Potent NETosis inducers do not show synergistic effects in vitro

Introduction
NETosis is a process whereby neutrophils release chromatin into the surrounding extra­cellular matrix to form neutrophil extracellular traps (NETs). Under physiological conditions NETosis can be initiated by a variety of stimuli, including immune complexes, complement activation products, and a milieu of proinflammatory cytokines. Because overproduction of NETs is often related to the promotion or aggravation of autoimmune responses, we decided to assess how simultaneous activation of NETosis by different stimuli affects NET production.

Material and methods
NET formation was initiated by using combinations of three different NETosis inducers: phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (fMLP) and calcium ionophore (CaI). We measured fluorometry in real time, while microscopic visualisation served as an additional control for NET release. In total, 30 subjects free from infections or chronic diseases were enrolled in this study.

Results
We were able to demonstrate that in all cases NETosis induced by a combination of two stimuli resulted in diminished NETs production when compared to PMA and CaI single stimulations (p ≤ 0.001). The only cases in which double stimulation showed similar results to single stimulation were when we compared fMLP + CaI stimulation with fMLP single stimulation. Furthermore, when neutrophils were exposed to all three stimuli NETosis was almost entirely inhibited, compared to any single stimulation (p ≤ 0.001).

Conclusions
Our results show that simultaneous stimulation of neutrophils by different NETosis-inducing agents results in diminished formation of NETs compared to a single stimulation. This indicates that cells may possess an internal regulatory mechanism that prevents overgeneration of NETs among healthy people.