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.
Highlights
Neutrophils are usually the first responders to an infection, and their primary role is killing invading pathogens [1]
In the current study we provide evidence that peripheral blood neutrophils from healthy donors require two simultaneous stimuli for optimal activation resulting in degranulation, mediator release and extracellular vesicle (EV) release
reactive oxygen species (ROS) production is more dependent on the type of stimulation than on dual stimulation, while phagocytosis appears already close to its maximum capacity when only one type of stimulus is present
Summary
Neutrophils are usually the first responders to an infection, and their primary role is killing invading pathogens [1]. Neutrophils influence other immune cells and thereby can modulate both innate and adaptive immune responses [2,3]. Neutrophils are present in a resting state but in response to invading microbes, neutrophils migrate and become activated at the site of infection. Neutrophils employ a variety of mechanisms to eliminate pathogens and modulate the function of surrounding cells [5,6]. These mechanisms include degranulation, phagocytosis, reactive oxygen species (ROS)
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