et al. 2009 ). It has been recently shown that
antibiotics can also modulate the formation of
neutrophil extracellular traps by bovine
granulocytes (Jerjomiceva et al. 2014 ).
Neutrophil extracellular traps (NETs) have
been described several years ago (Brinkmann
et al. 2004 ) as a novel strategy used by
neutrophils to fight and control infections.
NETsareweb-likestructurescomposedof
DNA and antimicrobial proteins, which entrap
pathogens and create space with a high local
concentration of antimicrobial factors. NETs
are an efficient weapon released in response to
wide range of microorganisms – bacteria,
viruses, parasites and fungi. They are of critical
role especially when pathogens are too large to
be phagocytized (parasites, hyphae form of
fungi), as granulocytes can size up pathogens
and selectively release DNA and antimicrobial
content to the environment (Branzk et al. 2014 ).
However, uncontrolled neutrophil extracellular
traps release entails some risk for the host.
NETs content is highly immunogenic, normally
stored within the cells, and when presented to
the cells of immune system, can exacerbate
autoimmune diseases, such as lupus, rheuma-
toid arthritis, or small vessel vasculitis (Pieterse
et al. 2015 ; Pratesi et al. 2014 ; Sangaletti
et al. 2012 ). Thus, it is crucial to keep the strict
balance between NETs formation and
clearance.
The aim of the present study was to investi-
gate whether cefotaxime and gentamicin, which
are among antibiotics known to affect phagocyte
function, can influence NETs formation in
humans.
2 Methods
2.1 Neutrophil Isolation
The study was approved by the Ethics Com-
mittee of the Medical University of Warsaw in
Poland. Human blood was collected from healthy
individuals. Informed, written consent was
obtained from all subjects in the study.
Neutrophils were isolated using a density cen-
trifugation method (Skrzeczynska-Moncznik
et al. 2013 ). Rich-platelet plasma was discarded
and replaced with 0.9 % solution of NaCl.
Diluted buffy coat was then layered onto
Histopaque 1077. After centrifugation, top
layers (mononuclear cells and isolation media
Histopaque 1077 layers) were carefully removed
and the layer containing polymorphonuclear
cells and red blood cell pellet was mixed with a
1 % solution of polyvinyl alcohol. Subsequently,
after a 20-min incubation at room temperature,
the upper phase containing alcohol and neutro-
phils was collected and centrifuged. The
remaining red blood cells were lysed by hypo-
tonic lysis and granulocytes were washed twice.
The supernatant was removed and the cell pellet
was suspended in a cell culture medium (RPMI
1640) without phenol red, supplemented with
10 mM HEPES (Gibco, Waltham, MA)).2.2 NETs QuantificationFreshly isolated neutrophils were seeded into
96-well black plates (10^5 cells/well) and
incubated for 2 h in 5 % CO 2 atmosphere at
37 C with 1 μl/ml gentamicin, 10 μg/ml
cefotaxime (G 1379, Sigma Aldrich, Saint
Louis, USA), or the Roswell Park Memorial
Institute (RPMI) 1640 Medium alone. Then,
NETs formation was stimulated with phorbol
myristate acetate (PMA, 100 nM) or calcium
ionophore (CI, 4 μM), and the cells were
incubated for 3 h in 5 % CO 2 atmosphere at
37 C. Unstimulated neutrophils were used
as controls. After incubation, 500mIU/ml of
micrococcal nuclease (ThermoFisher Scientific,
Waltham, MA) was added to detach DNA from
the cell surface, and the reaction was stopped
with 5 mM EDTA after 20 min incubation at
37 C. The cells were centrifuged, the superna-
tant was collected and DNA release was
measured in the presence of 100 nM Sytox
green fluorescent dye (Life Technologies,
Waltham, MA) in a FLUOstar OMEGA plate
reader (BMG Labtech, Ortenberg, Germany).48 A. Manda-Handzlik et al.