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Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

	Abstract

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In macrophages and monocytes, lipopolysaccharide (LPS) triggers the production of pro-inflammatory cytokine through Toll-like receptor (TLR) 4. Although major TLR signalling pathways are mediated by serine or threonine kinases including IKK, TAK1, p38 and JNKs, a number of reports suggested that tyrosine phosphorylation of intracellular proteins is involved in LPS signalling. Here, we identified several tyrosine-phosphorylated proteins using mass spectrometric analysis in response to LPS stimulation. Among these proteins, we characterized C-terminal Src kinase (Csk), which negatively regulates Src-like kinases in RAW 264.7 cells using RNAi knockdown technology. Unexpectedly, LPS-induced CD40 activation and the secretion of pro-inflammatory cytokine such as IL-6 and TNF-, was down-regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4-mediated activation of IB-, Erk and p38 but not of JNK, were also down-regulated in Csk knockdown cells. The protein expression levels of a tyrosine kinase, Fgr, were reduced in Csk knockdown cells, suggesting that Csk is a critical regulator of TLR4-mediated signalling by modifying the levels of Src-like kinases.

	Introduction

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Macrophages and monocytes are involved in the phagocytosis of bacteria, antigen processing and presentation, and the secretion of pro-inflammatory cytokines such as tumour necrosis factor- (TNF-), interleukin (IL)-1, and IL-6 (Morrison & Ryan 1979). Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, has many immunological effects on macrophages (Ulevitch & Tobias 1999). The activation of macrophages by LPS is mediated by Toll-like receptor 4 (TLR4). The stimulation of TLR4 results in the recruitment of Myeloid differentiation 88 (MyD88), Mal (MyD88-adaptor-like) and Trif (TIR-containing adaptor-inducing IFNs) to the cytoplasmic domain of TLR4. The MyD88/Mal recruits IL-1 receptor-associated kinase (IRAK) and the activated IRAK then dissociates them from the receptor complex, followed by association with tumor necrosis factor-associated factor 6 (TRAF6). This triggers the activation of several different pathways involving the Rel family transcription factor NF-B and the mitogen-activated protein kinase (MAPKs) including Erk, JNK and p38 (Akira 2003; Kopp & Medzhitov 2003). Trif (also known as TICAM-1) is shown to mediate the MyD88-independent pathway, which activates IRF-3 and NF-B (Akira 2003; Kopp & Medzhitov 2003). These signal pathways lead to the production of pro-inflammatory cytokine in macrophages. Although the inflammatory cytokines are essential for triggering the host defense, excessive cytokine production can provoke life-threatening conditions (Ulevitch & Tobias 1995). The balance of TLR signalling is necessary; however, the regulatory mechanisms are largely unknown.

Many cellular signal transductions from external stimulation are coordinated and regulated by tyrosine phosphorylation-dependent protein to protein interactions. Previous studies have suggested that tyrosine kinases (PTKs) may be involved in LPS signalling. PTKs have been shown to be required for the induction of pro-inflammatory cytokines (IL-1, IL-6 and TNF-) in response to LPS in murine macrophages (Geng et al. 1993), in addition to B cell proliferation (Dearden-Badet & Revillard 1993). Mainly, Src-family tyrosine kinases Hck, Lyn, and Fgr, have also been implicated in the biological response of LPS-activated macrophages (Ziegler et al. 1988; Boulet et al. 1992; Stefanova et al. 1993). Consistent with this notion, the inhibitors of tyrosine kinases (PP1) block LPS-induced biological responses in monocytes (Novogrodsky et al. 1994). Furthermore, it has been shown recently that Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, interacts with the TIR domain of TLR4 and is deeply involved in TLR4 signalling (Jefferies et al. 2003). Although these observations suggest that tyrosine kinases play a role in the response of LPS-activated macrophages, the underlying mechanisms of activation of tyrosine kinases and their substrates remain uncertain. Therefore, identification of tyrosine kinases, these substrates, or both in LPS-activated macrophages is an important step to define the role of the protein tyrosine phosphorylation of the TLR4 signalling pathway.

