1 3
Cancer Immunol Immunother (2017) 66:77–89
DOI 10.1007/s00262-016-1921-7
ORIGINAL ARTICLE
Neutrophils from chronic lymphocytic leukemia patients exhibit
an increased capacity to release extracellular traps (NETs)
Enrique Podaza
1
· Florencia Sabbione
2
· Denise Risnik
1
· Mercedes Borge
1
·
María B. Almejún
1
· Ana Colado
1
· Horacio Fernández‑Grecco
3
· María Cabrejo
3
·
Raimundo F. Bezares
4
· Analía Trevani
2
· Romina Gamberale
1
· Mirta Giordano
1
Received: 16 February 2016 / Accepted: 23 October 2016 / Published online: 28 October 2016
© Springer-Verlag Berlin Heidelberg 2016
its depletion reduced plasma capacity to enhance NETs
release. Finally, we found that culture with NETs delayed
spontaneous apoptosis and increased the expression of acti-
vation markers on leukemic B cells. Our study provides
new insights into the immune dysregulation in CLL and
suggests that the chronic inflammatory environment typi-
cal of CLL probably underlies this inappropriate neutrophil
priming.
Keywords Chronic lymphocytic leukemia · Neutrophil ·
Neutrophil extracellular traps · Interleukin 8 (IL-8) ·
Infections
Abbreviations
CLL Chronic lymphocytic leukemia
DHR Dihydrorhodamine 123
DPI Diphenyleneiodonium chloride
FSC Forward scatter
HD Healthy donors
MNase Micrococcal nuclease
MPO Myeloperoxidase
NETs Neutrophil extracellular traps
PFA Paraformaldehyde
PI Propidium iodide
ROS Reactive oxygen species
TMB 3,3′,5,5′ tetramethylbenzidine
Introduction
Chronic lymphocytic leukemia (CLL) is characterized by
the progressive accumulation of clonal CD5
+
B cells in
lymphoid tissues, bone marrow and blood [1]. The clini-
cal course of CLL is highly heterogeneous, ranging from
an indolent disease that will not require treatment for many
Abstract Chronic lymphocytic leukemia (CLL) is char-
acterized by immune defects that contribute to a high rate
of infections and autoimmune cytopenias. Neutrophils are
the first line of innate immunity and respond to pathogens
through multiple mechanisms, including the release of neu-
trophil extracellular traps (NETs). These web-like struc-
tures composed of DNA, histones, and granular proteins are
also produced under sterile conditions and play important
roles in thrombosis and autoimmune disorders. Here we
show that neutrophils from CLL patients are more prone to
release NETs compared to those from age-matched healthy
donors (HD). Increased generation of NETs was not due
to higher levels of elastase, myeloperoxidase, or reactive
oxygen species production. Instead, we found that plasma
from CLL patients was able to prime neutrophils from
HD to generate higher amounts of NETs upon activation.
Plasmatic IL-8 was involved in the priming effect since
Electronic supplementary material The online version of this
article (doi:10.1007/s00262-016-1921-7) contains supplementary
material, which is available to authorized users.
* Mirta Giordano
giordanomirta@gmail.com
1
Laboratorio de Inmunología Oncológica, Instituto de
Medicina Experimental (CONICET), Academia Nacional
de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires,
Argentina
2
Laboratorio de Inmunidad Innata, Instituto de Medicina
Experimental (CONICET), Academia Nacional de Medicina,
Buenos Aires, Argentina
3
Servicio de Hematología, Sanatorio Municipal Dr. Julio
Méndez, Buenos Aires, Argentina
4
Servicio de Hematología, Hospital Municipal Dr. Teodoro
Alvarez, Buenos Aires, Argentina
78 Cancer Immunol Immunother (2017) 66:77–89
1 3
years to a rapid progression and poor response to therapy
[2]. A major feature of CLL is a profound immune dys-
regulation that worsens as the disease progresses and after
antineoplastic therapy [3]. As a consequence, infectious
complications are still the main cause of morbidity and
mortality in CLL patients [4, 5]. On the other hand, the
breakdown of tolerance to self-antigens leads to autoim-
mune cytopenias in up to 30% of CLL patients during the
course of the disease [6]. Although the mechanisms respon-
sible for immune defects in CLL are not completely under-
stood, increasing evidence demonstrates that leukemic cells
are able to impair acquired and innate immunity through
the release of soluble factors and cell-to-cell contact [3].
