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1 The Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan
2 The Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
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Abstract |
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(TNF-) or interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [poly(I : C)] in RAW264.7 macrophages. In contrast, expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region did not promote such responses. Expression of SHPS-1–4F promoted the LPS- or poly(I : C)-induced activation of NF-
B. LPS and poly(I : C) each induced the tyrosine phosphorylation of SHPS-1 through a Src family kinase and the association of SHPS-1 with SHP-1 and SHP-2. These results suggest that LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with SHP-1 and SHP-2 in a manner dependent on a Src family kinase. SHPS-1 then negatively regulates TLR4- or TLR3-dependent cytokine production through inhibition of NF-B-dependent signaling. |
Introduction |
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(TNF-) and interleukin (IL)-6 as well as type-1 interferon (Liew et al. 2005). Such cytokine production by macrophages is promoted by Toll-like receptors (TLRs), which recognize conserved components of microbes such as lipopolysaccharide (LPS), double-stranded RNA and bacterial DNA (Barton & Medzhitov 2003; Akira & Takeda 2004; Liew et al. 2005). In response to binding of such ligands, most members of the TLR family, with the notable exception of TLR3, initiate a signaling cascade that includes myeloid differentiation 88 (MyD88), IL-1 receptor-associated kinase, TNF-associated factor 6 and transforming growth factor-β-activated kinase-1 (TAK1) (Barton & Medzhitov 2003; Akira & Takeda 2004; Liew et al. 2005). This latter kinase (TAK1) is thought to promote activation of downstream signaling molecules including nuclear factor-B (NF-B) as well as mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (Erk), p38 and c-Jun NH2-terminal kinase (JNK), thereby resulting in the production of proinflammatory cytokines. Moreover, TLR4 (a receptor for LPS) and TLR3 (a receptor for double-stranded RNA) recruit Trif (Toll/IL-1 receptor domain-containing adapter-inducing interferon-β) to their cytoplasmic domains and thereby promote production of proinflammatory cytokines in a MyD88-independent manner (Oshiumi et al. 2003; Yamamoto et al. 2003). Although the signal transduction underlying the TLR-stimulated production of proinflammatory cytokines by macrophages has been relatively well characterized, the mechanism for negative regulation of such signaling, including the possible role of tyrosine phosphorylation and dephosphorylation, has remained unknown.
Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) (Fujioka et al. 1996), also known as signal-regulatory protein (Kharitonenkov et al. 1997; van Beek et al. 2005) or BIT (Ohnishi et al. 1996), is a transmembrane protein whose extracellular region comprises three immunoglobulin (Ig)-like domains and whose cytoplasmic region contains immunoreceptor tyrosine-based activation motifs that mediate association with the protein tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors and cytokines as well as by integrin-mediated cell adhesion to extracellular matrix proteins (Timms et al. 1998; Oshima et al. 2002). SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli. Moreover, SHPS-1, through its extracellular region, interacts with the transmembrane protein CD47 (Jiang et al. 1999; Seiffert et al. 1999). CD47, which was originally identified in association with vβ3 integrin, is also a member of the Ig superfamily of proteins, possessing an Ig-V-like domain in its extracellular region (Brown & Frazier 2001). Among hematopoietic cells, SHPS-1 is especially abundant in macrophages, dendritic cells and neutrophils, being barely detectable in T or B lymphocytes (Adams et al. 1998; Veillette et al. 1998; Seiffert et al. 1999, 2001; Latour et al. 2001).
SHPS-1 plays negative roles in the hematopoietic and immune systems. Indeed, SHPS-1 expressed on the surface of macrophages, through its interaction with CD47 on red blood cells, is thought to prevent phagocytosis by the former cells of the latter through activation of SHP-1 (Oldenborg et al. 2000; Okazawa et al. 2005; Ishikawa-Sekigami et al. 2006). Such regulation is thought to be a determinant both of the life span of individual red blood cells and of the number of circulating erythrocytes. We have recently shown that mice that express a mutant version of SHPS-1 that lacks most of the cytoplasmic region (Okazawa et al. 2005; Ishikawa-Sekigami et al. 2006) are markedly resistant to experimental autoimmune encephalomyelitis as well as to contact hypersensitivity to 2,4-dinitro-1-fluorobenzene (Fukunaga et al. 2006; Tomizawa et al. 2007). Moreover, dendritic cells derived from these SHPS-1 mutant mice are defective in priming of CD4+ T cells for generation of IL-17-producing CD4+ T cells, suggesting that SHPS-1 is essential for priming of T cells by dendritic cells and for the development of autoimmune diseases. However, the role of SHPS-1 in the TLR-stimulated production of proinflammatory cytokines by macrophages has not previously been investigated.
