Differential activation of RAGE by HMGB1 modulates neutrophil-associated NADPH oxidase activity and bacterial killing

Abstract

The receptor for advanced glycation end products (RAGE) plays an important role in host defense against bacterial infection. In the present experiments, we investigated the mechanisms by which RAGE contributes to the ability of neutrophils to eradicate bacteria. Wild-type (RAGE(+/+)) neutrophils demonstrated significantly greater ability to kill Escherichia coli compared with RAGE(-/-) neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE(-/-) as compared with RAGE(+/+) mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and enhanced killing of E. coli, and intraperitoneal injection of AGE enhanced bacterial clearance during peritonitis. However, incubation of neutrophils with high mobility group box 1 protein (HMGB1), which also binds to RAGE, diminished E. coli-induced activation of NADPH oxidase in neutrophils and bacterial killing both in vitro and in vivo. Deletion of the COOH-terminal tail of HMGB1, a region necessary for binding to RAGE, abrogated the ability of HMGB1 to inhibit bacterial killing. Incubation of neutrophils with HMGB1 diminished bacterial or AGE-dependent activation of NADPH oxidase. The increase in phosphorylation of the p40(phox) subunit of NADPH oxidase that occurred after culture of neutrophils with E. coli was inhibited by exposure of the cells to HMGB1. These results showing that HMGB1, through RAGE-dependent mechanisms, diminishes bacterial killing by neutrophils as well as NADPH oxidase activation provide a novel mechanism by which HMGB1 can potentiate sepsis-associated organ dysfunction and mortality.

Fig. 1.

Effects of advanced glycation end products (RAGE) deficiency on bacterial killing in vitro and in vivo. A: bone marrow neutrophils (0.5 × 10⁶ / ml) obtained from RAGE^+/+ or receptor for RAGE-deficient mice (RAGE^−/−) were pretreated with AGE (0 or 100 μg/ml) for 15 min and then incubated with Eschericia coli (10⁶/ml) for an additional 90 min. Means ± SD values were obtained from three independent experiments. **P < 0.01 comparing RAGE^+/+ with RAGE^−/− or *P < 0.05 comparing control RAGE^+/+ with AGE-treated RAGE^+/+ neutrophils. The numbers of surviving bacteria were calculated as the percentage of E. coli numbers obtained after culture without neutrophils. B and C: wild-type (WT) (RAGE^+/+) or RAGE^−/− mice were subjected to intraperitoneal injection of AGE (0 or 300 μg) and E. coli (10⁴) with peritoneal lavage 3 h later. The numbers of viable E. coli (B) and total numbers of neutrophils (C) obtained from peritoneal lavages are shown. The number of E. coli (9.1 ± 1.8 × 10⁴ CFU/ml) obtained from peritoneal lavage of RAGE^+/+ mice was used as a control (100% viability) to calculate percent viability of E. coli recovered from AGE-treated and from RAGE-deficient mice. Values are means ± SD (n = 3). **P < 0.01 comparing RAGE^+/+ with RAGE^−/− or *P < 0.05 comparing control RAGE^+/+ with AGE-treated RAGE^+/+ mice.

Fig. 2.

Effects of high mobility group box 1 protein (HMGB1) on bacterial killing. A: neutrophils (0.5 × 10⁶/ml) were incubated with HMGB1 (0 or 300 ng/ml) for 15 min and then cultured with E. coli (10⁶/ml) for 90 min, or E. coli were incubated without neutrophils. Means ± SD values were obtained from three independent experiments. **P < 0.01 comparing RAGE^+/+ with RAGE^−/− or comparing HMGB1-treated RAGE^+/+ with RAGE^+/+ neutrophils. In B and C, mice were given E. coli (10⁴ ip) with or without HMGB1 (0 or 300 ng ip), and then peritoneal lavages were obtained 3 h later. The percentage of viable bacteria (B) and total numbers of neutrophils (C) in peritoneal lavage are shown. The number of E. coli (10.4 ± 1.9 × 10⁴ CFU/ml) obtained from peritoneal lavage of RAGE^+/+ mice not given HMGB1 was used as a control (100% viability). Values are means ± SD (n = 3). **P < 0.01 compared with untreated RAGE^+/+ controls. D–F: deletion of the COOH-terminal region of HMGB1 (ΔC-HMGB1) diminishes the ability of HMGB1 to inhibit bacterial killing. D: number of E. coli obtained after 90 min of culture with neutrophils that were pretreated with HMGB1 or ΔC-HMGB1 (0 or 300 ng/ml) for 15 min. Values are means ± SD (n = 3). *P < 0.05 comparing untreated to HMGB1-treated cells. E and F: percentages of remaining viable bacteria (E) and numbers of neutrophils (F) in peritoneal lavages that were obtained 3 h after mice received E. coli (10⁴ ip) with or without full-length HMGB1 or mutant ΔC-HMGB1. The number of E. coli (9.2 ± 1.4 × 10⁴ CFU/ml) obtained from peritoneal lavage of mice not exposed to HMGB1 or ΔC-HMGB1 was used as a control (100% viability). Values are means ± SD (n = 3). *P < 0.05 compared with untreated cells.