Despite the specific reports of dysfunction in T cells [7],
monocytes [8], macrophages [9], NK cells [10], and non-
leukemic B cells [11] in the context of CLL, little is known
about neutrophils, the most abundant leukocyte population
in peripheral blood.
Neutrophils constitute a first line of defense against
invading pathogens [12]. They are recruited to sites of
infection where they can destroy microorganisms using dif-
ferent strategies such as phagocytosis and degranulation. A
newly discovered feature of the biology of neutrophils is
their ability to release neutrophil extracellular traps (NETs)
[13, 14]. Composed of a chromatin meshwork decorated
with antimicrobial peptides and enzymes, NETs are gener-
ated through an active form of cell death known as NETosis
[15]. Not only microorganisms but also a variety of soluble
and particulate stimuli can trigger NETosis including high
mobility group B1 (HMGB1) [16], TNFα [17] and choles-
terol crystals [18]. In fact, NETs can also be formed under
sterile conditions and have been reported to play relevant
roles in pathologies as diverse as thrombosis [19], systemic
lupus erythematosus [20], diabetes [21] and cancer [22].
The aim of the present study was to evaluate the capac-
ity of neutrophils from CLL patients to generate NETs.
Given the frequency of respiratory infections by capsulated
bacteria in CLL patients [5] and the reported relevance of
NETs to confine the pneumococcal infection and reduce its
virulence [23], we hypothesized that the capacity to form
NETs would be impaired in CLL patients. By contrast, we
found that neutrophils from CLL patients are more prone to
release NETs in vitro compared to HD and that these NETs
might contribute to leukemic cells survival.
Materials and methods
Reagents
Culture medium (RPMI 1640) was purchased from Life
Technologies (Grand Island, NY); FBS from Natocor
(Argentina), penicillin and streptomycin from GIBCO
Laboratories (Grand Island, NY) and Ficoll from GE
Healthcare (Munich, Germany). Micrococcal nuclease
(MNase) was from Worthington Biochemical (Lake-
wood, NJ). One-step Ultra TMB (3,3′,5,5′-tetramethylb-
enzidine) was from Thermo Fisher (Massachusetts, MA).
Anti-human elastase Ab was obtained from Calbiochem
(Massachusetts, MA), and rabbit total IgG and DyLight-
488-anti-rabbit were purchased from Jackson ImmunoRe-
search Laboratories (West Grove, PA). Dihydrorhodamine
123 (DHR) and SYBRGold were from Life Technologies
(Carlsbad, CA). TNFα was from R&D Systems (Minneap-
olis, MN), PE-anti-human CD69, FITC-anti-human CD80,
and human IL-8 ELISA Kit were purchased from BD Bio-
sciences (Franklin Lakes, NJ). PCy5-anti-human CD19
was obtained from Beckman-Coulter (Brea, CA). PE-anti-
human-CD86, FITC-anti-human CXCR1, PE-anti-human
CXCR2, and recombinant IL-8 were from Biolegend (San
Diego, CA). Aqua-Poly/mount coverslipping medium was
purchased from Polysciences (Warrington, PA). Annexin-
V-FITC was obtained from ImmunoTools (Friesoythe,
Germany). All other reagents were acquired from Sigma-
Aldrich (St. Louis, MO).
CLL patients and HD samples
Peripheral blood samples were obtained from CLL patients
(age range 50–87), healthy age-matched and healthy young
(age range 21–50) donors after informed consent in accord-
ance with the Declaration of Helsinki. These studies were
approved by the Institutional Review Board of the Institutes
of the National Academy of Medicine, Buenos Aires. At the
time of the analysis patients were free from clinically rel-
evant infectious complications and were either untreated
or had not received antineoplastic treatment for ≥3 months
before the study began.
Cell separation procedures and culture
Peripheral blood leukocytes were isolated by centrifuga-
tion over a Ficoll–Triyosom layer. PBMC were recovered
from the interface, washed twice with saline and sus-
pended in complete medium (RPMI 1640 supplemented
with 10% FBS and antibiotics). Neutrophils were recov-
ered from the bottom pellet after dextran sedimentation
and contaminating erythrocytes were removed by hypo-
tonic lysis as previously reported [24]. After washing with
saline, cells (>96% viable neutrophils) were resuspended
in RPMI 1640 without phenol red and supplemented with
10% heat-inactivated FBS and antibiotics. To minimize
neutrophil spontaneous activation, cells were used imme-
diately after isolation.
