Supplemental Digital Content 1: detailed material and methods.
Neutropenic or severely immunosuppressed patients were excluded from the study.
Neutrophil viability and maturation stages
Neutrophils were cultured in RPMI (Gibco Products, Invitrogen Corporation, Grand Island,
NY) with 10% fetal calf serum (FCS, Amimed, Basel, Switzerland) and incubated at 37°C, in
a 5% CO2 atmosphere. Neutrophils were sampled each day in culture media, washed and
resuspended in Annexin V- binding buffer. Apoptosis and necrosis were quantified using
FACS by PE-conjugated Annexin V, and 7-amino-actinomycin D (7-AAD, BD Biosciences,
Franklin Lakes, NJ). CD16 surface expression was also quantified by FACS (FITCconjugated anti-CD16 mAb, BD Biosciences) every day in sampled neutrophils to determine
the stage of neutrophil maturation over time. Neutrophils were gated according to their
forward and side-scatter characteristics. More than 10,000 events were acquired for each
measurement.
Receptors expression
Cells were incubated for 1 hr at 4°C in the dark with unlabeled anti-CD14 (28C5, a gift from
P.S. Tobias, La Jolla, CA), anti-CD16 (BD Biosciences, Franklin Lakes, NJ), anti-HLA-DP, DQ, -DR (DakoCytomation, Glostrup, Denmark), anti-MD-2 (18H10 MAb, a gift from G.
Elson, NovImmune SA, Geneva, Switzerland) MABs or with isotype control IgG1κ and IgG2b
MAbs (BD Biosciences) depending on the isotype of the specific MAb in PBS containing 5%
human plasma. Cells were then incubated for 30 min with secondary APC-labeled goat antimouse IgG (Invitrogen, Eugene, OR) and analyzed by FACS. For some surface receptors,
we used directly conjugated MAbs: PE-labeled mouse anti-human TLR2 and anti-TLR4 mAb
(eBioscience, San Diego, CA) and PE-labeled mouse anti-human CXCR2 (IL-8 receptorB)
MAb (R&D systems, Minneapolis, MN) with corresponding PE-labeled mouse IgG2a κ
isotype control (eBioscience).
1
Chemotaxis
To measure the capacity of mature vs. immature neutrophils to migrate through a filter, we
used a modified Boyden chamber (ChemoTXR System from Neuro Probe, Inc. 96-well
format; model 101-5; 5 µm pores, Gaithersburg, MD). Neutrophils (20 µL) were deposited on
the top of the filter at the concentration of 2 x 106cell/mL in RPMI/10% FCS. Serial dilutions
of IL-8 (a gift from C. Power, MerckSerono, Geneva, Switzerland) and fMLP (Sigma, St.
Louis, MO) served as chemoattractants in the lower chamber. Neutrophils which have
migrated to the lower chamber after 1 hr at 37°C were fluorescently stained with DRAQ5TM
(Biostatus Limited, Leicestershire, UK) and counted using the Applied Biosystems 8200
Cellular Detection System (Applied Biosystems, Foster City, CA). Cell migration in % was
calculated as the number of neutrophils which migrated through the filter divided by the
number of neutrophils deposited above the filter x 100.
Phagocytosis
Mature and immature neutrophils were incubated with fluorescently-labeled Gram-negative
and Gram-positive particles (E.coli and S.aureus Bodipy FL BioParticles, Molecular Probes,
Eugene, OR). Before incubation with neutrophils, bacteria were opsonized for 1 hr at 37°C
with 2% autologous decomplemented plasma (plasma heated 30 min at 56°C). Opsonized
bacteria were then centrifugated at 1000g for 15 minutes at 4°C and washed once in PBS.
To test phagocytosis, 3 x 106 fluorescent opsonized bacteria were added to 106 neutrophils,
corresponding to a bacteria-to-cell ratio of 3:1. Phagocytosis was performed at 37°C for 10
minutes, in the same buffer as that used during opsonization. Cells were then put on ice,
centrifugated at 300 g for 4 minutes at 4°C, and washed with ice-cold PBS. To only quantify
internalized bacteria, extracellular fluorescence was quenched using 0.05% Trypan blue (Life
Technologies, Carlsbads, CA), and neutrophils were analyzed by FACS. The negative
2
control consisted in neutrophils pretreated 30 min with 100 µM cytochalasin D, a blocker of
phagocytosis (Sigma-Aldrich, St. Louis, MO). Phagocytosis was quantified using a
“phagocytic index” (PI) as defined by the percentage of cells becoming green (positive)
multiplied by their geometric mean of fluorescence, as described elsewhere (1).
