Supplementary Results:
CD48 mediates eosinophil increased survival by SA exotoxins. We explored the
possibility that CD48 may also be involved in the exotoxins effect on eosinophil
viability. SEB, PtA and PGN induced eosinophil survival (data not shown). The
increase in viability was evident and significant (~1.5-fold, p<0.05) starting 18 hrs
post incubation with each of the exotoxins used, and increased at 48 and 72 hrs.
These increased viability values were lower than the one obtained with GM-CSF
(~3.6-fold, p<0.01), although the exotoxins did not further augment GM-CSF induced
survival. Moreover, blocking CD48 significantly reduced PtA effect on eosinophils
viability (~25%), while the other two exotoxins effects were not significantly
decreased (~5%).
Supplementary Figure S1
Figure S1. Eosinophil expression of CD48 is increased in the presence of SA
exotoxins
FC analysis of (a) CD48, (b) 2B4, (c) ICAM-1 and (d) TLR-2 expression on human
eosinophils incubated with SEB, PtA or PGN for 18 hrs. Results are shown as a
histogram (grey bar: isotype control; black line: medium incubated; and green line:
eosinophils incubated with the exotoxins) and are representative of seven independent
experiments.
Supplementary Figure S2
Figure S2. CD48 is involved in exotoxin signal transduction molecules
phosphorylation in
human eosinophils. (a) FC analysis (histogram) of
phosphorylation of Fyn, Lyn and Erk1,2 of eosinophils upon incubation with
SEB, PtA or PGN: (black line) and of the same cells pre-incubated with antiCD48 (dashed line). (b) MFI quantification of histograms shown in a. Isotype
control and the total un-phosphorylated proteins (matched to each kinase tested)
did not change between the different exotoxins and/or in the presence of antiCD48 (not shown). Data are representative of four independent experiments.
Supplementary Materials and Methods:
Data S1
Human peripheral blood eosinophil purification. Human eosinophils were purified
as previously described [1, 2] from the peripheral blood of mildly atopic volunteers
(blood eosinophil levels 5%-10%), who were asymptomatic and therefore not taking
any drug for their condition. Written informed consent was obtained according to the
guidelines of the Hadassah-Hebrew University Human Experimentation Helsinki
Committee. Eosinophils collected at a purity of >98% (Kimura staining), with a
viability of >98% (trypan blue staining), were re-suspended (1x106 cells/ml) in EM
consisting of RPMI 1640 supplemented with 10% heat-inactivated FBS, and
penicillin-streptomycin solution (100 u/ml) (Biological Industries, Beit Haemek,
Israel) and GM-CSF (20 ng/ml; Peprotech, Rocky Hill, NJ).
SA culture conditions and production of heat killed SA. SA (ATCC 25923)
maintained at –70°C in skim milk-glycerol was subcultured onto blood agar and
incubated at 37°C for 18 hrs. A sweep of colonies was inoculated into tryptic soy
broth (TSB; Difco, USA) and incubated at 37°C for 24 hrs. The culture density was
estimated by the measurement of the optical density at 650 nm. SA was heat killed (30
min, 90°C) and then washed three times with phosphate-buffered saline (PBS), reconstituted in PBS (1x106/ml) and kept at 4°C. Bacteria were diluted to achieve a
multiplicity of infection (MOI) of 10 before use.