79Cancer Immunol Immunother (2017) 66:77–89
1 3
NETs induction and quantification
To induce NETs formation, neutrophils (2 × 10
6
cells/ml)
were seeded in 48-well plate and stimulated with PMA
25 ng/ml during 4 h at 37 °C under a humidified atmos-
phere with 5% CO
2
. Alternatively, neutrophils were stim-
ulated with ionomycin (1 µM), TNFα (10 ng/ml), LPS
(100 ng/ml), or IL-8 (0.15 or 10 ng/ml) as described in the
corresponding figure legends. NETs-containing superna-
tants were recovered after treatment with low dose MNase
(1 U/ml) to detach NETs from cell debris and 30 min later,
EDTA (5 mM) was added to stop MNase reaction.
NETs were quantified by determining DNA concentra-
tion and elastase activity in supernatants. DNA was quan-
tified by SYBRGold (1:2000) staining and fluorometric
detection (BioTeK Instruments, Winooski, VT, USA). DNA
values were corrected by subtracting those obtained when
the same sample was pretreated with a high concentration
of MNase (10 U/ml) for 30 min to degrade DNA. Elastase
activity in supernatants was assessed by spectrophotometry
using the specific substrate N-methoxysuccinyl-Ala-Ala-
Pro-Val as described [25].
Analysis of the NETotic process by confocal microscopy
Visualization of NETs by confocal microscopy was
performed using an adapted version of the protocol
described by Brinkmann et al. [13]. Briefly, neutrophils
(2 × 10
6
cells/ml) were seeded on poly-l-lysine-coated
microscope slides and stimulated with PMA for 2–4 h.
After incubation, the samples were fixed with 4% para-
formaldehyde (PFA), permeabilized with 0.5% Triton
X-100 in PBS for 1 min and blocked with 5% heat-inac-
tivated FBS in PBS for 1 h. Then samples were incubated
with rabbit anti-human elastase Ab (1 µg/ml) or the cor-
responding isotype control for 1 h, washed, and exposed
to DyLight488-conjugated goat anti-rabbit IgG Ab for
an additional hour. For DNA staining, samples were
incubated with PI for 5 min and mounted using Aqua-
Poly/mount medium. Images were acquired by using a
FluoView FV1000 confocal microscope (Olympus, Tokyo,
Japan) equipped with a Plapon 60×/1.42 objective and
analyzed with Olympus FV10-ASW software.
Analysis of ROS production
For measurement of intracellular ROS production, the fluo-
rescent dye DHR was used. Neutrophils from HD or CLL
patients were stained by incubation with 2 µM DHR for
30 min at 37 °C, washed once and exposed to PMA (25 ng/
ml) or saline. After 20 min of incubation, cells were placed
on ice and immediately analyzed by flow cytometry.
Evaluation of elastase and MPO activity in neutrophils
Neutrophils (5 × 10
5
) were lysed with PBS 1% Triton
X-100 and centrifuged, and supernatants were collected for
the immediate determination of enzyme activity. Elastase
activity was measured as described above for quantification
of NETs. Peroxidase activity was determined using TMB
as substrate. Briefly, 25 µl of sample (increasing dilutions
in PBS) was combined with 25 µl 1-Step TMB solution
and incubated at 37 °C for 5 min. The reaction was stopped
by adding 50 µl H
2
SO
4
and absorbance was measured at
450 nm.
Modulation of CLL‑B cell apoptosis and activation
markers by incubation with NETs
PBMC (>93% leukemic B cells) from CLL patients were
suspended in complete medium and placed into 48-well
plates at a concentration of 2 × 10
6
/ml. NETs were
obtained by stimulating neutrophils with ionomycin for
1 h, then cells were washed twice, and reincubated for an
additional 3 h at 37 °C. Alternatively, neutrophils were
stimulated with LPS (100 ng/ml) plus IL-8 (0.15 ng/ml).
Supernatants containing NETs were recovered as described
above except that mechanical disruption instead of MNase
was used to separate NETs from cell debris. Supernatants
were added to PBMC at 1:3 final dilutions. PBMC were
cultured with or without NETs at 37 °C. The expression of
surface activation markers were assessed on CD19
+
CD5
+
cells at 24 h (CD69), 48 h (CD86) or 72 h (CD80) by flow
cytometry. These time points were chosen based on optimal
measurements for each marker. Leukemic B cell viability
was evaluated at 48 h by Annexin-V/PI staining and flow
cytometry analysis.
