Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence.
High pressure frozen (HPF), cryo-substituted microtome sections of 2 μm thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1-2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis.
Department of Analytical Chemistry, Ghent University, Ghent, Belgium.
Björn
http://orcid.org/0000-0002-6759-4195
B
De Samber
Department of Clinical Microbiology / MIMS, Umeå University, Umeå, Sweden.|Microbial Immunology Research Group, Hans Knöll Institute / Leibniz-Institute for Natural Product Research and Infection Biology, Jena, Germany.
Maria J
MJ
Niemiec
Department of Analytical Chemistry, Ghent University, Ghent, Belgium.
Brecht
B
Laforce
DESY, Hamburg, Germany.
Jan
J
Garrevoet
Department of Analytical Chemistry, Ghent University, Ghent, Belgium.
Eva
E
Vergucht
Inflammation Research Centre, VIB and Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.|Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
Riet
R
De Rycke
European Synchrotron Radiation Facility, Grenoble, France.
Peter
P
Cloetens
Department of Clinical Microbiology / MIMS, Umeå University, Umeå, Sweden.
Constantin F
CF
Urban
Department of Analytical Chemistry, Ghent University, Ghent, Belgium.
Laszlo
L
Vincze
D002118:Calcium / Q000378:metabolism; D065206:Extracellular Traps / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology*; D013052:Spectrometry, X-Ray Emission / Q000295:instrumentation*; D017356:Synchrotrons; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism
D007501:Iron; D015032:Zinc; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium