Mechanisms of Siglec-F-Induced Eosinophil Apoptosis: A
Role for Caspases but Not for SHP-1, Src Kinases, NADPH
Oxidase or Reactive Oxygen
Hui Mao1¤, Gen Kano2, Sherry A. Hudson1, Mary Brummet1, Nives Zimmermann2, Zhou Zhu1,
Bruce S. Bochner1*
1Division of Allergy and Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America,
2Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of
Background: Siglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and
human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS)
generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the
objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that
Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms
involved in cell death via these receptors would differ.
Methods: Consequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and
by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology
domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and
Src family kinase activity were also used.
Results: Engagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on
caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases
in this apoptotic process.
Conclusions: These data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when
compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that
models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8
targeting in vivo in humans.
Citation: Mao H, Kano G, Hudson SA, Brummet M, Zimmermann N, et al. (2013) Mechanisms of Siglec-F-Induced Eosinophil Apoptosis: A Role for Caspases but
Not for SHP-1, Src Kinases, NADPH Oxidase or Reactive Oxygen. PLoS ONE 8(6): e68143. doi:10.1371/journal.pone.0068143
Editor: Patricia T. Bozza, Fundac ¸a ˜o Oswaldo Cruz, Brazil
Received November 1, 2012; Accepted May 26, 2013; Published June 28, 2013
Copyright: ? 2013 Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants AI72265, HL107151 (to BSB) and P30 DK078392 (pilot grant to NZ). Dr. Bochner also received support as a
Cosner Scholar in Translational Research from Johns Hopkins University. Publication of this article was funded in part by the Open Access Promotion Fund of the
Johns Hopkins University Libraries. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: Dr. Bochner is a co-inventor on existing and pending Siglec-8-
related patents. Dr. Bochner may be entitled to a share of royalties received by the University on the potential sales of such products. Dr. Bochner is also a co-
founder of, owns stock in, and is on the scientific advisory board of Allakos, Inc., a company focusing on the development of Siglec-8- directed therapies, which
makes him subject to certain restrictions under University policy. The terms of this arrangement are being managed by the Johns Hopkins University in
accordance with its conflict of interest policies. None of the other authors have any competing interests. There are no further patents, products in development or
marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the
guide for authors. Patents issued: US Patent #6,146,845 (issued 11/14/2000) ‘‘Polynucleotides encoding a Sialoadhesin Family, Member-2 (SAF-2)’’, US Patent
#7,214,772 (issued 5/8/2007) ‘‘Sialoadhesin Factor-2’’, US Patent #7,557,191 (issued 7/7/2009) ‘‘Sialoadhesin Factor-2 Antibodies’’, US Patent #7,745,421 (issued
6/29/2010) ‘‘Methods and compositions for treating diseases and disorders associated with Siglec-8 expressing cells’’, US Patent #7,871,612 (issued 1/18/2011)
‘‘Methods of use of Sialoadhesin Factor-2 Antibodies’’, Japan Patent #4860877 (issued 11/11/2011) ‘‘Sialoadhesin Factor-2 Antibodies’’, US Patent #8,178,512
(issued 5/15/2012) ‘‘Methods and compositions for treating diseases and disorders associated with Siglec-80, US Patent #8,197,811 (issued 6/12/2012)
‘‘Sialoadhesin Factor-2 Antibodies’’, US Patent #8,207,305 (issued 6/26/2012) ‘‘Sialoadhesin Factor-2 Antibodies’’. Patents pending: Pub. No.: WO/2001/066126;
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submitted 3/7/2000 ‘‘Sialoadhesin Factor-2 Antibodies.
* E-mail: email@example.com
¤ Current address: Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu, China
Eosinophils have been implicated in a variety of disorders
ranging from asthma and allergic diseases to helminth parasite
immunity to hematologic disorders [1,2]. Biologics that target
eosinophils by neutralizing the major eosinophil hematopoietic
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cytokine IL-5, or target the IL-5 receptor to facilitate eosinophil
depletion, are advancing in clinical trials [3–7].
