Nitric Oxide-Dependent Regulation of Cytokines Release in Type-II Diabetes Mellitus

The mechanism of release of proinflammatory cytokines by blood granulocytes in diabetes is unknown. We investigated whether diabetes mellitus affects the production of cytokines by granulocytes (PMN) and mononuclear cells (PBMCs) and whether this is modulated by NO. Isolated PMN and PBMC from with or without type-II diabetes mellitus were incubated at 37°C for 6？h with S-nitroso-N-acetylpenicillamine (SNAP) at 0, 1, and 100？μM with or without lipopolysaccharides (LPS) stimulation (1？μg/mL). Supernatants were assayed for tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) by sandwich ELISA. Significant increases in TNF-α and IL-8 were observed only in PMN from diabetic subjects with or without LPS stimulation and that exogenous NO inhibited further production of cytokines in a concentration-dependent manner. However, activity of PBMC when stimulated with LPS was greatly enhanced by diabetes, but not affected by NO production. Hence, suggesting that granulocytes activation and participation in diabetes related complications is modulated by NO bioavailability. 1. Introduction Production of basal levels of superoxide ( ) and nitric oxide (NO) radical by granulocytes is known to be enhanced in patients with type-II diabetes [1]. Recently it was reported in healthy subjects that NO production by granulocytes is mediated via cyclic AMP and cGMP pathways [2]. However, cyclic AMP and cGMP-elevating agents inhibited the production of NO in granulocytes isolated from human subjects with type-II diabetes, thus suggesting a possible disease-induced adapted metabolic response [2]. The consequences of this altered metabolic response on host defense and inflammation hence is unknown. Previously we demonstrated that NO-mediated pathway induce a dose-dependent release of proinflammatory cytokines in human monocytes through the modulation of nuclear factor-kappa-B-DNA binding activity [3] However, the mechanism of release of proinflammatory cytokines by blood granulocytes especially in diabetes yet remains not clear. In this study, we tend to investigate whether diabetes mellitus similarly affects the production of cytokines by granulocytes (PMN) and whether NO modulates this. 2. Materials and Methods 2.1. Reagents The following reagents were all obtained from Sigma Biochemicals (Dorset, UK): Hanks balanced salt solution, S-nitroso-N-acetylpenicillamine (SNAP), dextran sedimentation solution, Histopaque-1077 medium, and lipopolysaccharides (LPS). 2.2. Granulocytes Separation PMNs were purified by dextran sedimentation followed by gradient density centrifugation on