Immunohistochemistry (IHC) of AD skin biopsies. The frozen skin sections of
AD patients (lesional and non lesional n=5) (2 females and 3 males, ages 22-65,
moderate and severe) and healthy individuals (n=6) (4 females and 2 males, ages
20-55), were obtained according to the guidelines of the Duesseldorf University,
Medical Faculty Ethics Committee. Sections were fixed with 4% PFA (10 min,
RT), washed with PBS and treated for deactivation of endogenous HRP with 3%
H2O2. Slides were heated to 60°C to deactivate the endogenous alkaline
phosphatase, and blocked for endogeneous biotin with egg white. Then, slides
were blocked and permeabilized (1 hr, RT; 5% goat serum, 0.1% Triton, v/v in
PBS). Samples were stained with mouse anti-human-CD48 biotin Ab (1:100; 18
hr, 4°C; Abcam, Cambridge, UK) followed by the secondary Ab streptavidin
HRP (1:400; 1 hr, RT; R&D Systems, Minneapolis, MN, USA), and developed
by DAB (Vector, Burlingame, CA, USA). For specific eosinophil staining, slides
were co-stained with rabbit anti-human-EDN (1:50; 1 hr, RT) (a gift from Prof.
Gerald J. Gleich, University of Utah Health Sciences Center, Salt Lake City, UT,
USA) followed by secondary anti-rabbit histofine AP (1 hr, RT; Nichirei
Biosciences, Tokyo, Japan) and developed with AP (Vector, Burlingame, CA,
USA). Stained slides were observed and photographed by a light microscope
(Nikon TL, Japan).
Eosinophil immunofluorescence staining and confocal microscopy. Eosinophils
(1x105/100µl of EM) were incubated with fluorescein isothiocyanate (FITC) labeled
or unlabeled SA (MOI of 10), (37°C, 30 min, 5% CO2). For labeling, bacteria
(1x109/ml in PBS) were washed twice with PBS, suspended in FITC (1 μg/ml;
Sigma-Aldrich, Rehovot, Israel) dissolved in PBS, and incubated for 30 min under
constant shaking at 37°C and washed three times with PBS prior to use. Eosinophils
were then seeded on poly-L-Lysine covered slides (37°C, 30 min), stained by DAPI
(1:500; 10 min; Sigma Aldrich, Rehovot, Israel) and fixed with 4% PFA (15 min,
RT). Slides were then washed three times by PBS, treated with fluoromount
(Southern Biotech, Birmingham, AL), and evaluated by confocal microscopy (x630
magnification, zoom 2, Zeiss LSM 710, Germany).
Human peripheral blood leukocytes double staining. Blood from 14 AD patients
(9 females and 5 males, ages 24-80, mild, moderate and severe (SCORAD) [3])
and 18 healthy volunteers (4 females and 14 males, ages 23-61) were obtained
according to the guidelines of the Hadassah-Hebrew University Human
Experimentation Helsinki Committee. Isolated fractions of granulocytes and
peripheral blood mononuclear cells (2x105/100µl of 0.1% BSA/PBS) were incubated
in 96 U-shape wells (Nunc, Roskindle, Denmark) on ice. For FC double staining
analysis cells were blocked (5% goat serum, 0.1% BSA/PBS) and stained with the
specific anti-human CD48 mAb (1 µg/ml; MEM-102; BioLegend, San Diego, CA,
USA) or mIgG1 (1 µg/ml; R&D Systems, Minneapolis, MN, USA) for 1 hr on ice.
Thereafter, cells were washed twice (5 min, 250 g, 4°C) with 0.1% BSA/PBS and
incubated with FITC-goat anti-mouse IgG (1:500; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) for 40 min on ice. Cells were washed twice and incubated with
specific conjugated Abs (markers) for each cell population, i.e. FITC anti-human
CD3 (HIT3a; BioLegend, San Diego, CA, USA) for T cells, FITC anti-human CD20
(2H7; BioLegend) for B cells, FITC anti-human CD14 (HCD14; BioLegend) for
monocytes, FITC anti-human CD16 (CD16, 368; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) for neutrophils, PE anti-human CD56 (positive) + FITC anti-human
CD3 (negative) for NK cells (CD56, NCAM; BioLegend), PE anti-human CD203c
for basophils (E-NPP3, NP4D6; BioLegend), and PE anti-human CCR3 for
eosinophils (R&D Systems, Minneapolis, MN, USA), or the appropriate isotypes
FITC mouse IgG1 (MOPC-21; BioLegend), PE mouse IgG1 (MOPC-21; BioLegend)
or PE rat IgG2a (R&D Systems), for 40 min on ice. Cells were washed twice. A
representative cell sample was also stained using Propidium Iodide (PI) (10% v/v in
PBS) (Sigma-Aldrich, Rehovot, Israel) in order to verify viability of these cells and
gate them. Analysis was performed according to the specific Abs staining using
FACSCalibur and CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA).