Siglecs (sialic acid-binding, immunoglobulin-like lectins) are a
family of single-pass transmembrane cell surface proteins found
predominantly on leukocytes [8,9]. Siglec-8 was discovered as part
of efforts initiated about a decade ago to identify novel human
eosinophil proteins; subsequent studies also detected its expression
on mast cells [10–12]. The closest functional paralog in the mouse
is Siglec-F, which is also selectively expressed by eosinophils but
not on mast cells [12–15]. Both Siglec-8 and Siglec-F preferentially
recognize the glycan 69-sulfo-sialyl Lewis X (69-sulfo-sLeX) [16–
18], and studies in mice implicate a sialylated glycoprotein made
by airways epithelium as an endogenous ligand [19–21].
Engagement of Siglec-8 or Siglec-F with Abs and/or artificial
ligands in vitro causes eosinophil death [18,19,22–24].
Mechanistic studies with Siglec-8 implicated both caspases and
reactive oxygen species (ROS) generation resulting in mitochon-
drial injury in eosinophil death . Interestingly, Siglec-8-
induced cell death could not be overridden by survival-inducing
cytokines such as IL-5, GM-CSF and IL-33. In fact, Siglec-8-
induced eosinophil apoptosis was enhanced by these cytokines
[22,23,26,27]. Similar results were obtained using human eosin-
ophils primed in vivo following allergen bronchoprovocation, and
primed cells no longer used caspases in the apoptosis process,
instead relying exclusively on ROS generation and mitochondrial
Most siglecs, including Siglec-8 and Siglec-F, have immuno-
receptor tyrosine-based inhibitory motifs (ITIMs) in their intra-
cellular domain, suggesting an inhibitory role for signaling
[8,9,28,29]. The membrane proximal motif contains a well-
recognized 6-amino acid sequence described as (I/L/V)xYxx(L/
V)  that would putatively act with src homology domain-
containing protein tyrosine phosphatases (SHP) such as SHP-1
following phosphorylation by Src kinases, such as Lyn [31,32].
Indeed, ITIMs in various siglecs can recruit SHP-1 when
phosphorylated , and modulate cellular activity in an
inhibitory manner upon cross-linking with antibodies . The
function of the membrane distal CD150-like tyrosine motifs found
in both Siglec-8 and Siglec-F are less well explored, but it has been
suggested that such motifs could represent immuno-receptor
tyrosine-based switch motifs (ITSMs) that could mediate either
inhibitory or activating signals [29,35].
Conclusions from studies of Siglec-F have generally paralleled
those of Siglec-8. For example, antibody cross-linking of Siglec-F
on mouse eosinophils causes apoptosis [24,36,37], albeit not as
robust as has been seen with human eosinophils via Siglec-8
[22,25]. Administration of Siglec-F antibodies in mouse models of
chronic allergic asthma and blood and gastrointestinal eosinophilia
normalizes inflammatory responses and abrogates many aspects of
tissue remodeling [24,36–38]. Separate from effects on cell
survival, many siglec proteins undergo endocytosis following
engagement in an ITIM-dependent manner . For example,
Siglec-F becomes internalized when engaged with ligands, and
internalization was dependent on its cytoplasmic ITIM motif .
However, the exact pathways of signaling for Siglec-8 and Siglec-F
are still incompletely understood.
Given the potential similarities and differences involved in
Siglec-8 and Siglec-F mediated apoptosis, the goal of this work was
to define apoptosis pathways mediated by Siglec-F and explore the
role of ROS, NADPH oxidase activity, SHP-1 and caspases
involved in Siglec-F-induced cell death. Our data suggest that at
least for studies of eosinophil apoptosis, examination of Siglec-F
does not always provide the same mechanistic conclusions as for
Materials and Methods
All experiments were performed in accordance with the
National Institutes of Health guidelines for the humane treatment
of animals, and were approved by the Johns Hopkins Institutional
Animal Care and Use Committee under protocol #MO10M417
and the Cincinnati Children’s Hospital Medical Center Institu-
tional Animal Care and Use Committee under protocol
#OD11085. All mice were of the C57BL/6 background strain.
