Activation of mononuclear phagocytes in diabetes mellitus

Diabetes mellitus is a metabolic disease characterized at the immunological level by functional abnormalities of monocytes and dysregulation of inflammation. The aim of this study was to define the functional and biochemical abnormalities of diabetic monocytes to characterize the phenotype associated with poor diabetic control. Two basic functions of mononuclear phagocytes, oxidative burst and proinflammatory cytokine secretion were evaluated in 34 diabetic patients and 26 healthy non-diabetic individuals. Diabetic cells exhibited an extreme proinflammatory phenotype characterized, in part, by excessive superoxide (O2-) generation (p < 0.01) and tumor necrosis factor a (TNF-alpha) secretion (p < 0.05). Investigation of the hyper-inflammatory phenotype revealed a casual link to the priming by advanced glycated endproducts (AGE) through receptor for AGE (RAGE) and activation of protein kinase C (PKC). AGE has long been considered to play a major role in the pathogenesis of diabetic complications. RAGE ligands were found to be potent activators of PKC as evidenced by its elevated activity in the plasma membrane, increased phosphorylation of PKC isoforms and PKC substrates, and translocation of several PKC isoforms to the plasma membrane. Activation of PKC and induction of p47phox translocation by RAGE ligand provide the molecular basis of primed oxidative burst observed in diabetic monocytes. One particular PKC substrate, induced by RAGE activation, was markedly up-regulated in diabetic mononuclear phagocytes, suggesting RAGE activation might play a role in altered PKC signaling in diabetes. This protein was later identified as pleckstrin by mass spectrometry and immunoprecipitation. To investigate the importance of different PKC isoforms and pleckstrin in proinflammatory cytokine release induced by RAGE, expression of each isoform or pleckstrin was suppressed by RNA interference. Suppression of protein expression (PKC alpha, beta and pleckstrin) was performed in THP-1 monocytic cell-line. The data demonstrated that PKC beta and pleckstnn play a critical role in the RAGE signaling transduction leading to cytokine secretion. Taken together, the data demonstrate that monocyte priming in diabetes is mediated, in part, by AGE binding to RAGE, which results in proinflammatory phenotype. Further, RAGE signal through a newly identified PKC pathway that involves phosphorylation of pleckstrin, which is chronically active in poorly controlled diabetes.