Statistical analysis
Data were analyzed using Kruskal–Wallis test, Friedman
test, Mann–Whitney test or Wilcoxon signed rank test.
When appropriate, multiple comparisons Dunn’s posttest
was used. For correlation analysis, nonparametric Spear-
man’s correlation coefficient was calculated. Data were
analyzed using GraphPad Prism software version 6.01.
Results
Neutrophils from CLL patients release higher levels
of NETs compared to HD
We first evaluated the capacity of circulating neutrophils
from CLL patients to form NETs in vitro. To that aim,
80 Cancer Immunol Immunother (2017) 66:77–89
1 3
purified neutrophils were stimulated with PMA, a potent
NET inducer, for 4 h. NET formation was visualized by
confocal microscopy by evaluating the colocalization of
DNA with elastase (Fig. 1a) and quantified by measuring
the concentration of DNA and the enzymatic activity of
elastase in supernatants (Fig. 1b). Given that PMA-induced
NETosis depends on the generation of ROS by NADPH
oxidase [26], we used DPI to corroborate NETs release. As
Unstimulated
HD neutrophils
PMA stimulated
HD neutrophils
PMA stimulated
CLL neutrophils
ab
c
e
Unstimulated
CLL neutrophils
Unst.PMA
Elastase DNA Merge
d
0.0
0.1
0.2
0.3
0.4
PMA
++
-
-
Age-matched
HD
CLL
*
*
p<0.05
Elastase activity (AU)
0
2
4
6
8
PMA
++
-
-
Age-matched
HD
CLL
*
*
p<0.05
DNA (µg/ml)
0.0
0.1
0.2
0.3
0.4
Ionomycin
TNF-
α
+LPS
-
--
--
--
-
+
+
++
+
Aged-matched
HD
CLL
*
*
*
*
p<0.05
Elastase activity (AU)
0
2
4
6
8
-
--
--
--
-
+
+
++
+
DNA (
µ
g/ml)
*
*
*
*
p<0.05
Ionomycin
TNFα+LPS
Aged-matched
HD
CLL
Fig. 1 Neutrophils from CLL patients are more prone to release
NETs compared to those from HD. a Neutrophils (2 × 10
6
cells/
ml) from CLL patients were incubated with PMA or medium (unst.)
for 4 h. Preparations were stained with anti-neutrophil elastase Ab
(green) and PI (red), and analyzed by confocal microscopy. Shown
are representative fluorescence images (n = 5). Scale bar 25 μm. b
Elastase activity and DNA concentration measured in supernatants of
neutrophils from CLL patients stimulated or not with PMA. Where
indicated, DPI (10 µM) was added 30 min before PMA to inhibit
NADPH oxidase. Results are the mean ± SEM of 5 independent
experiments. Significance was determined using Friedman test fol-
lowed by Dunn’s multiple comparison posttest. c Elastase activity and
DNA concentration measured in supernatants of unstimulated (black
dots) or PMA-stimulated (white dots) neutrophils. Individual val-
ues and mean for each condition are shown. d Comparison of NETs
release induced by TNFα + LPS or Ionomycin. Significance was
determined using Kruskal–Wallis test followed by Dunn’s multiple
comparison posttest. *p < 0.05 unstimulated versus PMA-stimulated
neutrophils. e Representative images of NETotic neutrophils showing
co-localization of elastase and DNA (arrow heads). Scale bar 25 μm.
The percentages of NETotic cells in HD and CLL samples are shown
(mean ± SEM of 5 experiments). Significance was determined using
Mann–Whitney test
81Cancer Immunol Immunother (2017) 66:77–89
1 3
shown in Fig. 1b, inhibition of NADPH oxidase with DPI
prevented NET formation.
Next, we compared the capacity of neutrophils from
CLL patients and age-matched HD to form NETs. Clini-
cal features of CLL patients are summarized in supplemen-
tary Table 1. We found that neutrophils from CLL patients
released significantly higher levels of NETs compared to
those from HD (Fig. 1c). This increased response was also
observed when stimulation was performed with the calcium
ionophore ionomycin or by priming cells with TNFα fol-
lowed by LPS exposure (Fig. 1d).
