98.    UDP –G activates oxidative stress and respiratory burst in human                                          polymorphonuclear cells

Previously we have demonstrated that uridine diphosphate glucose (UDP-G) activates chemotaxis of human blood neutrophils (PMN). The aim of this study is to show that one of the mechanisms implicated in PMN activation by UDP-G is oxidative stress. Methodology: Oxidative stress and free radical production was evaluated by measuring spontaneous chemiluminescence (CL) of cells with a scintillation photon counter, and phagocyte respiratory burst by measuring oxygen consumption with an oxygen electrode type Clark at 37 ºC, at basal condition or after 15 min of PMN activation with lipopolysacharides (LPS, 2μg/mL), phorbol myristate acetate (PMA, 20 ng/mL) or UDP-G (0.1 mM). To establish the activation effect of the three agonists, the stimulation index (SI) was calculated for each different treatments as the ratio (quotient) between the mean of the parameter evaluated in activated cells and non-activated cells. PMN without stimulation were used as control. Results: The optimal cell concentration to evaluate oxidative stress in PMN was 1.5 to 15 x 104 PMN/mL when LPS was used as stimuli, and over 15 x 104 PMN/mL when were exposure to PMA, because an enhancement in CL of 35% (p<0.05) and 56% (p<0.01) takes place with SI of 1.56 and 2.20 for both LPS and PMA respectively (15 x 104 PMN/mL, control: 8.8 ± 1.4 cps/cm2). The optimal cell concentration to evaluate the oxygen consumption in activated PMN with PMA was: 10 x 104 cells/mL, showing a significantly increase of 40% (p<0.001), and 50 x 104 PMN/mL, with increment of 100% (p<0.05). When UDP-G was used as stimuli, an increase of 80 to 100% in the respiratory burst was observed (p<0.001) with 10 or 50 x 104 cells/mL (control: 7.2 ± 1 and 1.8 ± 0.6 nmol O2/min/ 106 cells, respectively), and SI of 1.2, 1.4 and 1.8 for LPS, PMA and UDP-G respectively for 10 x 104 cells/mL. When working with 2 x 104 cells/mL, no significantly differences of SI was observed for the agonists separately however, a synnergic effect was observed when the PMN were incubated sequentially with LPS plus UDP-G, with increment in the PMN oxygen consumption of 26% (p<0.05) respect to the control cells (55 ± 3 nmol O2/min/ 106 cells) and SI of 1.3. Discussion: Our results indicate that UDP-G UDP-G is able to activate oxidative stress and respiratory burst in PMN, and LPS has a synergistic effect with it.