Recently, affinity chromatography-combined HPLC/MS/MS analysis is leading to the identification of post-translational modification (Ibarrola et al. 2004; Zhao et al. 2004). Therefore, we attempted to identify tyrosine-phosphorylated proteins in LPS-activated macrophages using a proteomic approach, namely, immunopurification by an anti-phosphotyrosine antibody and followed by identification using automated nanoflow liquid chromatography or tandem mass spectrometry. Of the identified proteins, we were particularly interested in C-terminal src kinase (Csk) because this regulates Src-like kinases and is poorly characterized to date. We established Csk over-expressing and knockdown cells and demonstrated that the level of Csk expression could modulate LPS-induced macrophage activation.

	Results

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

 
Identification of tyrosine-phosphorylated proteins in LPS-activated RAW 264.7 cells

To identify tyrosine-phosphorylated proteins in LPS-activated RAW 264.7 cells, we used NHS-activated sepharose coupled with monoclonal anti-phosphotyrosine antibody (4G10). RAW 264.7 cells were treated with LPS for 30 min, 1 h or 3 h, and cell lysates were immunoprecipitated with the 4G10-conjugated beads. Bound proteins were subjected to SDS-PAGE, and tyrosine phosphorylated proteins were visualized by immunoblot with 4G10 (Fig. 1A). Immunoblot was compared to a silver staining gel to identify phosphorylated proteins on tyrosine residues (Fig. 1B). Clearly visualized bands were cut from the gel, subjected to in-gel trypsin digestion, and analyzed by HPLC/MS/MS as exemplified in Fig. 1C. With this approach, we identified 14, 13 and 19 proteins from 30 min, 1 h, and 3 h LPS-treated cell lysates, respectively (see Supplementary Table at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC839/GTC839.htm). Of them, tyrosine phosphorylation of SHIP, PLC2, tyrosine kinase Pyk2 and Syk has already been reported (Crowley et al. 1996; Shinji et al. 1997; Williams & Ridley 2000; Fang et al. 2004). The Fes/Fps has also been implicated in the function of macrophages from the analysis of knockout mice (Zirngibl et al. 2002).

View larger version (37K): [in this window] [in a new window]   Figure 1  Identification of tyrosine-phosphorylated proteins in RAW 264.7 cell lysates. (A, B) Cell lysates from 100 ng/mL LPS-stimulated RAW 264.7 cells for 30 min, 1 h, and 3 h were added to NHS Sepharose coupled with anti-phosphotyrosine monoclonal antibody 4G10. Bound proteins were separated by SDS-PAGE, visualized by silver staining and then immunoblotted by anti-phosphotyrosine antibodies. (C) Mass spectrum of tryptic peptides. The indicated peptide peak, peptide 1 corresponded to a peptide from mouse Csk. (D) MS/MS spectrum from peptide 1. The sequence was derived from the mass difference of the nested set of peptide fragments. The positions to assigned series of Y ions are marked. (E) RAW 264.7 cells were stimulated with 100 ng/mL LPS for 3 h, and the cell lysates were immunoprecipitated with anti-phosphotyrosine antibody. The immunoprecipitates were divided and immunoblotted with the indicated antibodies.

Generation of Csk knockdown cells

To elucidate the function of Csk in macrophages, we established RAW 264.7 stable transformants, in which Csk expression was knocked down by small interfering RNA (si-Csk). We also established two stable transformants expressing Csk containing a C-terminal 3 x FLAG epitope (F-Csk). As shown in Fig. 2A, immunoblotting with anti-Csk revealed a significant reduction of Csk in two independent siRNA transfected clones, si-Csk 1 and si-Csk 2 cells, while Stat5 levels were not different. The reduction of Csk levels by siRNA was greater in si-Csk 2 than in si-Csk 1 cells. In the FLAG-tagged Csk transformants, the expression levels of exogenous Csk were not extreme. Total levels of Csk in F-Csk 1 cells were almost similar to those in parental WT RAW cells and those of F-Csk 2 cells were about two times higher than those in WT RAW cells. The expression level of TLR4 was not significantly different among parental RAW 264.7 cells and transformants (Fig. 2B).

View larger version (29K): [in this window] [in a new window]   Figure 2  Generation of Csk over-expression cells and knockdown cells. (A) Whole cell lysates were analyzed by immunoblot with the indicated antibodies. (B) Total RNAs were extracted from cells. RT-PCR was performed as described in Experimental procedures.