(CD3d-IL5)NJ.1638Nal transgenic mice (IL-5+/+mice) displaying
splenomegaly and marked blood eosinophilia  were kindly
provided by Drs. James and Nancy Lee (Mayo Clinic, Scottsdale,
AZ), while B6(Cg)-Ncf1m1J/J mice with defective NCF1/p47phox
protein, resulting in reduced NADPH oxidase activity and ROS
production (Ncf2/2mice), viable motheaten mice deficient in Src-
homology 2-domain phosphatase (SHP)-1 (mev(Ptpn6me-v) mice)
and wild-type mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). For some experiments, (CD3d-IL5)NJ.1638Nal
transgenic mice were backcrossed with B6(Cg)-Ncf1m1J/J mice and
then interbred to create IL-5 transgenic mice with defective
NCF1/p47phoxprotein (Ncf/IL-50/+mice). Lyn-deficient mice
were obtained from Dr. Juan Rivera (National Institutes of Health,
Bethesda, MD) and wild type littermates were used as control.
Depending on the purpose of the experiment, cells from 8–22
week old mice were used.
Rat mAbs directed against murine FAS (CD95, clone RMF2,
IgG1, Beckman Coulter, Brea, CA), Siglec-F (Clone E50–2440,
rat IgG2a, BD Biosciences), CCR3 (Rat IgG2a, R&D Systems,
Minneapolis, MN), and irrelevant isotype-matched rat mAb (BD
Biosciences) were purchased from the sources indicated.
Isolation of Mouse Eosinophils from Blood, Spleen and
Blood was obtained via cardiac puncture from IL-5+and Ncf/
IL-50/+mice. Spleens were also simultaneously obtained and single
cell suspensions were generated. Erythrocytes were then lysed
hypotonically. This yielded eosinophils of purity ranging from
28% to 55%, with all contaminating cells being mononuclear cells.
In some experiments, immunomagnetic negative selection of
EDTA-anticoagulated whole blood buffy coats was performed to
generate blood eosinophils of .96% purity by depleting mono-
nuclear cells with a mixture of CD90.2 and CD45R coated beads
(Miltenyi Biotec, Auburn, CA) as previously described . In
other experiments, eosinophils were obtained from the peritoneal
cavity 48–72 hours after 4% thioglycollate injection, as described,
with purity ranging from 25% to 62% (average 36.6%, SD
11.6%), the contaminating cells being neutrophils and monocytes
Culture-derived Generation of Eosinophils from Bone
Ex vivo generation by culture of mouse bone marrow-derived
eosinophils was performed essentially as previously described by
Dyer et al . Briefly, bone marrow cells were collected from the
femurs and tibias of wild-type, Ncf2/2and mevmice by flushing
the bone marrow cavity with RPM 1640 medium (Gibco BRL,
Grand Island, NY). After red cell lysis, the bone marrow cells were
cultured at 106/ml in medium containing RPMI 1640 with 20%
fetal bovine serum (FBS, Cambrex, East Rutherford, NJ), 100 IU/
ml penicillin, 10 mg/ml streptomycin, nonessential amino acids,
Mechanism of Siglec-F-Induced Eosinophil Apoptosis
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Mechanism of Siglec-F-Induced Eosinophil Apoptosis
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1 mM sodium pyruvate (all from Invitrogen-Life Technologies,
Grand Island, NY), and 50 mM 2-ME (Sigma-Aldrich, St. Louis,
MO) supplemented with 100 ng/ml stem cell factor (SCF) and
100 ng/ml FLT3 ligand (FLT3-L) (PeproTech, Rocky Hill, NJ)
from days 0 to 4. On day 4, the cells were moved to new flasks and
the medium containing SCF and FLT3-L was replaced with
medium containing 10 ng/ml recombinant mouse IL-5 (R&D
Systems, Minneapolis, MN). Every other day, from this point
forward until cells were used, one-half of the media was replaced
with fresh media containing IL-5, and the concentration of the
cells was adjusted each time to 106/ml. Cells were enumerated in a
hemocytometer and viability (consistently .90%) and purity were
determined as mentioned above for human eosinophils. These
methods yield eosinophils of normal morphology expressing
proteins seen in mature eosinophils, including normal cell surface
levels of Siglec-F .