MFIs were determined by dividing the fluorescence intensity of the specific Abs from
that of the isotype control. PI-excluding cells were considered viable
Eosinophil survival. Eosinophils (1x105/100µl) in medium without GM-CSF, were
incubated with either one of the 3 bacterial exotoxins (10µg/ml each) in 96-well Ushape plates for 24, 48 and 72 hr (37°C, 5% CO2). At end-points, samples were
washed twice with 0.1% BSA/PBS (5 min, 250 g, 4°C), stained by PI (10% v/v in
PBS) and analyzed by FC as above. PI-excluding cells were considered viable.
Eosinophil degranulation. Eosinophils (2x105/100µl) were re-suspended in the
specific medium/buffer according to the degranulation assay performed (see below).
The cells were incubated with SA (MOI of 10) or either one of the 3 bacterial
exotoxins (10µg/ml each) in 96-well F-shape plates (37ºC, 45 min, 5% CO2). As
positive control, eosinophils were primed with GM-CSF (20ng/ml) (37°C, 20 min,
5% CO2) prior to their incubation with PAF (10-6M; Sigma-Aldrich, Israel), under the
same culture conditions.
As markers of eosinophils degranulation, the following granule mediators were
evaluated: EPO, β-Hex and ECP.
For EPO release, eosinophils were re-suspended in 0.1% BSA/PBS and incubated
with the bacterial exotoxins. EPO release was measured by a colorimetric assay using
a standardized assay with purified human EPO (a kind gift of Prof. Gerald J. Gleich,
University of Utah Health Sciences Center, Salt Lake City, UT, USA) and freshly
prepared peroxidase substrate solution containing o-phenylenediamine (OPD) as
described elsewhere [4]. Data are expressed as EPO concentration, as extrapolated
from the standard curve.
For β-Hex release, eosinophils were re-suspended in Tyrode’s gelatin-calcium buffer
as previously described [5], and incubated with the SA. Following incubation, cells
were centrifuged (3 min, 600 g, 4 ºC), separated from the supernatant, and lysed by
three freeze/thaw cycles. β-Hex released in the supernatant and retained in the cells
was determined by an enzymatic method as previously described [6]. Data are
expressed as percentage of release [mediator in the supernatant/ (mediator release in
the supernatant + mediator in the cells)x100].
For ECP release, eosinophils were re-suspended in medium without GM-CSF and
incubated with the SA. Following incubation, cells were centrifuged (3 min, 600 g, 4
ºC), and supernatants were used to determine ECP release by a commercial enzymatic
immunoassay kit (Mesacup ECP test, MBL, Nagoya, Japan) according to the
manufacturer’s instructions.
Evaluation of eosinophil cytokine release. Eosinophils (1x105 /100µl of EM) were
incubated with SA (MOI of 10) or the bacterial exotoxins (10µg/ml each) for 18 hrs
(37ºC, 5% CO2) in 96-well U-shape plates. Levels of IL-8 and IL-10 in the
supernatants were determined by using commercial enzymatic immunoassay kits
according to the manufacturer's instructions (Peprotech, Rocky Hill, NJ, USA).
