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 ϩ/ϩ ) neutrophils demonstrated significantly greater ability to kill Eschericia coli compared with RAGE Ϫ/Ϫ neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE Ϫ/Ϫ as compared with RAGE ϩ/ϩ mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NA-DPH) 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. coliinduced 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.receptor for advanced glycation end products; nicotinamide adenine dinucleotide phosphate oxidase; peritonitis; sepsis; inflammation; Eschericia coli NEUTROPHILS play central roles in acute inflammatory and innate immune responses through producing anti-bacterial peptides, cytokines, and other proinflammatory mediators, including reactive oxygen intermediates, and contributing to the formation of extracellular traps (8,22,33). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a high-output, superoxide-generating system, is an important source of the reactive oxygen intermediates produced by activated neutrophils. Deficiency in NADPH oxidase is associated with diminished ability of neutrophils to effectively kill bacteria. There is increased susceptibility to infection with Staphylococci and other extracellular bacteria in patients with chronic granulomatous disease, a condition associated with a hereditary defect of NADPH oxidase (14,15,17,18).The receptor for advanced glycation end products (RAGE) recognizes a diverse spectrum of ligands, including nonenzymatically modified glycoproteins such as advanced glycation end products (AGE) (31, 32), as well as high mobility group box 1 protein (HMGB1) and calgranulin (calcium binding cellular factors, S100B) (19,20). Cellular activation through RAGE engagement i...

HMGB1 promotes neutrophil extracellular trap formation through interactions with Toll-like receptor 4

“…Custom antibody mixtures (Abs) and negative selection columns for neutrophil isolation were from StemCell Technologies (Vancouver, British Columbia, Canada). Recombinant HMGB1 and mutant HMGB1 lacking the COOHterminal tail (⌬C-HMGB1) were prepared as previously described (3,42). Polyclonal antibodies to neutralize HMGB1 were prepared as previously described (12).…”

“…Bone marrow neutrophils were purified using a negative selection column purification system, as previously described (42,50). Briefly, bone marrow cell suspensions were isolated from the femur and tibia of a mouse by flushing with RPMI 1640 medium with 5% FBS.…”

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

“…The efficiency of bacterial eradication in vivo was performed as described previously (42,43). Mice were subjected to administration of metformin (125 mg/kg of body weight; IP) for 12 h, compound C (3 mg/kg of body weight; IP) or vehicle (PBS) for 2 h before IP injection of ampicillin-resistant E. coli (2 × 10 8 ).…”

“…This possibility was examined using a murine model for peritonitis-induced sepsis (42,43). As shown in Figures 5E and 5F, there were significant decreases in the numbers of viable bacteria recovered from mice that received metformin as compared with controls.…”

Ligands of RAGE-Proteins: Role in Intercellular Communication and Pathogenesis of Inflammation

“…После связывания с RAGE или TLR4 HMGB1 активирует клетки сосуди-стого эндотелия и макрофаги/моноциты, которые на-чинают экспрессировать провоспалительные цитокины, хемокины и молекулы адгезии. Находясь во внеклеточ-ном пространстве, он является сильным провоспалитель-ным медиатором при эндотоксемии, артритах и сепсисе [62]. Кроме того, HMGB1 может секретироваться сти-мулированными макрофагами и моноцитами.…”