We then examined the effect of Csk on downstream signalling in LPS-activated macrophages. First, we analyzed LPS-induced pro-inflammatory cytokine production in RAW 264.7 cells and Csk transformants. As shown in Fig. 3, LPS-induced TNF-, IL-6 and IL-12 production from two Csk knockdown cells, was strongly suppressed (Fig. 3B,C,D). NO synthesis was also greatly reduced in si-Csk transformants (Fig. 3A). The suppression of TNF- production in the Csk knockdown cells was because of the reduction of mRNA synthesis (Fig. 3E). We also observed reduced IL-1ß mRNA induction in Csk knockdown cells (Fig. 3E). These data indicate that Csk positively regulates pro-inflammatory cytokine production and NO synthesis. However, interestingly, Csk over-expression slightly yet significantly reduced TNF- and IL-6 as well as NO synthesis in F-Csk 2 cells. However, the mRNA level of TNF- and IL-1ß was not significantly reduced in F-Csk 2 cells (Fig. 3E). Little difference was observed in F-Csk 1 cells, where the Csk levels were not greatly different from those in parental RAW 264.7 cells. However, the total Csk levels were about two times higher in F-Csk2 cells than in parental RAW 264.7 cells (Fig. 2A). Our data are consistent with a previous report showing that over-expression of Csk in a macrophage cell line resulted in the production of pro-inflammatory cytokines in response to LPS (Iwabuchi et al. 1997). These data indicated that LPS-induced pro-inflammatory cytokine production was tightly regulated by the Csk expression level.

View larger version (27K): [in this window] [in a new window]   Figure 3  Csk modulates LPS-induced macrophage activation. (A) Cells were stimulated with 1 µg/mL LPS for 24 h, then the supernatants of nitrite levels were measured as described in Experimental procedures. (B–D) Cells were stimulated with 100 ng/mL or 1 µg/mL LPS for 6 h. TNF-, IL-6 and IL-12 in cell culture supernatants were determined using ELISA. (E) RAW 264.7 cells and Csk transformants were stimulated with 100 ng/mL LPS for 1 h, then total RNAs were extracted from cells.

View larger version (22K): [in this window] [in a new window]   Figure 4  Effect of Csk on LPS-induced CD40 expression. Cells were stimulated with 1 µg/mL LPS for 24 h, and CD40 expression on cells analyzed by flow cytometry.

TLR4-mediated signal transduction of Csk transformants

LPS regulates gene transcription via NF-B, AP-1 and other transcription factors through the activation of p42/44 ERK, c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein (MAP) kinase family, as well as IKK complexes (Akira 2003; Kopp & Medzhitov 2003). First, we examined LPS-induced up-regulation of NF-B transcriptional activity using a reporter gene assay (Fig. 5A). LPS-induced up-regulation of NF-B activity was severely impaired in si-Csk 2 cells, while transcriptional activity increased in F-Csk 2 cells (Fig. 5A, left panel). Similar results were obtained using transient expression of siRNA and cDNA of Csk (Fig. 5A, right panel), which exclude the possibility of clonal variation of our stable transformants. Reduction of NF-B activation in si-Csk 2 cells is consistent with the reduction of pro-inflammatory cytokines and NO (Fig. 3A–E), but the enhanced NF-B activity in F-Csk 2 cells was unexpected. Reduction of inflammatory cytokine production from F-Csk 2 cells may not be to the result of reduced NF-B activity, which is consistent with similar TNF- and IL-1ß mRNA induction in these cells (Fig. 3E).

View larger version (35K): [in this window] [in a new window]   Figure 5  Effect of Csk on TLR signalling. (A) In left panel, RAW 264.7 cells and Csk stable transformants were transfected with 0.5 µg of the NF-B reporter plasmid and the control LacZ plasmid. After stimulation with or without LPS 1 µg/mL LPS for 8 h, luciferase activity was measured. In right panel, parental RAW264.7 cells were transfected with 0.5 µg of the NF-B reporter plasmid and the control LacZ plasmid together with siRNA or cDNA in the expression vector (0.5 µg). (B) Parental RAW 264.7 cells and two individuals both over-expressing and knockdown Csk transformants were stimulated with 100 ng/mL LPS. The levels of phospho-IB- and IB- were determined 15 min after the treatment. Likewise, indicated times after stimulation and those of p-JNK, JNK, p-p38, p-38, p-Akt, and Akt were measured 30 min after the treatment. (C) RAW 264.7 cells and respective Csk transformants were stimulated with 1 µg/mL CpG DNA for 15 or 30 min. The CpG-induced IB- and Erk activation were analyzed as described in B.