Fig. 3.

Effects of AGE, HMGB1, S100B, and ΔC-HMGB1 on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in E. coli-stimulated neutrophils. A: NADPH oxidase activity was determined in WT (RAGE^+/+) and RAGE-deficient (RAGE^−/−) neutrophils after incubation of the cells with AGE (0 or 100 μg/ml) or HMGB1 (0 or 300 ng/ml) for 15 min followed by culture with E. coli (10⁶/ml) for an additional 15 min. Values are means ± SD (n = 3). *P < 0.05, NS, not significant. B: NADPH oxidase activity in neutrophils treated with HMGB1 or ΔC-HMGB1. Cells were cultured with HMGB1 (0 or 300 ng/ml) or ΔC-HMGB1 (0 or 300 ng/ml) for 15 min and then exposed to E. coli (0 or 10⁶/ml) for 15 min. Values are means ± SD (n = 3). *P < 0.05, ***P < 0.001, NS, not significant. C: neutrophils were treated with HMGB1 (0, 3, 30, or 300 ng/ml) for 15 min and then incubated with AGE (0 or 100 μg/ml) and E. coli (10⁶/ml) for a further 15 min. NADPH oxidase activity was then measured. Values are means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. D: NADPH oxidase activity was measured in neutrophils treated with AGE (0 or 100 μg/ml) or simultaneously with AGE (100 μg/ml) and HMGB1 (300 ng/ml) for 15 min. Values are mean ± SD (n = 3). *P < 0.05 compared with untreated cells or #P < 0.05 compared with neutrophils treated with AGE alone. E: NADPH oxidase activity was determined after incubation of neutrophils (RAGE^+/+) with S100B (0 or 50 μg/ml) for 15 min. Values are means ± SD (n = 3). F: ability of neutrophils to eradicate E. coli was determined after exposure of neutrophils (0.5 × 10⁶/ml) to S100B (0 or 50 μg/ml) for 15 min followed by inclusion of E. coli (0 or 10⁶/ml) in the cultures for an additional 120 min. Values are means ± SD (n = 3). *P < 0.05 compared S100B treated with control. G: representative images show expression of RAGE on the cell surface of control neutrophils and neutrophils treated with HMGB1 or AGE for 2 h. Green, RAGE; blue, nuclei.

Fig. 4.

Inactivation of NADPH oxidase diminishes eradication of bacteria in vitro and in vivo. A: effects of HMGB1 or AGE on the ability of WT and p47^phox mutant neutrophils to kill bacteria. Neutrophils (WT or p47 mutant) were incubated with HMGB1 (0 or 300 ng/ml) for 15 min or with AGE (0 or 100 μg/ml) for 15 min followed by culture with E. coli (10⁶/ml) for an additional 15 min. NADPH oxidase activity was then measured. Values are means ± SD (n = 3). *P < 0.05. B and C: wild-type (C57BL/6) mice and mice deficient in NADPH oxidase activity (C57BL/6J-Ncf1m1J/J) were subjected to intraperitoneal administration of E. coli (10⁴), and then the numbers viable E. coli (p47 WT 8 ± 7 × 104 CFU/ml and p47 mutant 1.45 ± 0.31 × 104 CFU/ml) (B) and total number of neutrophils (C) determined in peritoneal lavages were obtained 3 h later. Values are means ± SD (n = 3). ***P < 0.001 compared with WT mice. D and E: representative images show the level of DCF fluorescence in p47^phox WT or mutant neutrophils treated with PMA (0 or 50 nM) for 30 min (D) or after exposure RAGE^+/+ or RAGE^−/− neutrophils to E. coli (E).

Fig. 5.

HMGB1 diminishes p40^phox phosphorylation in E. coli-stimulated neutrophils. Representative Western blots show p40^phox phosphorylation and actin levels obtained from neutrophils treated with E. coli (10⁶) for 0, 10, 20, or 30 min. Cells were cultured with HMGB1 (0 or 300 ng/ml) for 15 min before addition of E. coli. Values are means ± SD (n = 3). *P < 0.05 comparing neutrophils cultured with E. coli alone to control (untreated) neutrophils or to neutrophils treated with HMGB1 and E. coli.