Evaluation of Mouse Eosinophil Phenotype and
Binding of antibodies and appropriate isotype controls (all used
at saturating or equivalent concentrations) was determined on
eosinophils by use of single or dual color immunofluorescence and
flow cytometry with appropriate gating (e.g., CCR3+granulocytes)
employing a FASCalibur flow cytometer (BD Biosciences) as
described previously [24,44].
Eosinophil apoptosis was assessed for freshly isolated cells or
mature cells grown from bone marrow-derived cells using flow
cytometry following labeling with Annexin-V as described .
Briefly, apoptosis-inducing antibodies (Siglec-F, FAS and other
controls) were added and cells were incubated for 18–24 hr in
10 ng/ml mouse IL-5 (R&D Systems). In some experiments, cells
were preincubated for 5–30 minutes with the pan-caspase
inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-
methyl]- fluoromethylketone, 10 mM), Src family kinase inhibitor
PP2, its inactive control PP3, Src family/ABL tyrosine kinase
inhibitor dasatinib or the ROS production inhibitor diphenyle-
neiodonium (DPI, 20 mM) (each obtained from EMD Chemicals,
San Diego, CA except for dasatinib which was from Bristol-Myers
Squibb, Princeton, NJ) before adding the proapoptotic antibodies
as previously described [22,25].
Measurement of Eosinophil ROS Production
The release of ROS was quantified using a lucigenin-
dependent, 96-well based chemiluminescence assay exactly as
described . Briefly, eosinophils (100,000/well) suspended in
Hanks’ balanced salt solution (pH 7.4) containing 10 mM HEPES
and 1 mg/ml bovine serum albumin were added to 96 well tissue
culture plates. For some wells, 1 ng/ml phorbol myristate acetate
(PMA, Sigma-Aldrich) was used as a positive control, while others
contained 10 mg/ml anti-Siglec-F or control IgG2a rat antibody.
Chemiluminescence readings were repeatedly determined in
triplicate using a Veritas Microplate Luminometer (Turner
Biosystems, Inc., Sunnyvale, CA) for up to 150 min at 37uC.
Data are reported as chemiluminescence intensity (arbitrary units)
All data are presented as means 6 SD. Statistical analysis was
conducted using ANOVA, followed by post-hoc multiple com-
parison tests. Values were considered significant at P,0.05.
Figure 2. Eosinophils from Ncf2/2mice undergo Siglec-F-
induced apoptosis to the same degree as those of wild type
mice. Eosinophils were grown from bone marrow precursors of wild
type and Ncf2/2mice. Purity of eosinophils at day 10 of culture was
52623% and 50622% for Ncf2/2mice and wild type mice respectively
(n=4). Antibody exposure conditions are provided in the legend. Open
bars: no mAb; black bars: Siglec-F mAb; striped bars: FAS mAb; gray
bars: irrelevant rat IgG1 mAb (n=2, to control for the FAS mAb) and
irrelevant rat IgG2a mAb (n=2, to control for the Siglec-F mAb). As no
differences in effects were see with the two isotype-match control
mAbs, data were pooled and shown as ‘‘IgG control’’; n=4. *p,0.05;
Figure 3. Effects of the pan-caspase inhibitor Z-VAD-FMK
(10 mM) on Siglec-F-induced eosinophil apoptosis. Eosinophils
were generated from bone marrow of wild type, Ncf2/2and mevmice
and tested at day 10 of culture (purity was 46620%, 52618% and
41623%, respectively, n=3–6). Cells were preincubated for 30 minutes
with or without Z-VAD-FMK followed by co-culture with Siglec-F or
control irrelevant rat IgG2a mAb for an additional 18 hr before
determining Annexin positivity. Various treatment conditions are
provided by the legend. * p,0.05.