Next, we examined whether Csk was involved in another TLR signalling. Unmethylated 2'-deoxyribo (cytidine-phosphate-guanosin) CpG DNA motifs are commonly found in bacterial and viral genomes, which activate NF-B via TLR9/Myd88 (Akira 2003; Kopp & Medzhitov 2003). We tested the involvement of Csk expression and activation of IB- and Erk by CpG-oligonucleotide (ODN) stimulation. Both IB- and Erk activation were markedly down-regulated in Csk knockdown cells (Fig. 5C). Csk over-expressing cells, F-Csk 2, but not F-Csk 1 cells, enhanced CpG-ODN-induced phosphorylation of IB- and Erk (Fig. 5C). These data suggest that TLR9 signalling was also modulated by Csk expression level.

Effect of Csk expression on Stats and total tyrosine phosphorylation

Indirectly, the autocrine or paracrine of secreted cytokines by LPS-activated macrophages, such as IL-6 and IFNß, activates Jak/Stat pathway (Crespo et al. 2000; Toshchakov et al. 2002). Therefore, we examined the tyrosine phosphorylation of Stat1 and Stat3. LPS-induced phosphorylation of Stat1 and Stat3 was severely impaired in si-Csk 2 cells (Fig. 6A). However, Stat1 and Stat3 phosphorylation was enhanced in F-Csk 2 cells, in which cytokine production such as IL-6 was reduced. These data suggest that Csk regulates the phosphorylation of Stat1 and Stat3 through direct and indirect mechanism.

View larger version (48K): [in this window] [in a new window]   Figure 6  Effect of Csk expression on c-Fgr degradation and tyrosine phosphorylation of Stats and multiple cellular proteins. (A) The cells were stimulated with 100 ng/mL LPS for 3 h, and cell extracts were then analyzed by immunoblot with the indicated antibodies. (B–D) Whole-cell lysates were analyzed by immunoblot with the indicated antibodies. (D) Cells were pretreated with PP2 (10 µM) for 15 min and then stimulated with 100 ng/mL LPS for another 15 min.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

 
In this study, we identified tyrosine-phosphorylated proteins in LPS-activated RAW 264.7 cells using anti-phosphotyrosine antibodies and LC-MS/MS. Among these proteins, SHIP and PLC2, Syk and Pyk2 were known to be tyrosine-phosphorylated in macrophages after LPS stimulation (Crowley et al. 1996; Shinji et al. 1997; Williams & Ridley 2000; Fang et al. 2004). Thus, our approach is proved to be suitable for finding novel molecules that may participate in TLR signalling. We are particularly interested in the phosphorylation of Csk, because this kinase is shown to be essential for regulating all Src-like kinases. This is the first documentation that Csk is tyrosine-phosphorylated in response to LPS. Recently, it was demonstrated that conditional Csk gene targeting in granulocytes (Csk-Gecre mice) causes acute inflammation and hypersensitivity to LPS. However, TNF- levels produced from bone marrow-derived or peritoneal macrophages in response to LPS did not differ from the controls (Thomas et al. 2004). Thus, hypersensitivity to LPS in Csk mutant mice did not seem to be derived from mutant macrophages to LPS (Thomas et al. 2004). Therefore, the role of Csk in LPS-activated macrophages has remained uncertain.

