Cytokine Network & NETs.

The process of forming and releasing neutrophil extracellular traps (NETs) can be regulated by exogenous and endogenous factors, including cytokines. The study aimed to assess the impact of proinflammatory cytokines, IL-15, IL-17, IL-18, and anti-inflammatory IL-10 on the formation of NETs, all in comparison to IL-8 and pathogenic factors: LPS, fMLP. Also, the expression of myeloperoxidase (MPO), one of the main elements of neutrophil traps, was evaluated. After isolating the neutrophils with Polymorphprep™, the cells were sorted using CD16 MACS® microbeads and incubated with selected factors. The formation of NETs was registered using a BD Pathway 855 microscope system and the expression of MPO was evaluated using flow cytometry. The amounts of circulating DNA in cell supernatants was fluorescently quantified. Microscopic photographs indicated that rhIL-15, rhIL-17, rhIL-18 and fMLP induce formation and release of NETs at a similar timespan, while in the presence of rhIL-10, the formation of the traps was delayed. The presence of the studied cytokines indicated two populations of neutrophils displaying differing MPO expression (MPOlow and MPOhigh). Moreover, stimulation of neutrophils with LPS and fMLP revealed two populations of these cells that differed not only in the expression of MPO, but also in size.