Bacterial killing
In order to measure directly the bacterial killing by neutrophils, mature vs. immature
neutrophils were incubated with live E.coli and S.aureus. Live E.coli and S.aureus (107
bacteria) were osponized for 30 min at 37°C with 10% autologous plasma in pH 7.4 HBSS
containing 1% HSA and 20mM HEPES. After washing with PBS, bacteria were given to 2 x
106 neutrophils (immature vs. mature) for 30 min in RPMI containing 10% autologous
plasma. To kill extracellular, unphagocytosed bacteria, 25 µg/mL gentamicin was then added
to neutrophil/E.coli cultures and 4 µg/mL lysostaphin (AMBI products LLC, Lawrence, NY) to
neutrophil/S.aureus cultures. To measure phagocytosed bacteria, cultures were kept for an
additional 30 min, centrifuged, and then neutrophils were lysed in 0.01% Triton X-100
(Sigma, St. Louis, MO). To measure bacterial killing, neutrophils were kept in culture for an
additional 2 hours, centrifuged, and then lyzed. After neutrophil lysis and release of
intracellular bacteria, serial dilutions of the cell lysate were plated on agar plates and put
overnight at 37°C. Bacteria colonies were then counted and comparisons were made
between mature and immature neutrophils from patients with septic shock, and with
neutrophils from healthy subjects.
Production of reactive oxygen species
To quantify the capacity of mature vs. immature neutrophil to generate reactive oxygen
species (ROS), we incubated cells with bacteria or PMA and a fluorescent substrate
sensitive to the generation of H2O2 (2’, 7’ dichlorofluorescein, DCFH, Sigma-Aldrich, St.
3
Louis, MO) as described elsewhere (2, 3). Briefly, neutrophils (107/mL) were preincubated for
15 min with 5 µM of DCFH-DA at 37°C, and then incubated for 10 min with the following
agonists: 500 nM PMA (Sigma), heat-killed E.coli and S.aureus at 2.5 x 108 cfu/106 cells (4),
and PBS as a negative control. Oxidized DCFH (DCF) fluorescence was measured by
FACS. Results were expressed as the ratio of fluorescence intensity of stimulated cells
divided by the fluorescence of nonstimulated cells.
Intracellular cytokine content
The basal intracellular content of tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 was
measured by FACS in permeabilized mature and immature neutrophils. Neutrophils (107/ml)
were fixed with IC fixation buffer and permeabilized using IC permeabilization buffer
(Invitrogen, Carlsbad, CA). Cells were incubated for 30 min with PE-mouse anti-human TNFα and anti-human IL-10 MAb (R&D systems, Minneapolis, MN) and with corresponding PEmouse IgG1 isotype control (R&D systems), and quantified using FACS, and results were
expressed as mean fluorescence index (MFI).
Statistical analysis
Data were presented as box-plot graphs, scatter dot-plots, curves depending on the assays,
using the GraphPad Prism software 5.0. (La Jolla, CA). Variables were expressed as median
with 25-75% interquartile range (IQR). Comparisons between independent groups were
performed with the Mann-Whitney U test and paired data with the Wilcoxon test. The
evolution with time during the first 3 d was analyzed by a mixed effect linear regression in
order to take into account paired measurement. Predictors were the time and the group, and
an interaction was introduced because the evolution over time was different between groups.
The regression analyses were performed with the Splus 8.0 software (Insightful Corp.,
Seattle, WA). P values were considered significant when ≤ 0.05.
4
References
1.
Ramet M, Manfruelli P, Pearson A, et al: Functional genomic analysis of phagocytosis
and identification of a Drosophila receptor for E. coli. Nature 2002; 416:644-648.
2.
Bass DA, Parce JW, Dechatelet LR, et al: Flow cytometric studies of oxidative
product formation by neutrophils: a graded response to membrane stimulation. J Immunol
1983; 130:1910-1917.
3.
Cassidy RA, Burleson DG, Delgado AV, et al: Effects of heme proteins on nitric oxide
levels and cell viability in isolated PMNs: a mechanism of toxicity. J Leukoc Biol 2000;
67:357-368.
4.
Elson G, Dunn-Siegrist I, Daubeuf B, et al: Contribution of Toll-like receptors to the
innate immune response to Gram-negative and Gram-positive bacteria. Blood 2007;
109:1574-1583.
5