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.
Highlights
Neutrophils, the most frequent type of white blood cells, are circulating cells of the innate immune system serving as first line of defense against microbial pathogens [1]
The maximum intensity of Ca, Zn and Fe for the neutrophils from control culture was set as an upper threshold, which was preserved for rendering the 1 h and 2 h phorbol myristate acetate (PMA)-stimulated neutrophil RGB composite maps
Ca/Fe and Fe-containing hotspots appeared in the cytoplasm after 2 h PMA stimulation
Summary
Neutrophils, the most frequent type of white blood cells, are circulating cells of the innate immune system serving as first line of defense against microbial pathogens [1] They are terminally differentiated, non-proliferate cells armed with a large antimicrobial arsenal [2]. The formation of NETstructures is believed to be in close connection with the removal of essential trace elements from the pathogens, which is referred to as ‘nutritional immunity’, possibly involving proteins with metal chelating properties. In this way, NETs form a defense mechanism against microbes using chelating proteins, removing crucial trace elements from the pathogen. Metals such as Mn, Fe, Co, Ni, Cu and Zn are high-probability candidates for interaction with NETs
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