TLRs-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor-alpha by Human Peripheral Blood Neutrophils
necrosis factor-alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand) and lipopolysaccharide (TLR4 ligand). Luminoland lucigenin-dependent chemiluminescence was used to detect reactive oxygen species production. The concentration of tumor necrosis factor-alpha was measured by enzyme immunoassay. The plots of dependences of the sum of light emission of luminol- and lucigenin-dependent chemiluminescence on concentration of each ligand were viewed as saturation curves. Comparison of the sum of light emission of lucigenin-dependent chemiluminescence (production of superoxide anion radical) and the the sum of light emission of luminol-dependent chemiluminescence (total production of reactive oxygen species) showed that the contribution of NADPH-oxidase to the total TLRsmediated production of oxidants can reach 40-50%. To compare the ability of ligands of TLRs stimulate the production of reactive oxygen species and tumor necrosis factor-alpha by neutrophils, stimulation indices were calculated. It has been established that activation of neutrophils with zymosan leads to higher production of reactive oxygen species (8-fold higher) rather than tumor necrosis factor-alpha production. Unlike zymosan, lipopolysaccharide stimulated in a greater degree (2-fold higher) the production of tumor necrosis factor-alpha than reactive oxygen species production. Peptidoglycan is intermediate in position between these ligands. Thus, the production of effector molecules, reactive oxygen species and tumor necrosis factor-alpha, by human peripheral blood neutrophils depends on the nature of the TLR ligand.