We have thus now examined the effects of forced expression of mutant versions of SHPS-1 that lack either the tyrosine phosphorylation sites (SHPS-1–4F) or almost the entire cytoplasmic region (SHPS-1-
cyto) on TLR-dependent production of proinflammatory cytokines in RAW264.7 mouse macrophage cells.
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Results |
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cyto on TLR-induced production of TNF- or IL-6 by RAW264.7 cells
To investigate the role of SHPS-1 in regulation of TLR-stimulated production of proinflammatory cytokines, we studied RAW264.7 cell lines that we had generated previously (Ikeda et al. 2006). These cell lines express either SHPS-1–4F (4F cells) or SHPS-1-cyto (cyto cells); all four tyrosine residues (Tyr436, Tyr460, Tyr477, Tyr501) in the cytoplasmic region are replaced by phenylalanine in SHPS-1–4F, whereas SHPS-1-cyto (amino acids 1–404) lacks almost the entire cytoplasmic region (Fig. 1A). Several cell lines expressing SHPS-1–4F or SHPS-1-cyto were obtained, from which we chose two 4F lines (clones 12 and 28) as well as two cyto lines (clones 8 and 11) for further analysis. Immunoblot analysis of cell lysates with the p84 mAb to the extracellular region of SHPS-1 revealed that the total amount of mouse SHPS-1 protein (both endogenous and exogenous) in 4F cells or cyto cells was markedly greater than that in parental RAW264.7 cells (Ikeda et al. 2006) (data not shown). In addition, the abundance of SHP-1 or SHP-2 was not altered by forced expression of either SHPS-1–4F or SHPS-1-cyto (Ikeda et al. 2006) (data not shown).
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and IL-6 in a concentration-dependent manner (Fig. 1B). The LPS-induced production of TNF- in the two 4F cell lines was markedly greater than that in the parental cells (Fig. 1B). In contrast, the LPS-stimulated production of TNF- in cyto cells was similar (clone 11) or slightly decreased (clone 8), compared with that apparent in the parental cells (Fig. 1B). The LPS-induced production of IL-6 in the two 4F cell lines was similar to that in parental cells, whereas that in cyto cells was similar (clone 11) or slightly decreased (clone 8) (Fig. 1B).
We next examined the effects of poly(I : C) or CpG ODN on the production of TNF- and IL-6 by the various cell lines. The poly(I : C)-stimulated production of TNF- and IL-6 in the two 4F cell lines was substantially greater than that in the parental cells (Fig. 2A). Such enhanced responses to poly(I : C) were not apparent in the two cyto cell lines (Fig. 2A). The CpG ODN-induced production of TNF- in the two 4F cell lines was slightly greater than that in the parental cells, whereas the CpG ODN-stimulated production of TNF- in cyto cells was also similar (for clone 11) or slightly decreased (for clone 8), compared with that apparent with the parental cells (Fig. 2B). The CpG ODN-stimulated production of IL-6 was decreased in the two 4F cell lines and unchanged in the two cyto cell lines compared with that apparent in the parental cells (Fig. 2B). Given that LPS and poly(I : C) are ligands for TLR4 and TLR3, respectively (Poltorak et al. 1998; Hoshino et al. 1999; Alexopoulou et al. 2001), these data suggested that expression of SHPS-1–4F promotes TLR3-dependent production of TNF- and IL-6 as well as TLR4-dependent production of TNF- by RAW264.7 cells.
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cyto cells were similar to those in parental cells (data not shown), suggesting that the increase in TLR3- or TLR4-dependent production of proinflammatory cytokines observed in 4F cells is not simply attributable to an increased expression level of TLR3 or TLR4 genes.
