previously that LPS and IL-1 induce cross-tolerance in mouse macrophages (44), yet IL-1 pretreatment does not decrease TLR4-MD-2 cell surface expression (54). As IL-1 and LPS signal through different receptors but share many downstream signaling intermediates (71), it suggests the existence of additional mechanisms of endotoxin tolerance. Furthermore, if the mechanism of LPS tolerance were due solely to a decrease in the number of TLR4 and/or MD-2, then all LPS responses should be inhibited in endotoxin-tolerant cells, as TLR4 has been unambiguously demonstrated as the principal LPS receptor in mammals (18, 19, 20, 21, 22, 23, 24, 25, 26, 27). This is not the case, because a number of cellular responses are not affected or even enhanced in LPS-tolerant cells (reviewed in Ref. 49). In contrast, induction of LPS cross-tolerance by mycobacterial products could result from a decrease in cell surface expression of endogenous TLR4 and/or MD-2 following pretreatment of 3E10/TLR2 cells with LAM or STF, providing that cross-talk between the TLR2 and TLR4 signaling pathways exists. However, Sato et al. (65) found that prior exposure of mouse macrophages to TLR2-specific MALP-2 failed to decrease the expression of TLR4-MD-2 complex. In addition, overexpression of MD-2 in RAW 264.7 macrophages did not affect the ability of the TLR2-reactive mycobacterial component LAM to elicit cellular responses, while greatly enhancing cell sensitivity to LPS (72). Furthermore, a LPS-unresponsive CHO cell clone that expresses a mutant MD-2 protein was found to respond normally to Mtb (72). Therefore, although MD-2 may promote optimal signal transduction by LPS and PGN (23, 25, 26, 27, 31), it does not appear to influence TLR2-mediated cellular responses to mycobacterial components. Taken together, our results further argue against the hypothesis that tolerance to mycobacterial pathogens occurs due to down-regulation of TLR2 or MD-2 expression.

We propose that tolerance to bacterial products targets intracellular signaling intermediates downstream of TLRs. Following activation of TLR2 or TLR4, a common set of signaling intermediates participates in mediating NF-B activation, including MyD88, IRAK-1, TRAF-6, evolutionary conserved signaling intermediate in Toll pathways, NF-B-inducing kinase, and IB kinases (24, 29, 39, 40, 41, 73, 74). Decreased activity of IRAK-1 has been reported in LPS-tolerant THP-1 human monocytic cells (47) and mouse Kupffer cells (46). Furthermore, IRAK-1 failed to associate with MyD88 in THP-1 cells pretreated with LPS, whereas the association of phosphorylated IRAK-1 with TRAF-6 was not affected (47), suggesting IRAK-1 as an attractive target for down-regulation as a consequence of LPS tolerance. Expression and functions of other downstream signaling molecules, e.g., MyD88, TRAF-6, and evolutionary conserved signaling intermediate in Toll pathways, could also be inhibited in cells rendered tolerant to LPS or TLR2-specific mycobacterial ligands. In contrast, some responses that are not dependent on MyD88 or IRAK-1 (41) could be even enhanced in tolerant cells, whereas MyD88- and IRAK-1-dependent responses may be down-regulated. Studies are in progress to evaluate this theory and to identify downstream signaling intermediates involved in the mechanisms of tolerance to LPS and mycobacterial components.

	Acknowledgments

 
We are thankful to Dr. Sachiko Akashi and Kensuke Miyake (Saga Medical School, Saga, Japan) for kindly providing us with the anti-TLR4 mAb HTA125 and the MD-2 expression vector.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HL55681 (to M.J.F.), GM54060 (to D.T.G.), and AI-18797 (to S.N.V.), and by Deutsche Forschungsgemeinschaft (HE 3127/1-1 to P.H. and DFG Schr 621/1-1 and SFB to A.S.).

² The opinions or assertions contained within are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense.

³ Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address: vogel{at}bob.usuhs.mil

⁴ Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; Mtb, Mycobacterium tuberculosis; LAM, lipoarabinomannan; STF, soluble tuberculosis factor; MD-2, myeloid differentiation protein-2; LTA, lipoteichoic acid; PGN, peptidoglycan; IRAK, IL-1R-associated kinase; TRAF, TNFR-associated factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; MALP-2, macrophage-activating lipopeptide, 2 kDa; CHO, Chinese hamster ovary.

Received for publication January 5, 2001. Accepted for publication June 21, 2001.

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