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. 2005 Jul;17(7):827-36.
doi: 10.1093/intimm/dxh264. Epub 2005 May 20.

Association of SIGNR1 with TLR4-MD-2 enhances signal transduction by recognition of LPS in gram-negative bacteria

Koji Nagaoka  1 , Kazuhiko Takahara, Kay Tanaka, Hideo Yoshida, Ralph M Steinman, Shin-Ichiro Saitoh, Sachiko Akashi-Takamura, Kensuke Miyake, Young Sun Kang, Chae Gyu Park, Kayo Inaba
Affiliations

Association of SIGNR1 with TLR4-MD-2 enhances signal transduction by recognition of LPS in gram-negative bacteria

Koji Nagaoka et al. Int Immunol. 2005 Jul.

Abstract

SIGNR1, a member of a new family of mouse C-type lectins, is expressed at high levels in macrophages (Mphi) within the splenic marginal zone, lymph node medulla, and in some strains, in peritoneal cavity. We previously reported that SIGNR1 captures gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, as well as Candida albicans. We have now investigated the precise ligands and innate responses that involve SIGNR1. The interaction of SIGNR1 with FITC-dextran and E. coli was completely inhibited by LPS from E. coli and Salmonella minnesota. Using LPS from various types of rough mutants of Salmonella, we found that SIGNR1 primarily recognizes oligosaccharides in the non-reductive end of the LPS core region. In transfectants, expression of SIGNR1 enhanced the oligomerization of Toll-like receptor (TLR) 4 molecules as well as the degradation of IkappaB-alpha after stimulation with E. coli under low-serum conditions. The enhanced TLR4 oligomerization was inhibited by pre-treatment of the cells with anti-SIGNR1 mAb or with mannan. A physical association between SIGNR1 and the TLR4-MD-2 complex was also observed by immunoprecipitation. Finally, we found that transfection of SIGNR1 into the macrophage-like RAW264.7 cells resulted in significant augmentation of cytokine production. These results suggest that SIGNR1 associates with TLR4 to capture gram-negative bacteria and facilitate signal transduction to activate innate M responses.