In the present study, we demonstrated that Csk plays an important role in LPS-induced signal transduction and pro-inflammatory cytokine production in RAW 264.7 macrophage-like cells. Knockdown of Csk suppressed tyrosine phosphorylation of intracellular proteins. On the other hand, enhanced expression of Csk resulted in the hyperactivation of signalling of TLR4 and TLR9, although production of TNF- and IL-6 was reduced. These findings suggested that the level of Src family kinases were correlated with tyrosine phosphorylation in a variety of intracellular proteins. In general, Csk is believed to negatively regulate Src family tyrosine kinases by phosphorylating the C-terminal regulatory tyrosine residue conserved among the Src family (Nada et al. 1991). Our data do not agree with this general concept. However, it has been reported that the lack of C-terminal tyrosine phosphorylation of Src-like kinases induces ubiquitination and degradation by proteasomes (Harris et al. 1999; Shima et al. 2003). Therefore, it is quite likely that Csk positively regulates Src-like kinases under certain conditions. Our data are consistent with this hypothesis, as the reduction of Csk caused a reduction of the total cellular tyrosine phosphorylation level and a major tyrosine kinase Fgr in RAW 264.7 cells. In addition, our data revealed a novel mechanism of the reduction of cytokine production by Csk over-expression. A previous study showed that over-expression of Csk in macrophages resulted in reduced TNF-, IL-1, IL-6 and NO production in response to LPS (Iwabuchi et al. 1997); however, this mechanism has not been clarified. We demonstrated that Csk over-expression increased the overall tyrosine phosphorylation of cellular proteins, which supports the idea that Csk positively regulates tyrosine kinases in macrophages. We showed that the transcription of TNF- and IL-1 mRNA was not affected by Csk over-expression. Therefore, we speculate that the reduction in cytokine production is a result of post-transcriptional modification by hyper tyrosine phosphorylation.

The biological significance of the Src family tyrosine kinases in LPS-activated macrophages should be explored. A previous report shows that the Src family tyrosine kinase, Hck, Fgr, and Lyn are rapidly activated after LPS stimulation (Ziegler et al. 1988; Boulet et al. 1992; Stefanova et al. 1993). The hck^–/–, fgr^–/–, lyn^–/– triple mutant macrophages have defects in integrin-mediated cell adhesion and spreading (Meng & Lowell 1998). Csk-mediated regulation of Src family kinases is also essential for cell spreading and migration (Shima et al. 2003). These findings demonstrated an important role of the regulatory system of Src family kinases in cell adhesion signalling. However, triple mutant macrophages, hck^–/–, fgr^–/–, lyn^–/–, exhibit no discernible defects in LPS-induced cytokine production and signal transduction such as NF-B and JNK activation (Meng & Lowell 1997). These observations suggest that tyrosine kinases other than Hck, Fgr, and Lyn may be involved in the signalling pathways regulating inflammatory cytokine production in response to LPS. However, molecular mechanism of the activation of tyrosine kinase through TLR is still unclear. Hsu & Wen (2002) reported that reactive oxygen (ROS) induced by LPS is involved in tyrosine kinase activation. ROS may inhibit protein tyrosine phosphatases, which leads to activation of Src-like kinases.

In contrast, Csk over-expression enhanced TLR4 signalling and caused a significant induction of tyrosine phosphorylation of multiple cellular proteins. In addition, JNK and AP-1 were up-regulated by LPS stimulation in Csk over-expressing cells (Kizaki et al. 2001). Over-expression of Csk appears to up-regulate the Src family kinases or other tyrosine kinases in macrophages. Tyrosine phosphorylation of the signal components of TLR may enhance the efficiency of signal transduction. However, we could not detect the interaction of Csk to TLR4 signalling molecules, such as IRAK, Myd88, and TRAF6, or the tyrosine phosphorylation of those molecules. Nevertheless, our study demonstrates that Csk is a critical regulator of the LPS/TLR4 signalling pathway. The identification of target molecules, which are phosphorylated by Csk and related kinases, may reveal a novel regulatory mechanism of the TLR4 signal cascade.

	Experimental procedures

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Cell culture

The murine macrophage cell line RAW 264.7 cells were obtained from the RIKEN Cell Bank (Japan). The RAW 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 1% nonessential amino acids solution (Gibco), penicillin and streptomycin. The Stable RAW 264.7 cell transformants were established as described (Kinjyo et al. 2002).