Figure 1. Exposure of mouse eosinophils to Siglec-F mAb does not result in detectable ROS production. Eosinophils were isolated from
IL-5+mice (panels A and B) or from Ncf/IL-50/+mice (Panel C) and exposed as indicated in the legend to Siglec-F mAb or an irrelevant isotype-
matched rat IgG2a mAb. PMA (1 ng/ml) was used as the positive control. Panel A represents data using spleen cells (4468% pure eosinophils, n=4).
Panel B represents data from peripheral blood (3063% purity, n=4). Panel C represents pooled data from two mice in which blood eosinophils and
spleen eosinophils were tested separately with similar results, yielding a total of 4 separate experiments (34612% purity).
Mechanism of Siglec-F-Induced Eosinophil Apoptosis
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Previous studies of Siglec-8-induced apoptosis using inhibitors of
ROS generation in human eosinophils suggested that this pathway
is particularly important in IL-5-activated human eosinophils
[23,26]. Therefore, the contribution of ROS to Siglec-F-induced
murine eosinophil activation was initially explored. As shown in
Figure 1, murine eosinophils isolated from the spleen or blood of
IL-5+mice produced ROS in response to PMA but not in response
to Siglec-F mAb exposure (panels A and B). Furthermore,
eosinophils isolated from the blood or the spleen of IL-5+mice
crossbred with the Ncf2/2mice (so-called Ncf/IL-50/+mice)
produced very little ROS, even in response to PMA treatment
(panel C). This was not due to differences in Siglec-F surface
expression among cell populations, because eosinophils from all
sources used expressed similar surface levels of Siglec-F, and use of
eosinophils cultured for up to 24 hours in the absence of IL-5
yielded similar results (data not shown). Furthermore, this was not
due to an effect of contaminating cells, because other preparations
containing blood eosinophils from IL-5 transgenic mice enriched
to .96% purity also failed to produce any detectable ROS in
response to anti-Siglec-F antibody, even though similar levels of
ROS to those made by impure cells were detected in response to
stimulation with PMA (data not shown).
Despite the lack of detectable ROS production following Siglec-
F engagement, eosinophils grown from bone marrow-derived
precursors (Figure 2), underwent apoptosis when exposed to either
Siglec-F mAb or anti-FAS mAb as a control, and virtually identical
levels of apoptosis were seen under these conditions, whether or
not the eosinophils were derived from wild type, Ncf2/2mice.
Note that the magnitude of this modest apoptotic response was
remarkably similar to that seen with identical methods employed
with higher purity eosinophils derived from IL-5 transgenic mice
as we and others have previously published, even when a
secondary polyclonal anti-rat secondary antibody was used
[19,24]. These data suggest that the use of culture-derived
eosinophils provides a suitable model for studying mechanisms
of Siglec-F-mediated eosinophil apoptosis.
