Septic Shock Is Associated with Receptor for Advanced Glycation End Products Ligation of LPS

Yasuhiko Yamamoto, Ai Harashima, Hidehito Saito, Koichi Tsuneyama, Seiichi Munesue, So Motoyoshi, Dong Han, Takuo Watanabe, Masahide Asano, Shin Takasawa, Hiroshi Okamoto, Satoshi Shimura, Tadahiro Karasawa, Hideto Yonekura and Hiroshi Yamamoto
J Immunol March 1, 2011, 186 (5) 3248-3257; DOI: https://doi.org/10.4049/jimmunol.1002253

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Binding of LPS to RAGE. A, SPR assays of LPS binding to the extracellular domain of RAGE immobilized on a sensor chip. LPS from E. coli 055:B5, E. coli 0127:B8, E. coli 0111:B4, Klebsiella pneumoniae, and Salmonella enterica serotype typhimurium and KDO2-lipid A were used. Kinetic analyses by SPR were done with LPS E. coli 055:B5 (2.5, 5, 10, and 20 ng/ml) and KDO2-lipid A (0.313, 0.625, 1.25, 2.5, and 5 ng/ml). SPR assay was performed as described under Materials and Methods. After the injection (60 s), the mobile phase was changed back to the buffer without LPS. Normalized successive curves are shown from the lowest to highest concentrations. 10, 10 ng/ml; 20, 20 ng/ml. B, SPR assay of esRAGE binding to LPS (E. coli 0111:B4) immobilized on a sensor chip. Purified esRAGE (0.5 and 1.0 μg/ml) was injected. C, Competition assay by SPR. Five hundred nanograms per milliliter esRAGE was preincubated with indicated concentrations of AGE-BSA for 30 min, and the preincubation mixtures were injected into the sensor chip on which LPS (E. coli 0111:B4) had been immobilized.

  • FIGURE 2.
    FIGURE 2.

    Binding of LPS to RAGE and LPS activation of NF-κB and secretion of TNF-α. A, Plate competition assay. One hundred nanograms per milliliter esRAGE was preincubated with indicated concentrations of LPS (E. coli 055:B5) for 30 min, and the preincubation mixtures were added into wells on which AGE-BSA had been immobilized. The formation of AGE–RAGE complexes was detected with Eu-conjugated anti-RAGE Ab. The ordinate indicate percentage AGE-RAGE binding with the value without LPS being 100%. B, Plate competition assay. One hundred nanograms per milliliter esRAGE proteins were preincubated with indicated concentrations of AGE-BSA for 30 min, and the preincubation mixtures were added into wells on which LPS (E. coli 055:B5) had been immobilized. The formation of LPS–RAGE complexes was detected with Eu-conjugated anti-RAGE Ab. The ordinate indicates percentage of LPS-RAGE binding with the value without LPS being 100%. C, Flow cytometry. Peritoneal macrophages from RAGE+/+ mice, but not RAGE−/− mice, expressed RAGE protein. RAGE expression was not correlated with TLR2 or TLR4. One representative result from a total three repeats is shown. D, TNF-α secretion from peritoneal macrophages. After 24 h incubation of LPS 50 ng/ml, TNF-α level in the cell culture media was assayed. *p < 0.0001 compared with RAGE−/− mice exposed with LPS. E, NF-κB promoter assay. C6 glioma cells that had been transformed by a mammalian expression vector pCI-neo with human full-length RAGE cDNA and by a reporter gene carrying an NF-κB promoter and firefly luciferase gene were treated with indicated concentrations of LPS and sRAGE. Immunoblot in the inset shows diminished RAGE proteins under siRNA and expression of dominant-negative RAGE under dnRAGE. Data are presented as mean ± SEM. *p < 0.0001 compared with LPS alone. F, NF-κB promoter assay with the RAGE expressing C6 glioma cells. Data are presented as mean ± SEM. *p < 0.03 compared with LPS alone and siCNT. AU, arbitrary unit; dnRAGE, the C6 glioma cells further transformed by pCI-neo expressing dominant-negative RAGE that lacked the cytoplasmic domain; nd, not detected; siCNT, control siRNA; siRAGE, RAGE siRNA; siRNA, the C6 glioma cells further transformed by RAGE siRNA expression vector; siTLR2, TLR2 siRNA; siTLR4, TLR4 siRNA; siTrip, a combination of RAGE siRNA, TLR2 siTNA, and TLR4 siRNA.

  • FIGURE 3.
    FIGURE 3.

