TY - JOUR
T1 - Efficient neutrophil activation requires two simultaneous activating stimuli
AU - Mol, Sanne
AU - Hafkamp, Florianne M. J.
AU - Varela, Laura
AU - Simkhada, Neena
AU - Taanman-Kueter, Esther W.
AU - Tas, Sander W.
AU - Wauben, Marca H. M.
AU - Kormelink, Tom Groot
AU - de Jong, Esther C.
N1 - Funding Information: Funding: This work was supported by a grant from the Dutch Arthritis Society No 17-1-403 and received funding from the EU’s H2020 research and innovation programme under Marie S. Curie cofund RESCUE grant agreement No 801540. Funding Information: This work was supported by a grant from the Dutch Arthritis Society No 17-1-403 and received funding from the EU?s H2020 research and innovation programme under Marie S. Curie cofund RESCUE grant agreement No 801540. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
PY - 2021/9/1
Y1 - 2021/9/1
N2 - 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.
AB - 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.
KW - Degranulation
KW - Extracellular vesicle release
KW - Mediator release
KW - NETosis
KW - Phagocytosis
KW - ROS production
UR - http://www.scopus.com/inward/record.url?scp=85115045520&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/ijms221810106
DO - https://doi.org/10.3390/ijms221810106
M3 - Article
C2 - 34576270
SN - 1661-6596
VL - 22
JO - International journal of molecular sciences
JF - International journal of molecular sciences
IS - 18
M1 - 10106
ER -