Effects of expression of CD8–4F on TLR3- or TLR4-induced TNF- production by RAW264.7 cells
Expression of SHPS-1–4F, but not that of SHPS-1-cyto, markedly promoted LPS- or poly(I : C)-stimulated production of proinflammatory cytokines in RAW264.7 cells. We thus next examined CD8–4F cells, which we previously generated and which express a chimeric protein comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1–4F (Ikeda et al. 2006) (Fig. 1A). The production of TNF- stimulated by LPS (Fig. 3A) or by poly(I : C) (Fig. 3B) was substantially greater in two CD8–4F cell lines than in the parental cells. These results suggested that expression of the cytoplasmic region of SHPS-1–4F is sufficient for the promotion of LPS- or poly(I : C)-stimulated production of TNF- in RAW264.7 cells.
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B and the activity of NF-B in LPS- or Poly(I : C)-stimulated RAW264.7 cells
The activation of NF-B in signaling downstream of TLRs is essential for the TLR-induced production of proinflammatory cytokines (Barton & Medzhitov 2003; Akira & Takeda 2004; Liew et al. 2005). In resting cells, NF-B is present in the cytosol as an inactive complex with its inhibitor protein, IB. Exposure of cells to TLR ligands induces the phosphorylation, ubiquitination and proteasome-mediated degradation of IB. The liberated NF-B then translocates to the nucleus, where it activates the transcription of cytokine genes (Barton & Medzhitov 2003; Akira & Takeda 2004; Liew et al. 2005). To investigate further the molecular mechanism by which SHPS-1–4F promotes LPS- or poly(I : C)-stimulated production of proinflammatory cytokines, we examined the abundance of IB in 4F cells or cyto cells stimulated by TLR ligands. In parental RAW264.7 cells, the amount of IB was greatly decreased 30 min after the onset of stimulation with LPS, suggesting that IB was degraded in response to LPS stimulation, but it had returned to its prestimulation level by 60 min after exposure to LPS (Fig. 4A and B). The level of IB in unstimulated 4F cells was decreased compared with that in parental cells, and the LPS-induced down-regulation of IB in 4F cells was sustained even at 60 min after the onset of stimulation (Fig. 4A and B). In contrast, the pattern of LPS-induced changes in the abundance of IB in cyto cells was similar to that in parental cells (Fig. 4A and B). Similarly, the poly(I : C)-induced decrease in the amount of IB was sustained even at 60 min after the onset of stimulation in 4F cells but not in cyto cells (Fig. 4A and B). These results suggested that the activity of NF-B would be increased in LPS- or poly(I : C)-stimulated or unstimulated 4F cells compared with that in parental cells.
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B in RAW264.7 cell lines. The activity of NF-B was evaluated by measurement of the DNA binding activity of the p65 subunit of NF-B in nuclear extracts with a plate-bound oligonucleotide containing a specific DNA sequence targeted by phosphorylated p65. The activity of NF-B in 4F cells was markedly increased compared with that in parental cells under both basal and LPS- or poly(I : C)-stimulated conditions (Fig. 4C). In contrast, the basal and TLR ligand-stimulated activities of NF-B in cyto cells were similar to those in parental cells (Fig. 4C). Together, these results suggested that expression of SHPS-1–4F increases the activity of NF-B under basal or LPS- or poly(I : C)-stimulated conditions in RAW264.7 cells.
TLR ligands such as LPS and poly(I : C) induce the activation of MAPKs as well as that of the protein kinase Akt (Weinstein et al. 1992; Han et al. 1993; Hambleton et al. 1996; Salh et al. 1998; Alexopoulou et al. 2001; Guillot et al. 2005). We thus next examined the effects of LPS or poly(I : C) on the activation of such signaling molecules. However, the extent of LPS-induced activation of Akt, p38, Erk or JNK did not differ substantially among 4F, cyto and parental cells (Fig. 5). The extent of activation of these signaling molecules induced by poly(I : C) was also similar among these three cell lines (data not shown).
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We next investigated whether stimulation with LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and SHPS-1 binding to SHP-1 or SHP-2. Stimulation with LPS indeed increased the tyrosine phosphorylation of SHPS-1 as well as the association of SHPS-1 with SHP-1 and SHP-2 in parental RAW264.7 cells (Fig. 6A). The maximal level of tyrosine phosphorylation of SHPS-1 was apparent 30 min after exposure to LPS. Poly(I : C) also induced tyrosine phosphorylation of SHPS-1 and the binding of SHPS-1 to SHP-1 and SHP-2 in parental RAW264.7 cells, with the maximal increase in the level of tyrosine phosphorylation of SHPS-1 being apparent at 15 min after the onset of stimulation (Fig. 6B).
