The Effect of LPS on Neutrophils from Patients with High Risk of Type 1 Diabetes Mellitus in relation to IL‐8, IL‐10 and IL‐12 Production and Apoptosis In Vitro

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Abstract
Innate immunity includes neutrophil inflammatory function, tissue destruction and regulatory cytokine production. Programmed cell death (apoptosis) is postulated to be a key mechanism for neutrophil elimination during inflammation. The aim of the present study was to evaluate the neutrophil apoptosis in relation to IL‐8, IL‐10 and IL‐12 production in vitro by neutrophils of patients suffering from diabetes mellitus (DM)1 and the first‐degree relatives of patients with DM1. The early stage of neutrophils apoptosis was assessed morphologically, and the later stage by DNA‐binding dye propidium iodide, both after treatment with lipopolysaccharide (LPS), insulin or anti‐CD95 antibody (Ab) as stimulators. CD16 (FcγRIII) receptor expression was also evaluated. Production of IL‐8, IL‐10, and IL‐12 cytokine was evaluated in supernatant after neutrophil incubation for 21 h in culture medium alone, in medium in the presence of LPS, insulin or anti‐CD95 antibody (Ab). Cytokine concentrations were measured by enzyme‐linked immunosorbent assay (ELISA) method using commercially available kits. Our study demonstrates that LPS inhibits the early stage of apoptosis (as evaluated morphologically) of healthy donors' neutrophils. The LPS‐dependent early apoptosis inhibition of neutrophil of patients with DM1 or in prediabetics was decreased in comparison with control. The later stage of apoptosis of neutrophils treated in vitro with anti‐CD95 Ab of patients suffering from DM1 was decreased in comparison with prediabetics and healthy donors (propidium iodide (PI) staining). LPS‐induced production of anti‐apoptotic cytokines IL‐8, IL‐10 by neutrophils of prediabetic and patients with DM1 was increased. The formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced proapoptotic reactive oxygen intermediates (ROI) production was significantly higher in DM1 patients. We have concluded that neutrophils from prediabetic and diabetic patients demonstrated the misbalance in anti‐apoptotic IL‐8 and IL‐10 cytokine and proapoptotic ROI production. LPS‐dependent IL‐12 overproduction by neutrophils is responsible for the switch in T helper Th1/Th2 balance to Th1 and in this way may participate in inflammation and autoimmune DM1 progression.