The next set of experiments was designed to directly explore the
role of NADPH oxidase, SHP-1, Src family kinases and caspases in
Siglec-F-induced eosinophil apoptosis. For these experiments,
eosinophils were derived from the bone marrow of wild type,
Ncf2/2and mevmice, the latter used to explore the role of SHP-1
in Siglec-F-mediated apoptosis. Apoptosis was determined in the
presence or absence of Siglec-F mAb and the pan-caspase
inhibitor Z-VAD-FMK. As shown in Figure 3, Siglec-F mAb
was equally effective in inducing apoptosis among eosinophils
derived from wild type, Ncf2/2and mevmice, and significant
inhibition by Z-VAD-FMK of Siglec-F-induced apoptosis in all
three types of mouse eosinophils was also seen, demonstrating that
Siglec-F-induced apoptosis does not require NADPH oxidase
activity or SHP-1 but is dependent on caspase activation. These
results are in contrast to published work showing that human
eosinophil apoptosis induced by Siglec-8 is only partially caspase-
dependent, and after exposure to IL-5 or IL-33 primarily involves
mitochondrial pathways and the generation of ROS [22,25–27].
Because the ITIM and ITIM-like domains may have functions
independent of SHP-1 activation, we tested the role of Src family
kinases that have been shown to phosphorylate ITIM/ITIM-like
Figure 4. Src family kinases are not required for Siglec-F-
induced eosinophil cell death. Eosinophils from wild type (panel A)
and Lyn-deficient (Lyn KO, panel B) mice were treated with anti-Siglec-F
(filled bars) or isotype-matched control antibody (open bars). In panel A,
eosinophils from peritoneal lavage were treated in the presence of PP2
(5 and 10 mM) and dasatinib (1 and 10 nM). Shown are data from a
single experiment representative of n=3 experiments with similar
results. In panel B, eosinophils were obtained from a Lyn deficient
mouse by peritoneal lavage (n=1, shown as the representative
experiment, to match the cell source of panel A) or from mouse bone
marrow-derived eosinophils (n=3, data not shown but with similar
results). Error bars in these representative experiments represent the
standard deviation of triplicates.
Figure 5. Effects of DPI on Siglec-F-induced apoptosis of
eosinophils. Wild-type mouse bone marrow-derived eosinophils
(n=4) were incubated with or without DPI (20 mM) for 5 min before
adding either a control mAb or Siglec-F mAb and then cells were
cultured in IL-5 for an additional 18 hr before determining Annexin
positivity. * p,0.05.
Mechanism of Siglec-F-Induced Eosinophil Apoptosis
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domains of inhibitory receptors, including siglecs. As seen in
Figure 4A, inhibition of Src family kinases with the compound PP2
in the absence of anti-Siglec-F (open bars) led to increased
eosinophil cell death compared to eosinophils incubated with
inactive control compound PP3, suggesting that Src family kinases
are required for eosinophil viability in general. However, Siglec-F-
induced eosinophil cell death (filled bars) was comparable in cells
incubated with and without Src-family inhibitor PP2 as well as a
broader kinase inhibitor dasatinib, suggesting that Src family
kinases are not required for Siglec-F-induced cell death. Further-
more, we used an independent genetic approach to assess
specifically the role for Lyn kinase, and found that Lyn-deficient
eosinophils underwent eosinophil cell death at levels comparable
to that of wild type cells (Figure 4B). Together, these data
demonstrate that Src kinases are not required for Siglec-F-induced
eosinophil cell death.
Lastly, mouse eosinophils were tested to determine whether
Siglec-F engagement might trigger generation of ROS within
mitochondria (perhaps at low-levels), and whether mitochondria-
derived ROS might be necessary or sufficient for Siglec-F-induced
apoptosis. For these experiments, a pharmacologic inhibitor of
ROS production was employed. As shown in Figure 5, the
mitochondrial electron transport inhibitor, DPI, did not prevent
Siglec-F-induced apoptosis. This was in contrast to prior studies in
human eosinophils, where DPI completely blocked Siglec-8-
induced apoptosis of cytokine-primed human eosinophils .