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Finally, we examined which tyrosine kinase might mediate the LPS- or poly(I : C)-induced tyrosine phosphorylation of SHPS-1. PP2, an inhibitor of Src family kinases, but not PP3, an inactive analog of PP2, markedly inhibited the tyrosine phosphorylation of SHPS-1 elicited by LPS (Fig. 7A) or poly(I : C) (Fig. 7B). These results suggested that a Src family kinase participates in the TLR4- or TLR3-induced tyrosine phosphorylation of SHPS-1.
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Discussion |
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as well as the poly(I : C)-induced production of TNF- and IL-6 by RAW264.7 cells. Moreover, expression of the CD8–4F chimera mimicked the effects of SHPS-1–4F. Given that SHPS-1–4F and CD8–4F each behave as dominant negative mutants of SHPS-1, these results suggest that SHPS-1 negatively regulates TLR4- or TLR3-induced production of proinflammatory cytokines by RAW264.7 cells. In contrast, expression of SHPS-1-cyto did not promote the production of proinflammatory cytokines induced by LPS or poly(I : C), suggesting that the cytoplasmic region as well as mutation of its tyrosine phosphorylation sites are required for the dominant negative effect of SHPS-1–4F. We also found that LPS or poly(I : C) induced the tyrosine phosphorylation of SHPS-1 and its association with either SHP-1 or SHP-2. Furthermore, such tyrosine phosphorylation of SHPS-1 was blocked by treatment with an inhibitor of Src family kinases, suggesting that a Src family kinase most likely contributes to the TLR-mediated tyrosine phosphorylation of SHPS-1. Indeed, a Src family kinase has been shown to be activated by TLR3 or TLR4 (English et al. 1993; Stefanová et al. 1993; Johnsen et al. 2006). We also found that LPS- or poly(I : C)-induced tyrosine phosphorylation of SHPS-1 induced the association of SHPS-1 with SHP-1 and SHP-2. Indeed, expression of CD8–4F inhibited also the LPS- or poly(I : C)-induced tyrosine phosphorylation of endogenous SHPS-1 or its association with SHP-1 and SHP-2. These results further indicate that CD8–4F behaves as a dominant negative mutant of SHPS-1. Given that both SHP-1 and SHP-2 are negative regulators of signaling downstream of TLRs (An et al. 2006; Hardin et al. 2006; Zhao et al. 2006), our data suggest that negative regulation by SHPS-1 of the TLR4- or TLR3-induced production of proinflammatory cytokines is mediated by these protein tyrosine phosphatases and their association with tyrosine-phosphoryated SHPS-1.
Although the decreased endogenous SHPS-1 phosphorylation in CD8–4F cells at 15 min after poly(I : C) stimulation was subtle, the impaired recruitment of SHP-1 and SHP-2 was evident (Fig. 6B). This result also suggests that the recruitment of SHP-1 and SHP-2 to SHPS-1 in response to poly(I : C) is not totally dependent on the tyrosine phosphorylation of SHPS-1. It is possible that SHPS-1 physically binds another SHP-1/SHP-2 docking protein, such as Gab2, in a phosphorylation of SHPS-1-independent manner in RAW264.7 cells. However, the molecular mechanism underlying such phosphorylation-independent recruitment of SHP-1 and SHP-2 to SHPS-1 remains unknown, and further analysis will be necessary.
