The formation of neutrophil extracellular traps (NETs) depends on the generation of reactive oxygen species (ROS). Previous studies revealed that both NADPH oxidase and myeloperoxidase (MPO) are required for NET release. However, the contribution of various ROS as well as the role of mitochondria-derived ROS has not been addressed so far. In the present study we aimed to investigate in a systematic and comprehensive manner the contribution of various ROS and ROS-generating pathways to the PMA-induced NET release. By using specific inhibitors, the role of both NADPH oxidase- and mitochondria-derived ROS as well as the contribution of superoxide dismutase (SOD) and MPO on the NET release was assessed. We could demonstrate that NADPH oxidase function is crucial for the formation of NETs. In addition, we could clearly show the involvement of MPO-derived ROS in NET release. Our results, however, did not provide evidence for the role of SOD- or mitochondria-derived ROS in NET formation.

Neutrophil extracellular traps (NETs) have been suggested to play a pathophysiological role in several autoimmune diseases. Since NET-formation in response to several biological and chemical stimuli is mostly ROS dependent, in theory any substance that inhibits or scavenges ROS could prevent ROS-dependent NET release. Therefore, in the present comprehensive study, several antioxidative substances were assessed for their capacity to inhibit NET formation of primary human neutrophils in vitro. We could show that the flavonoids (−)-epicatechin, (+)-catechin hydrate, and rutin trihydrate as well as vitamin C and the pharmacological substances N-acetyl-L-cysteine and 5-aminosalicylic acid inhibited PMA induced ROS production and NET formation. Therefore, a broad spectrum of antioxidative substances that reduce ROS production of primary human neutrophils also inhibits ROS-dependent NET formation. It is tempting to speculate that such antioxidants can have beneficial therapeutic effects in diseases associated with ROS-dependent NET formation.

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MP's therapeutic effects.

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