Abstract
BackgroundDuring sepsis or sterile tissue injury, the nuclear protein high mobility group box 1 (HMGB1) can be released to the extracellular space and ultimately into systemic circulation, where it mediates systemic inflammation and remote organ failure. The proinflammatory effects of HMGB1 can be suppressed by recombinant thrombomodulin (rTM), in part through a mechanism involving thrombin–rTM-mediated degradation of HMGB1. Given that HMGB1 is proinflammatory but the HMGB1 degradation product (desHMGB1) is not, an analytical method that discriminates between these two molecules may provide a more in-depth understanding of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency.MethodsA peptide that has a shared amino-terminal structure with desHMGB1 was synthesized. C3H/lpr mice were immunized with the desHMGB1 peptide conjugate, and antibody-secreting hybridoma cells were developed using conventional methods. The reactivity and specificity of the antibodies were then analyzed using antigen-coated enzyme-linked immunosorbent assay (ELISA) as well as antibody-coated ELISA. Next, plasma desHMGB1 levels were examined in a cecal ligation and puncture (CLP)-induced septic mouse model treated with rTM.ResultsThrough a series of screening steps, we obtained a monoclonal antibody that recognized desHMGB1 but did not recognize intact HMGB1. ELISA using this antibody specifically detected desHMGB1, which was significantly increased in CLP-induced septic mice treated with rTM compared with those treated with saline.ConclusionsIn this study, we obtained a desHMGB1-specific monoclonal antibody. ELISA using the novel monoclonal antibody may be an option for the in-depth analysis of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency.
Highlights
During sepsis or sterile tissue injury, the nuclear protein high mobility group box 1 (HMGB1) can be released to the extracellular space and into systemic circulation, where it mediates systemic inflammation and remote organ failure
These findings suggest the importance of analyzing HMGB1 levels in inflammatory diseases; a mixture of different HMGB1 subtypes is present in analytical samples, which poses a challenge for the in-depth understanding of HMGB1-induced pathogenicity (Xue et al 2021)
These findings indicate that HMGB1 and desHMGB1 are very different in terms of their roles in sepsis pathogenesis, and suggest that an analytical method that can discriminate between these two molecules is required for an in-depth understanding of HMGB1-induced pathogenicity
Summary
During sepsis or sterile tissue injury, the nuclear protein high mobility group box 1 (HMGB1) can be released to the extracellular space and into systemic circulation, where it mediates systemic inflammation and remote organ failure. Extracellular HMGB1 levels are increased in ischemia–reperfusion injury (Andrassy et al 2008; Yamamoto et al 2010), heatstroke (Tong et al 2011), acute coronary syndrome (Hashimoto et al 2012), and rheumatoid arthritis (Taniguchi et al 2003), and are associated with poor outcomes (Hatada et al 2005; Volz et al 2012) These findings suggest the importance of analyzing HMGB1 levels in inflammatory diseases; a mixture of different HMGB1 subtypes is present in analytical samples, which poses a challenge for the in-depth understanding of HMGB1-induced pathogenicity (Xue et al 2021). We have previously demonstrated that the proinflammatory effects of HMGB1 can be suppressed by rTM (Abeyama et al 2005), in part through a mechanism involving thrombin–rTM-mediated degradation of HMGB1 (Ito et al 2008). We screened for monoclonal antibodies that recognize desHMGB1 but do not recognize intact HMGB1 for the discriminative analysis of HMGB1 subtypes
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