Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli

Abstract

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli.

Neutrophils Require Activation to Express Functional Cell-Surface Complement Receptor Immunoglobulin

“…Thus, to increase the significance of our findings we examined whether engagement of CRIg promoted the release of NETs. In these studies, we used GM-CSF as a priming agent since this has been shown to increase the expression of CRIg ( Figure 3 ), but not the release of NETs ( 18 ). To view the release of NETs, the DNA dye PI and granule constituent, MPO, were measured in treated cells, both microscopically and by flow cytometry.…”

Vitamin D3 Priming of Dendritic Cells Shifts Human Neutrophil-Dependent Th17 Cell Development to Regulatory T Cells

“…As shown in our previous study, donor-to-donor variation exists in Th17 cell induction by neutrophils ( 9 ). This could depend on the ability of neutrophils to become activated in co-culture to secrete neutrophil elastase ( 9 ), among others, given that neutrophil activation in response to diverse stimuli varies between healthy donors as well ( 43 ). Our observations are in line with two studies demonstrating reduced Th17 cell development by VD3-treated DCs.…”

Enterococcus faecalisandStaphylococcus aureusmixed species infection attenuates pathogen-specific neutrophil responses and impairs bacterial clearance

“…ROS is an important regulator of NET formation [70]; however, E. faecalis -induced ROS does not lead to NETosis. This is not unprecedented, given that neutrophils stimulated with GM-CSF and LPS also produce intracellular ROS that does not coincide with NET formation [71]. One potential explanation is that E. faecalis -induced ROS accumulates within phagosome which, together with fusion of primary granules to the phagosome [72, 73], could result in sequestration of NETosis activators such as neutrophil elastase (NE) and myeloperoxidase (MPO) [74], eventually leading to the inhibition of S. aureus -induced NETosis in mixed-infection.…”