High-glucose-induced changes in macrophage secretome: regulation of immune response.

Secretory products from infiltrating macrophages have been thought to play crucial roles in development and progression of diabetic complications in various tissues/organs. Nevertheless, diabetes-induced changes in macrophage secretory products remained largely unknown. We thus analyzed high-glucose (HG)-induced changes in secretome of human macrophages derived from U937 human monocytic cell line after phorbol 12-myristate 13-acetate (PMA) activation. Serum-free culture supernatants were collected from macrophages exposed to 5.5 mM glucose (NG-M-sup) (normal control), 25 mM glucose (HG-M-sup), or 5.5 mM glucose + 19.5 mM mannitol (MN-M-sup) (osmotic control) for 16 h. After dialysis and lyophilization, secreted proteins were subjected to 2-DE analysis (n = 5 gels derived from 5 independent cultures per group). Quantitative analysis and statistics revealed 23 protein spots whose secretory levels significantly differed among the three conditions. These proteins were successfully identified by nanoLC-ESI-MS/MS analyses and changes in levels of heat shock protein 90 (HSP90), HSP70, HSP60, and β-actin were confirmed by Western blotting. Global protein network and functional enrichment analyses revealed that the altered proteins in HG-M-sup were involved mainly in regulation of immune response that might communicate with other bystander cells through the release of extracellular vesicles. These data may lead to a wider view of pathogenic mechanisms of diabetic complications.