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Figures

Fig. 1
Fig. 1
FITC–dextran binding by SIGNR1 on resident peritoneal Mφ is blocked by LPS. (A) Resident Mφ specifically express SIGNR1 and bind FITC–dextran. Resident, PP- or thioglycollate-elicited peritoneal cells were stained by PE–anti-CD11b and anti-SIGNR1 antibody, ER-TR9. SIGNR1 expression patterns of the CD11bhigh cells were indicated as histograms (middle panels). Open histograms showed staining patterns with control antibody. Binding of FITC–dextran by the CD11bhigh cells is also depicted (right panels). After PE–anti-CD11b staining, the peritoneal cells were incubated with FITC–dextran (80 μg ml−1) at 4° C for 1 h. (B) Resident peritoneal cells were pre-incubated with ER-TR9, control IgM (25 μg ml−1 each), mannan (1 mg ml−1), EDTA (20 mM), Escherichia coli LPS (80 μg ml−1) or Salmonella minnesota (80 μg ml−1) LPS for 30 min at 4° C before adding FITC–dextran. Histograms indicate FITC–dextran binding of the CD11bhigh cells.
Fig. 2
Fig. 2
Binding of E. coli via SIGNR1 and its inhibition by LPS and CPS. (A) Ba/F3 cells expressing SIGNR1 were incubated with Alexa488-labeled E. coli at 4° C for 4 h. Binding was analyzed by flow cytometry. For inhibition assays, the transfectants were pre-incubated with mannan (1 mg ml−1) at 4° C for 30 min. (B) Ba/F3 cells expressing SIGNR1 and parent cells were pre-incubated with LPS from E. coli or S. minnesota, or type 3 or type 14 CPS at 100 μg ml−1 as in (A). After washing, binding of Alexa488-labeled E. coli was analyzed 4 h later as described. (C) SIGNR1 transfectants and parental Ba/F3 cells were pre-incubated with the graded doses of LPS or CPS, and then binding of Alexa488-labeled E. coli was analyzed as in (B). Percent inhibition was calculated as described in Methods.
Fig. 3
Fig. 3
SIGNR1 recognizes the core polysaccharide portion LPS from gram-negative bacteria. (A) Structures of outer and inner core region of LPS from S. typhimurium. Sugar components deleted from LPS in various S. typhimurium rough mutants are shown. GlcNAc, N-acetyl-glucosamine; Glc, glucose; Gal, galactose; Hep, l-glycero-d-manno-heptose; KDO. (B) As in Fig. 3(A), transfectants were pre-incubated with 100 μg ml−1 of various types of LPS from rough mutants, KDO, lipid A or 1 mg ml−1 of mannan at 4° C for 30 min. Binding of Alexa488-labeled E. coli was analyzed.
Fig. 4
Fig. 4
SIGNR1 enhances TLR4 oligomerization upon stimulation with E. coli. (A) Ba/F3 transfectant expressing TLR4-Flag, TLR4-GFP, MD-2Flag and CD14 was transduced with SIGNR1 gene and stained with anti-SIGNR1 (ER-TR9), anti-TLR4–MD-2 (MTS510) or anti-CD14 (Sa2-8). Open histograms represent staining profiles with control antibodies. (B) Transfectants were stimulated with E. coli at various numbers of microbes for 30 min at serum concentrations indicated. The cell lysates were subjected to immunoprecipitation with anti-GFP for TLR4. Co-precipitation of TLR4-Flag was detected by western blotting with anti-Flag. Precipitation of TLR-GFP was confirmed by anti-GFP antibody. (C) The transfectants prepared in (A) were pre-treated with mannan, hamster IgG or anti-SIGNR1 (22D1), followed by stimulation with E. coli in the presence of 0.01% serum.
Fig. 5
Fig. 5
SIGNR1 enhances IκB-α degradation after stimulation with E. coli. Escherichia coli bacteria were added to cell cultures at 10 and 20 microbes per cell in (A) and (B), respectively. After incubation for various time periods in the presence of 0.01% serum, IκB-α and β-actin in whole-cell lysates were analyzed by western blotting.
Fig. 6
Fig. 6
Association of SIGNR1 with the TLR4–MD-2 complex. (A) Cell lysates of 293T transiently transfected with pEFBOS/mTLR4flaghis, pEFBOS/mMD-2flaghis and pcDNASIGNR1 (Xpress tagged) were subjected to immunoprecipitation with antibodies indicated, followed by western blot analyses by anti-Flag antibody against TLR4-Flag and MD-2Flag. (B) As in (A), cell lysates were precipitated with anti-Flag or anti-TLR4–MD-2 complex (MTS510), followed by blotting SIGNR1-Xpress using anti-Xpress antibody. (C) Cell lysates of stable Ba/F3 transfectants expressing TLR4-Flag, MD-2Flag and SIGNR1 was precipitated with anti-SIGNR1 (PAb-C13), followed by western blot analysis using anti-Flag antibody. Numbers indicate two independent clones.
Fig. 7
Fig. 7
Augmented cytokine production by the macrophage-like cell line, RAW264.7, via SIGNR1 in response to S. typhimurium. RAW264.7 transfectants (5 × 105 cells per well) were cultured overnight in serum-free medium, X-VIVO15, and stimulated with S. typhimurium (3 microbes per cell) for 1 h at 37°C. After washing, cells were further incubated for 7 h at 37°C. For the inhibition assay, cells were pre-treated with 25 μg ml−1 of anti-SIGNR1 (clone 22D1) or control hamster IgG for 30 min at 37°C before the addition of microbes. Then culture supernatants were collected and assessed for inflammatory cytokine production using Cytometric Bead Array.
Fig. 8
Fig. 8
SIGNR1-dependent cytokine production induced by E. coli. C3H/HeN- and C3H/HeJ-derived resident peritoneal Mφ enriched negatively by magnetic beads were stimulated with E. coli at 1 microbe per 10 cells in X-VIVO15 medium for 6 h, and cytokine activities in culture supernatants were assessed by Cytometric Bead Array. Inhibition assay was carried out as in Fig. 7.
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