Analysis of signal transduction pathways in human eosinophils. To analyze the
effects of the bacterial exotoxins on eosinophil signal transduction, the
phosphorylated forms of different kinases were evaluated by WB or FC. For WB,
eosinophils (1x106 /ml) were re-suspended in medium without GM-CSF, and
incubated with either one of the 3 bacterial exotoxins (10 µ/ml each) in sterile
Eppendorf tubes (Nunc, Roskindle, Denmark) (10 min, 37°C; 5% CO2). Cells were
then centrifuged (3 min, 600 g, 4˚C) and re-suspended in 50 µl M-PER lysis buffer
(Pierce Endogen, Rockford, IL, USA) supplemented with PMSF (1M) and protease
inhibitor cocktail (15 min, RT) (Sigma-Aldrich, Israel). Cell lysates were loaded to
10% SDS-PAGE gels and transferred to a nitrocellulose membrane (Invitrogen,
Carlsbad, CA, USA). Detection of specific ph. kinase was performed by blotting the
membranes with rabbit-anti-human ph. Fyn (1:1000; Abcam, Cambridge UK), rabbit
anti-human ph. GSK3β (1:1000; Cell Signaling, Beverly, MA, USA), rabbit-antihuman ph. Lyn (1:1000; Cell Signaling, Beverly, MA, USA), and goat-anti-human βactin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), (18 hrs, 4˚C,
constant shaking), followed by horseradish peroxidase (HRP)-conjugated goat-antirabbit (1:5000; Cell Signaling, Beverly, MA, USA), or bovine-anti-goat (1:5000;
Santa Cruz) Abs (1 hr, RT) and ECL-plus detection (GE Healthcare, Amersham,
UK).
For FC analysis, eosinophils (2×105 cells/100µl), were re-suspended in medium
without GM-CSF, and incubated with either one of the 3 bacterial exotoxins (10
µg/ml each) in 96-well U-shape plates (10 min, 37°C; 5% CO2). Cells were fixed for
10 min (4% PFA; in cold 0.1% BSA/PBS) and permeabilized for 30 min using icecold 90% methanol, followed by a blocking stage for 15 min in 0.1% BSA/PBS
containing 5% goat serum. Cells were washed with 0.1% BSA/PBS and stained
(1hr, on ice) using 1:100 dilution of rabbit anti-human ph. Lyn, rabbit antihuman total Lyn, rabbit anti-human ph. AKT, rabbit anti-human total AKT,
rabbit anti-human ph.GSK-3β, rabbit anti-human total GSK-3β, rabbit antihuman ph. Erk1,2, rabbit anti-human total Erk, rabbit anti-human total Fyn
(Cell Signaling, Beverly, MA, USA), rabbit anti-human ph. Fyn (Abcam,
Cambridge, UK), or rabbit IgG control (Jackson Laboratories, West Grove, PA,
USA) Abs. This step was followed by a 1 hr incubation with FITC-conjugated
goat anti-rabbit Ab (1:200; Jackson Laboratories, West Grove, PA, USA). Each
Ab-incubation step was followed by two washes in 0.1% BSA/PBS. Data were
acquired in a BD Biosciences FACScalibur flow cytometer and analyzed using
CellQuest software.
CD48-/- and WT mice and BMEos generation. C57BL/6 wild-type (WT) mice
were purchased from Harlan Laboratories Ltd. Israel. CD48-/- were a kind gift from
Prof. A. Sharpe (Harvard Medical School, Boston, MA, USA). Mice were housed and
bred at the animal housing facility of the Hebrew University of Jerusalem in a
specific pathogen-free environment. All experimental protocol involving animals and
primary animal cells were approved by the Animal Experimentation Committee of
The Hebrew University of Jerusalem.
Bone marrow cells obtained from femurs of CD48-/- and WT mice were cultured as
previously described [7]. On day 10-12 of the culturing, cells were cytocentrifuged (1
min, 650g, RT) (Cytospin 4, Thermo Scientific, USA), fixed and stained using a
modified Giemsa preparation (Sigma-Aldrich, Rehovot, Israel). Cells were also
examined by FC for the expression of their characteristic surface markers using
conjugated anti-mouse-Siglec-F (BD Biosciences, USA) to determine eosinophil
maturation. Cell viability was assessed by Trypan blue.
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