Plasmids

The cDNA encoding mouse Csk was generated from the total RNA of RAW264.7 cells by RT-PCR and inserted into pCMV14 (Sigma) containing FLAG-tag. The small interfering RNA sequence that efficiently targets mouse Csk was determined by InvivoGen siRNA wizard positions 653–673 (5'-AAGTTGCAGTCAAGTGCATCA-3'). The annealed nucleotides were inserted into psiRNA-hH1neo expression vector (InvivoGen).

Transient transfection and reporter gene analysis

The RAW264.7 cells were seeded onto 6-well plates on the day before transfection. The NF-B responsive promoter-luciferase reporter gene, a generous gift from Dr T. Fujita (The Tokyo Metropolitan Institute of Medical Science, Japan), has been described (Fujita et al. 1993) In brief, the NF-B luciferase plasmid was co-transfected using the ß-galactosidase control plasmid by FuGENE 6 (Roche). After incubation at 37 °C for 8 h, the DNA mixture was replaced with 2 mL of a culture medium. After 18 h, the cells were treated with 1 µg/mL LPS for 8 h, and the luciferase and ß-galactosidase activities were measured. The luciferase activity was normalized to ß-galactosidase activity.

Large scale immunoprecipitation and mass spectrometric analysis

For proteome analysis, RAW 264.7 cells (4 x 10⁸ cells) were treated with 100 ng/mL LPS for 30 min, 1 h and 3 h at 37 °C or left untreated. The cells were lyzed in a lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 150 mM NaCl, 1 mM Na₃VO₄ and a protease inhibitor cocktail (Roche-Boehringer)). The cell lysates were incubated in a lysis buffer with anti-phosphotyrosine antibody 4G10 immobilized to NHS-activated Sepharose^TM 4 Fast Flow for 3 h at 4 °C. The precipitated immune complexes were washed three times with washing buffer (0.5% NP-40, 150 mM NaCl, 25 mM Tris-HCl (pH 7.5)), the bound proteins were eluted with 10 mM phenyl phosphate and the eluted fractions were concentrated with ULTRAFREE-MC 10000 NMW Filter Unit (MILLIPORE). The proteins were finally separated by SDS-PAGE and visualized by silver staining and immunoblotting with 4G10. For mass spectrometric analysis, bands visualized with silver staining, which is also detected by immunoblotting, were cut from gels and digested with modified trypsin (sequencing grade, Promega) (Saeki et al. 2003), and the eluted peptides were loaded on automated nanoflow liquid chromatography or tandem mass spectrometry (FINNIGAN LCQ DECA; Finnigan). The peptide masses obtained by LC-MS/MS analysis were searched against the non-redundant protein sequence database of the National Centre for Biotechnology Information (NCBI) using the Mascot search engine (MATRIX SCIENCE).

Antibody and reagents

Rabbit polyclonal antibodies against IB- (C-21), Erk2 (C-14), Csk (C-20), Fes (N-19), Stat1 (E-23), Stat3 (C-20), Stat5 (C-17) and c-Fgr (C-1) were purchased from Santa Cruz Biotechnology. The Akt, SAPK/JNK, p38, phospho-Akt (S473), phospho-Stat1 (Y701), phospho-Stat3 (Y705) antibodies and mouse monoclonal antibodies against phspho-IB- (S32/36) phospho-p40/42 MAP kinase (T202/Y204), phospho-SAPK/JNK (T183/Y185), phospho-p38 (T180/Y182) were obtained from Cell Signalling Co. The phosphotyrosine antibody (4G10) was from Upstate Biotechnology and the Pyk2 antibody was from Signal Transductions Laboratory. The anti-FLAG M2 mouse monoclonal antibody was from Sigma Chemical Co. The FITC-anti-mouse CD40 (HM-40) was from BD PharMingen. The LPS (E. coli serotype 055:B5) was obtained from Sigma Chemical Co. The phosphorothioate-stabilized CpG oligodeoxynucleotide (ODN) (TCC ATG ACG TTC CTG ACG TT) was synthesized from Hokkaido Bioscience. The NHS-activated Sepharose^TM 4 Fast Flow was obtained from Amersham Pharmacia Biotech.