All TLRs activate conserved inflammatory signaling pathways that culminate in the activation of NF-B and activator protein-1 (AP-1) (Kawai & Akira 2006). Activation of this latter protein (AP-1) by TLR signaling is mediated mostly by MAPKs including Erk, p38 and JNK (Kawai & Akira 2006). We found that expression of SHPS-1–4F promoted the LPS- or poly(I : C)-induced degradation of IB and the consequent activation of NF-B. In contrast, expression of SHPS-1–4F did not enhance the activation of Akt or MAPKs induced by LPS. SHPS-1 may thus preferentially regulate the IB–NF-B pathway rather than the MAPK or Akt pathway, although the precise level at which SHPS-1 regulates TLR signaling remains to be determined. Expression of SHPS-1–4F enhanced the production of TNF- induced by CpG ODN to a much lesser extent than it did that elicited by LPS or poly(I : C). Whereas TLR3 promotes the production of proinflammatory cytokines by recruiting Trif to its cytoplasmic domain and in a MyD88-independent manner, the effect of TLR9 on proinflammatory cytokine production is MyD88 dependent but Trif independent (Häcker et al. 2000; Alexopoulou et al. 2001; Yamamoto et al. 2003). In contrast, TLR4 acts through both MyD88 and Trif (Kawai et al. 1999; Yamamoto et al. 2003). It is thus likely that SHPS-1 regulates predominantly the Trif-mediated signaling pathway downstream of TLR3 and TLR4 rather than signaling downstream of TLR9. Indeed, SHP-2 was recently shown to inhibit Trif-mediated signaling downstream of TLR3 (An et al. 2006). We have also shown that expression of a dominant negative mutant of SHP-2 promotes both poly(I : C)- and LPS-stimulated production of TNF- in RAW267.4 cells (Miyake et al., personal communication).
Engagement of SHPS-1 by its ligand CD47 regulates the phagocytic activity of macrophages as well as cell migration (Motegi et al. 2003; Okazawa et al. 2005; Ishikawa-Sekigami et al. 2006). Interaction of SHPS-1 with CD47 on neighboring RAW264.7 cells might thus contribute to the negative regulation by SHPS-1 of proinflammatory cytokine production. Expression of SHPS-1–4F might block such interaction of endogenous SHPS-1 with CD47 and thereby promote the TLR-dependent production of such cytokines. However, expression of SHPS-1-cyto did not promote TLR-induced proinflammatory cytokine production. Moreover, expression of CD8–4F promoted this TLR-dependent effect. The extracellular region of SHPS-1–4F therefore likely does not participate in the promotion by this mutant protein of the TLR-dependent production of proinflammatory cytokines.
In conclusion, we have shown that SHPS-1 negatively regulates the TLR4- or TLR3-dependent production of proinflammatory cytokines through a Trif- and NF-B-dependent pathway rather than a MyD88- and MAPK-dependent pathway in RAW264.7 macrophages. Further investigation is required to determine the precise site of SHPS-1 action in TLR signaling.
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Experimental procedures |
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Hybridoma cells producing a rat monoclonal antibody (mAb) to mouse SHPS-1 (p84) were kindly provided by C. F. Lagenaur (University of Pittsburgh, Pittsburgh, PA). Rabbit polyclonal antibodies (pAbs) to SHPS-1 (anti-SIRP) were obtained from UBI (Lake Placid, NY); rabbit pAbs to SHP-1, to SHP-2, or to IB were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit pAbs to Akt, to phospho-Akt, to p38, to phospho-p38, or to JNK as well as mouse mAbs to phospho-JNK were from Cell Signaling Technology (Danvers, MA); and rabbit pAbs to Erk or to phospho-Erk were from Promega (Madison, WI). Horseradish peroxidase–conjugated goat pAbs to mouse or rabbit IgG were from Jackson ImmunoResearch (Grove, PA). Rabbit pAbs specific for tyrosine-phosphorylated SHPS-1 were generated in response to a synthetic phosphopeptide corresponding to amino acids 496–506 (PSFSEpYASVQV) of mouse SHPS-1 and were purified from serum by affinity chromatography with the synthetic peptide covalently coupled to Epoxy-activated Sepharose 6B (GE Healthcare, Little Chalfont, UK). Details on the specificity of the pAbs to tyrosine-phosphorylated SHPS-1 will be described elsewhere (Ohnishi et al., personal communication). LPS (Escherichia coli, serotype 055:B5) and polyinosinic-polycytidylic acid [poly(I : C)] were obtained from Sigma-Aldrich, St. Louis, MO. The phosphorothioate-stabilized CpG oligodeoxynucleotide (ODN) 5'-TCCATGACGTTCCTGACGTT-3' was synthesized by JBioS (Saitama, Japan). PP2 and PP3 were from EMD Chemicals.
