Advanced oxidation protein products (AOPP) inhibit bovine neutrophil activity in vitro.

Advanced oxidation protein products (AOPP) are novel markers of protein oxidation that were first described and characterized in plasma of uremic patients. Their formation is mainly dependent from chlorinated agents related to neutrophil and monocyte activation, thus being also considered a good indicator of the inflammatory response. In addition, AOPP can activate both human neutrophils and monocytes in vitro. The observation that AOPP were higher in plasma of cows affected by late embryonic loss raised our interest in the biological role of AOPP in dairy cattle. The aim of this work was to study the effects of AOPP on bovine neutrophil activation in vitro. Standard AOPP-BSA was produced by oxidizing bovine serum albumin (BSA) with hypochlorous acid. Bovine neutrophils were isolated by Ficoll gradient from peripheral blood obtained from healthy dairy heifers aged 10-13 months (N=10). The effects of either BSA or AOPP-BSA in the culture medium were tested in neutrophils either stimulated or unstimulated with phorbol myristate acetate (PMA,1 ug/ml) over a 3-hour period. PMA-stimulated and unstimulated neutrophils incubated without proteins in the culture medium were used as the control. Myeloperoxidase (MPO)-dependent formation of ROS was measured by chemiluminescence using luminol as substrate during the whole incubation period at 5-minute intervals, and results were analysed as the area under the curve (AUC). Parallel experiments were conducted to assess cell viability using both LDH and MTT assays. AOPP-BSA failed to trigger any response in the unstimulated bovine neutrophils. The ROS generation in PMA-stimulated was significantly lower (P<0.05) in neutrophils cultured with AOPP-BSA (AUC: 4860±35 CPS*min) in comparison to those cultured with both BSA (AUC: 14528±53) and without protein in the culture medium (AUC:7342±48 CPS*min). Cell viability, measured by the MTT assay, was significantly lower in PMA-stimulated neutrophils incubated with AOPP-BSA (P<0.05). Potential effects of BSA and AOPP-BSA on luminol reaction were studied in a cell-free system, by activating the MPO enzyme with increasing concentration of hydrogen peroxide in presence of either BSA or AOPP-BSA. The AUC observed in presence of BSA (13004±70 CPS*min) was not significantly different from that measured in presence of AOPP-BSA (14993±65 CPS*min).The results of this study suggested that AOPP-BSA inhibits the response of bovine neutrophil to PMA activation possibly by affecting cell viability. Moreover, it is noteworthy to underline that the bovine neutrophil response to AOPP-BSA observed in this study was totally different than that reported for the human neutrophils.