Immunoprecipitation and immunoblotting analysis

Cells were solubilized for 30 min at 4 °C in a lysis buffer and then centrifuged at 13 000 g for 15 min, and the supernatant was collected. Protein concentrations were measured using a Bio-Rad protein assay kit. For immunoprecipitations, equal amounts of cellular proteins were incubated with antiphospho-tyrosine antibodies and protein G-sepharose (Amersham Pharmacia) for 3 h at 4 °C. The immunoprecipitates were collected by centrifugation, washed three times in a washing buffer (0.5% NP-40, 150 mM NaCl, 25 mM Tris-HCl, pH(7.5)), and analyzed with SDS-PAGE. After proteins were transferred to polyvinylidene difluoride membranes, the membranes were blocked with TBS containing 5% skim milk. The membranes were immunoblotted with specific antibodies and visualized with the appropriate horseradish peroxidase-conjugated secondary antibodies using the Super Signal West Pico Chemiluminescent Substrate (PIERCE).

Measurement of nitrite concentration

NO was measured as the accumulation of nitrite in the incubation medium. The cells were plated on 96-well plates on the day before LPS stimulation. Nitrite was determined spectrophotometrically by adding 50 µL Griess reagent (1% sulfanilamide/0.1%-N-(1-napthyl)-ethylenediamine dihydrochloride/2.5%) to 50 µL of culture medium (Salh et al. 1998) After a 5-min incubation at room temperature, the absorbance at 550 nm was measured and compared with that of a standard NaNO₂.

TNF-, IL-6 and IL-12 ELISA

The cells were plated on 6-well plates on the day before LPS stimulation. Cells were treated with LPS for 6 h at 37 °C. Culture supernatants were stored at –80 °C before performing assays. The TNF-, IL-6, and IL-12 protein levels were determined using ELISA (TECHNE Corporation) according to the manufacturer's instructions.

Flow cytometric analysis

For the cell surface expression analysis of CD40, FACS analysis was performed. The cells were plated on 6-well plates on the day before LPS stimulation. The cells were then left untreated or were treated with LPS for 12 h and washed with Hanks’ solution containing 0.1% NaN₃ and 1% foetal bovine serum and stained with appropriate antibodies. Analysis was performed using BD LSR.

RT-PCR

Total RNA was isolated from 10⁷ cells with ISOGEN (Wako). The cell pellets were lyzed with ISOGEN for 5 min at room temperature. After treatment with chloroform for 5 min, the samples were centrifuged for 15 min at 4 °C. RNA was precipitated from the aqueous phase with isopropanol for 10 min at room temperature and pelleted by centrifugation for 10 min at 4 °C. The pellet was washed with 70% ethanol and dissolved in diethylpyrocarbonate (DEPC)-treated water. RT-PCR was carried out using the one-step RT-PCR kit (Applied Biosystems) according to the manufacturer's instructions. The following oligonucleotides were used for mouse IL-1ß: 5'-AAGCTCTCCACCTCAATGGACAG-3' and 5'-CTCAAACTCCACTTTGCTCTTGA-3' (Caivano & Cohen 2000), mouse TNF-: 5'-GGCAGGCTACT TTGGAGTCATTGC-3' and 5'-ACATTCGAGGCTCCAGTGAATTCGG-3' (Gao et al. 2001), mouse TLR4 5'-AGTGGGTCAAGGAACAGAAGC-3', 5'-CTTTACCAGCTCATTTCTCACC-3' (Matsuguchi et al. 2000), and mouse glycerylaldehyde 3-phospho dehydrogenase (G3PDH) 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCCACCACCCTGTTG CTG TA-3' (Kamio et al. 2004). The amplified PCR products were analyzed on agarose gels containing ethidium bromide and bands were visualized under UV light.

	Supplementary material

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The following supplementary material is available at:http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC839/GTC839.htm

Supplementary Table

	Acknowledgements

 
We thank Ms. Y. Nishi for manuscript preparation. This work was supported by special grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Haraguchi Memorial Foundation, Yamanouchi Foundation for Research on Metabolic Disorders, Takeda Science Foundation, Mochida Memorial Foundation, the Kato Memorial Foundation, Japan Diabetes Foundation and the Uehara Memorial Foundation.

	Footnotes

 
Communicated by: Kozo Kaibuchi

^* Correspondence: E-mail: yakihiko{at}bioreg.kyushu-u.ac.jp

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Received: 4 November 2004
Accepted: 3 January 2005

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