For most stimuli, the release of NETs is the result of
a unique form of cell death known as NETosis. During
the process, the activated neutrophil first loses the clas-
sical lobulated nuclear morphology and later the inter-
nal membranes disappear allowing the mixing of nuclear,
cytoplasm and granular components [26]. To corroborate
the differences in NETs generation between neutrophils
from HD and CLL patients, we evaluated the morphologi-
cal changes of PMA-activated neutrophils before the final
phase of NETs extrusion. We found that at 120 min after
PMA challenge a higher proportion of neutrophils from
CLL patients showed colocalization of elastase and DNA
in expanded nuclei characteristic of NETotic cells (Fig. 1e)
and, as expected, they were no longer functional (supple-
mentary figure 1). Not only there were increased numbers
of NETotic cells in CLL samples, but they also showed fea-
tures of a more advanced NETosis compared to those from
age-matched HD.
Plasma from CLL patients increases the capacity
of neutrophils from HD to release NETs
During the formation of NETs, neutrophil elastase trans-
locates from the granules to the nucleus via a mechanism
that requires ROS and MPO [27]. In an attempt to deter-
mine whether differences in any of these molecules could
account for the increased generation of NETs in CLL
patients, we evaluated ROS production induced by PMA as
well as elastase and MPO activities. As shown in Fig. 2a,
there were no significant differences in any of these param-
eters between neutrophils from young or age-matched HD
and CLL patients.
Since neutrophils can be primed by cytokines as IL-8
or TNFα to become more susceptible to release NETs [28,
29], and these and other soluble factors have been reported
to be elevated in CLL [30], we decided to evaluate whether
plasma from CLL patients could enhance NET formation.
To that aim, neutrophils from young HD were incubated
with plasma from CLL patients or age-matched HD for
40 min, washed and suspended in fresh complete medium.
Then neutrophils were stimulated with PMA (25 ng/ml)
and cultured for an additional 3.5 h to allow the release
of NETs. We found that exposure of neutrophils to CLL
plasma significantly enhanced production of NETs induced
by PMA while plasma from HD did not (Fig. 2b). None of
the evaluated CLL plasma samples were able to stimulate
NET release in the absence of PMA challenge (not shown).
We corroborated the priming effect of CLL plasma on neu-
trophils by evaluating the early morphological changes that
are characteristic of death by NETosis [26]. The propor-
tion of NETotic cells were significantly increased in cul-
tures exposed to CLL plasma compared to cells activated in
medium alone (Fig. 2c). Together these results suggest that
soluble factors present in CLL plasma might be responsible
for priming neutrophils to release NETs.
IL‑8 depletion impairs the priming activity of CLL
plasma to release NETs
Among the potential candidates in CLL plasma capable
of favoring NETs production, we chose to explore the
involvement of IL-8 because it has been implicated in
NETs formation by different groups [17, 28] and, more
importantly, its plasmatic levels are significantly increased
in most CLL patients as we corroborated for our cohort
(supplementary figure 2). To determine whether IL-8
was responsible, at least in part, for the priming effect
of CLL plasma on NETs release, we reduced its levels
by incubating CLL plasma on anti-IL-8-coated plates
for 60 min (Fig. 3a) and compared the capacity of whole
and IL-8 depleted plasma to modulate NETs formation.
The results show that the priming effect of CLL plasma
on NETs release was markedly impaired after IL-8 deple-
tion (Fig. 3b). These findings prompted us to look for a
correlation between the plasmatic levels of IL-8 in our
CLL patient cohort and the capacity of their neutrophils
to release NETs upon activation. We found that both, the
activity of elastase and the amount of DNA released to
supernatants after PMA challenge significantly correlated
with the levels of IL-8 in plasma (Fig. 3c).
As it was reported that recombinant IL-8 is able to
directly induce NETs formation but we found that CLL
plasma did not stimulate NETs in the absence of PMA
challenge, we performed additional experiments comparing
two concentrations of IL-8 in their capacity to activate neu-
trophils for NETosis. In agreement with previous reports
[17, 28], we found that 10 ng/ml of recombinant IL-8
stimulated NETs release, while 0.15 ng/ml, the average
concentration of IL-8 that we detected in CLL plasma had
no effect, though it enhanced NETs formation induced by
LPS (supplementary figure 3). Interestingly, when added to
HD plasma, this low dose of recombinant IL-8 potentiated
NETs release induced by PMA (Fig. 3d). Overall, these
data suggest that increased levels of IL-8 in CLL plasma
play a key role in priming neutrophils for NETs formation.