Cells and cell culture
The mouse macrophage cell line RAW264.7 (kindly provided by Y. Kaneko, Gunma University, Gunma, Japan) was cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich). RAW264.7 cells stably expressing either the SHPS-1–4F mutant of mouse SHPS-1, in which all four tyrosine residues (Tyr436, Tyr460, Tyr477, Tyr501) in the cytoplasmic region were replaced with phenylalanine by site-directed mutagenesis; the SHPS-1-cyto mutant of mouse SHPS-1, which lacks almost the entire cytoplasmic region (amino acids 405–509); or a chimeric protein (CD8–4F) comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1–4F were generated by infection of the cells with retroviruses encoding these various proteins as previously described (Ikeda et al. 2006). The infected cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and puromycin (2 µg/mL) (Sigma-Aldrich).
RT-PCR
Total RNA was extracted from cells with an RNeasy Mini kit (Qiagen, Valencia, CA). The RNA was subjected to reverse transcription (RT) with Superscript III (Invitrogen, Carlsbad, CA), and the resulting cDNA was subjected to the polymerase chain reaction (PCR) with primers (forward and reverse, respectively) specific for mouse TLR4 (5'-AGTGGGTCAAGGAACAG-3' and 5'-CTTTACCAGCTCATTTCTC-3'), mouse TLR3 (5'-GAGGGCTGGAGGATCTCTTTT-3' and 5'-CCGTTCTTTCTGAACTGGCCA-3'), and mouse glyceraldehyde-3-phosphate dehydrogenase (5'-GAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CAGCTTTCCAGAGGGGCCATCCACA-3'). The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The stained products were scanned and the signal intensity was quantified with the use of an image analyzer (LAS-3000, Fujifilm, Tokyo, Japan) and Image Gauge software (Fujifilm). The intensity of the PCR products for TLR4 or TLR3 was normalized by that of the PCR product for glyceraldehyde-3-phosphate dehydrogenase.
Measurement of TNF- and IL-6 production
Cells (5 x 105 per well) were plated on 12-well plates, incubated for 12 h, and then stimulated with LPS (10 or 100 ng/mL), poly(I : C) (10 µg/mL), or CpG ODN (1 µg/mL) for 6 h (for TNF-) or 24 h (for IL-6). The levels of TNF- or IL-6 in culture supernatants were determined by enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN).
Measurement of DNA binding activity of NF-B p65
Cells (5 x 106) were stimulated with LPS (1 µg/mL) or poly(I : C) (10 µg/mL) for 6 h, washed with ice-cold phosphate-buffered saline, and isolated by centrifugation at 1000 g for 5 min at 4 °C. They were suspended in buffer A [10 mM Hepes–KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl2] by gentle pipetting and maintained on ice for 20 min. The resulting lysates were centrifuged at 10 000 g for 10 s at 4 °C, and the pellets were suspended in buffer B [20 mM Hepes–KOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol], incubated on ice for 20 min, and centrifuged at 10 000 g for 2 min at 4 °C. The protein concentration of the resulting nuclear extracts (supernatants) was measured with the use of the BCA protein assay (Pierce, Rockford, IL), and the DNA binding activity of the p65 subunit of NF-B in the extracts (10 µg) was determined with the use of a TransFactor NF-B p65 Colorimetric Kit (Clontech, Palo Alto, CA).
Immunoprecipitation and immunoblot analysis
Cells were washed with ice-cold phosphate-buffered saline and then lysed on ice with lysis buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 1% Nonidet P-40] containing 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF and 1 mM Na3VO4. The lysates were centrifuged at 15 000 g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis. For immunoprecipitation, the supernatants were incubated for 1 h at 4 °C with the p84 mAb to mouse SHPS-1, after which protein G-conjugated Sepharose beads (15 µL) (GE Healthcare) were added to the mixture and the incubation was continued for an additional 2 h. The beads were then washed 3 times with 1 mL of lysis buffer, suspended in Laemmli sample buffer, boiled for 5 min, and subjected to immunoblot analysis. Immune complexes were detected with an ECL detection system (GE Healthcare).
Statistical analysis
Data are presented as means ± SE and were analyzed by Student's t test. A P value of < 0.05 was considered statistically significant.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: matozaki{at}showa.gunma-u.ac.jp
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Accepted: 19 November 2007
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