Regulation of the autophagic machinery in human neutrophils.
The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.
Mitroulis
Ioannis
I
First Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece.
Kourtzelis
Ioannis
I
Kambas
Konstantinos
K
Rafail
Stavros
S
Chrysanthopoulou
Akrivi
A
Speletas
Matthaios
M
Ritis
Konstantinos
K
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002103:Cadaverine / Q000031:analogs & derivatives / Q000032:analysis; D002867:Chromones / Q000494:pharmacology; D004396:Coloring Agents / Q000032:analysis; D004926:Escherichia coli; D006151:Guanosine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007249:Inflammation / Q000276:immunology; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010588:Phagosomes / Q000502:physiology / Q000648:ultrastructure; D011070:Poly I-C / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000187:drug effects / Q000502:physiology; D014158:Transcription, Genetic; D014617:Vacuoles / Q000502:physiology
D002867:Chromones; D004396:Coloring Agents; D009025:Morpholines; D017382:Reactive Oxygen Species; D025841:Small Ubiquitin-Related Modifier Proteins; D051193:Toll-Like Receptors; D006151:Guanosine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C025946:3-methyladenine; C068562:loxoribine; C008542:monodansylcadaverine; D000225:Adenine; D002103:Cadaverine; D013755:Tetradecanoylphorbol Acetate; D011070:Poly I-C; D020123:Sirolimus