    Flow cytometry. A, RAGE expressed on neutrophils and monocytes. Dark shading, cells from RAGE+/+ mice; gray shading, cells from RAGE−/− mice. One representative result from a total three repeats is shown. B, Neutrophils have more abundant cell-surface RAGE than monocytes. Dark shading, neutrophils from RAGE+/+ mice; gray shading, monocytes from RAGE+/+ mice. *p < 0.013. C–F, LPS binding to immune cell surface. Whole blood was incubated with 50 μg/ml FITC-LPS or FITC alone for 2 h at 4°C. Dark shading, FITC-LPS incubation with cells from RAGE+/+ mice; gray shading, FITC alone incubation with cells from RAGE+/+ mice (C). Whole blood was incubated with 50 μg/ml FITC-LPS for 2 h at 4°C. Dark shading, cells from RAGE+/+ mice; gray shading, cells from RAGE−/− mice (D). Arrow indicates a neutrophil cluster highly associated with FITC-LPS. One representative result from a total three repeats is shown. Mean fluorescence intensity (MFI) of FITC-LPS signals from neutrophils and monocytes. *p < 0.05 compared with RAGE+/+ monocytes (E). Neutrophil population (%) highly associated with FITC-LPS as indicated in D with the arrow (F). Two hours incubation of 50 μg/ml FITC-LPS and whole blood with or without pretreatment of 50 μg/ml unlabeled LPS at 4°C. *p < 0.05 compared with RAGE+/+ without pretreatment of unlabeled LPS. G, Relationship between RAGE and TLR2 or -4 in neutrophils and monocytes. One representative result from a total three repeats is shown. H, TLR2- and -4–positive percentage in neutrophils and monocytes. Data are presented as mean ± SEM. *p < 0.05.

  • FIGURE 4.
    FIGURE 4.

    LPS-induced septic shock in mice (9.00 ± 0.25 wk of age and 41.1 ± 1.3 g in body weight). A, Mortality after LPS administration. LPS was injected i.p. at the dosage of 50 mg/kg body weight. Closed circle, RAGE+/+ mice (n = 18); open circle, RAGE−/− mice (n = 13). Statistical analysis was performed using log-rank test. *p < 0.0001 between RAGE+/+ and RAGE−/− mice. Serum concentrations of TNF-α (B), IL-6 (C), ET-1 (D), and HMGB1 (E) determined by ELISA. Serum samples were taken at the indicated time points after LPS administration. Closed circle, RAGE+/+ mice (n = 12); open circle, RAGE−/− mice (n = 6); closed bar, RAGE+/+ mice (n = 8); open bar, RAGE−/− mice (n = 5). Data are presented as mean ± SEM. *p < 0.001 between RAGE+/+ and RAGE−/− mice, **p < 0.05 between RAGE+/+ and RAGE−/− mice.

  • FIGURE 5.
    FIGURE 5.

    sRAGE protection against LPS-induced septic shock in mice (8.22 ± 0.07 wk of age and 36.0 ± 0.5 g in body weight). A, Mortality after LPS administration. LPS was injected i.p. at the dosage of 50 mg/kg body weight. Closed circle, RAGE+/+ mice without treatment (n = 11); open circle, RAGE−/− mice without treatment (n = 14). Statistical analysis was performed using log-rank test. Thirty-five micrograms sRAGE per mouse was administered for the treatment. Closed triangle, RAGE+/+ mice with sRAGE treatment (n = 15); open triangle, RAGE−/− mice with sRAGE treatment (n = 14). *p < 0.02 between sRAGE-treated and untreated RAGE+/+ mice, **p < 0.01 between sRAGE-treated and untreated RAGE−/− mice. B, Detection of LPS–sRAGE complex in plasma. Anti-mouse Ig, κ beads (Beads) of polystyrene particles (51-9006274, BD Biosciences), or the negative control beads (C-Beads) (51-9006227, BD Biosciences) with anti-RAGE Ab (αRAGE; clone 278-13G4) were used. The anti-mouse Ig, κ beads (Beads), but not control beads (C-Beads), incubated with the RAGE Ab (αRAGE) and sRAGE could make a complex with FITC-LPS (upper panel). Depletion of sRAGE in this reaction mixture did not show positive signals (upper panel), indicating the usefulness of this detection system. sRAGE–FITC–LPS complex was detected in the plasma from mice with i.p. injection of FITC-LPS and sRAGE (lower panel). C, Histopathological findings. H&E stain of lung (original magnification ×100 and ×400) and liver (original magnification ×400) of RAGE+/+ and RAGE−/− mice treated or not treated with sRAGE. Tissues were removed and fixed at 25 h after LPS injection. D, Neutrophil infiltration evaluated by immunohistochemistry with anti-MPO Ab. Brown signal indicates neutrophil. Serum concentrations of TNF-α (E) at 1 h (*p < 0.05, **p < 0.037), IL-6 (F) at 6 h (*p < 0.022, **p < 0.014, p = 0.051 between sRAGE-treated or nontreated RAGE−/− mice.), ET-1 (G) at 24 h and HMGB1 (H) at 24 h after LPS injection. Results are presented as mean ± SEM. Liver+/+, liver from RAGE+/+ mice; Liver−/−, liver from RAGE−/− mice; Lung+/+, lung from RAGE+/+ mice; Lung−/−, lung from RAGE−/− mice.

Additional Files

  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Figures S1-S6 (PDF, 498 Kb) - Description:
      Figure S1. A, SPR assays with anti-AGE antibodies...
      Figure S2. SPR assays with degradated LPS.
      Figure S3. SPR assays using synthetic peptides.
      Figure S4. Plasma concentrations of LPS.
      Figure S5. Serum levels of S100A8/A9 and S100B.
      Figure S6. Plasma concentrations of LPS.
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