Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence

PLoS One. 2016 Nov 3;11(11):e0165604. doi: 10.1371/journal.pone.0165604. eCollection 2016.

Abstract

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.

MeSH terms

  • Calcium / metabolism
  • Extracellular Traps / drug effects
  • Extracellular Traps / metabolism*
  • Humans
  • Intracellular Space / drug effects
  • Intracellular Space / metabolism*
  • Iron / metabolism
  • Neutrophils / cytology*
  • Spectrometry, X-Ray Emission / instrumentation*
  • Synchrotrons*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Zinc / metabolism

Substances

  • Iron
  • Zinc
  • Tetradecanoylphorbol Acetate
  • Calcium

Grants and funding

Björn De Samber acknowledges his postdoctoral research grant from FWO Vlaanderen (application nr. 12B3313N), FWO grant for a long stay abroad (application nr. V.4.114.10.N.00) and statistical consult from Ghent University FIRE (Fostering Innovative Research based on Evidence). Maria J. Niemiec acknowledges financial support from the J.C. Kempe Memorial Fund. Eva Vergucht, Brecht Laforce and Jan Garrevoet were funded by a PhD grant from the Flemish Institute for the Promotion of Scientific and Technological Research in Industry (IWT Flanders, Belgium). Constantin F. Urban acknowledges support from the Swedish Research Council (521-2014-2281), Medical Faculty Umeå University and the Åke Wiberg Foundation (M15-0108). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.