In this paper, we have found that there is no detectable role for
ROS in Siglec-F-mediated apoptosis. This conclusion was based
on an inability to detect any ROS production after Siglec-F mAb
exposure in normal or IL-5 primed mouse eosinophils derived
from blood, spleen or culture, and similar rates of apoptosis using
eosinophils derived from Ncf2/2mice deficient in NADPH
oxidase activity. Instead, Siglec-F-mediated apoptosis was com-
pletely blocked by the addition of a broad-spectrum caspase
inhibitor. While the latter has also been reported to play a role in
Siglec-8 function in non-cytokine primed human eosinophils
[22,25], the lack of a ROS-dependent pathway for Siglec-F-
induced eosinophil death, even after cytokine activation, repre-
sents a difference between Siglec-F signaling in mouse eosinophils
and Siglec-8 signaling in human eosinophils. Despite these
differences, it remains to be determined how Siglec-F, which
lacks any classical motifs for caspase activation, initiates this death
In additional experiments to explore the potential involvement
of the Siglec-F cytoplasmic ITIM domain using eosinophils
derived from mevmice deficient in SHP-1 function, it was
observed that SHP-1 was not required for Siglec-F-induced
eosinophil apoptosis. While one cannot rule out the possibility
that other tyrosine phosphatases may be involved in Siglec-F or
Siglec-8-mediated signaling, most prior reports of signaling via
CD33-family siglecs have implicated SHP-1 [8,29,33,34]. Since
the ITIM motif is often phosphorylated by Src family kinases
(which leads to binding of phosphatases such as SHP-1, or other
signal transduction molecules), we tested the role of Src family
kinases and specifically Lyn kinase. However, our data suggest Src
family kinases are not involved in the apoptosis induced by Siglec-
F. Our recent studies have implicated MAP kinases ERK-1/2 in
Siglec-8-mediated human eosinophil cell death (Kano et al.
Mechanism of Siglec-8-mediated cell death in IL-5-activated
eosinophils: Role for reactive oxygen species-enhanced MEK/
ERK activation. J. Allergy Clin. Immunol. in press), and thus
future studies will assess the role of this pathway in Siglec-F-
mediated cell death of mouse eosinophils. Given the presence of
the membrane distal ITSM motif conserved in both Siglec-8 and
Siglec-F, it is also possible that there is a differential role of this
cytoplasmic region in different cell types. Indeed, in human mast
cells the membrane proximal ITIM motif completely controls the
inhibitory effect of Siglec-8 engagement on FceRI-mediated
degranulation, yet these cells fail to undergo Siglec-8-dependent
apoptosis . More work is needed to explore the exact role of
the membrane distal ITSM motif in signaling via Siglec-F and
In conclusion, the work reported here has uncovered potential
differences between Siglec-F and Siglec-8 signaling and its
consequences. Despite the remarkably consistent benefits of
targeting Siglec-F in mouse models of hypereosinophilia, asthma
and gastrointestinal eosinophilia and the exaggerated eosinophilic
responses seen in mice deficient in Siglec-F when put through
various models of allergic inflammation , based on the data
presented herein, Siglec-F appears to have a different intracellular
mechanism of activity than Siglec-8 in that even under cytokine
priming conditions, cell death is always caspase dependent. Given
the fact that Siglec-F is a functional paralog rather than a true
ortholog of Siglec-8  and that below chimpanzees there is no
Siglec-8 ortholog [47,48] efforts to advance the targeting of Siglec-
8 as a potential therapeutic for human diseases will need to
consider other strategies besides simply relying on data from
studies of Siglec-F.
The authors would like to thank Nancy and Jamie Lee for providing the
IL-5 transgenic mice, and Dr. Juan Rivera for the Lyn-deficient mice. We
also thank Dr. Hirohito Kita and his lab members for helpful discussions.
Conceived and designed the experiments: HM NZ ZZ BSB. Performed the
experiments: HW GK SAH MB. Analyzed the data: HM GK SAH MB
NZ ZZ BSB. Contributed reagents/materials/analysis tools: ZZ. Wrote the
paper: HM GK NZ BSB.
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Mechanism of Siglec-F-Induced Eosinophil Apoptosis
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