Introduction RAGE is expressed by both alveolar epithelial and endothelial cells. The receptor can bind to pro-inflammatory ligands, including HMGB1 which is elevated in ALI. RAGE negative mice show a reduced inflammatory response when challenged with LPS. We hypothesised that RAGE activation in the pulmonary circulation propagates inflammation in ALI and that soluble RAGE (sRAGE) can act as a scavenger receptor, reducing RAGE-mediated inflammation.
Aim To investigate HMGB1 RAGE-dependent activation of pulmonary microvascular endothelial cells, and resultant production of inflammatory cytokines and tissue-destructive proteases in a model of ALI.
Methods Primary HPMECs (human pulmonary microvascular endothelial cells) were stimulated with clinically relevant concentrations of HMGB1. MAPK phosphorylation was assessed by Western Blot. Supernatants collected at 72 h were analysed by ELISA for IL-8, Tissue Inhibitors of Metalloproteinases (TIMPs)-1 and -2. Experiments were repeated both with a RAGE-blocking antibody and in the presence of sRAGE.
Results HMGB1 increased phosphorylation of ERK1/2 at 15 min and p38 at 30 min after stimulation HMGB1 increased IL-8 secretion (from 6.46 ng/ml to 8.75 ng/ml, p<0.01) and significant decrease in TIMP-1 secretion (from 19.79 ng/ml to 16.9 ng/ml, p<0.05) at 72 h. MAPK activation, IL-8 increase and TIMP-1 decrease was significantly reversed in the presence of sRAGE (p<0.05) (Abstract S52 Figure 1). Incubating cells with a RAGE blocking antibody inhibited MAPK phosphorylation by HMGB1.
Conclusions Data suggest that RAGE ligation leads to an increase in pulmonary endothelial cell activation and IL-8 release. In vivo this would increase inflammatory cell influx into the pulmonary environment, propagating inflammation. This is combined with a decrease in TIMP protection potentially increasing degradation of the basement membrane by functionally unopposed proteinases. That these changes can be partially rescued using sRAGE shows that it could potentially decrease inflammatory damage in ALI.
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