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Blood First Edition Paper, prepublished online October 8, 2013; DOI 10.1182/blood-2013-07-514497
COORDINATED AND UNIQUE FUNCTIONS OF THE E-SELECTIN LIGAND ESL-1 DURING
INFLAMMATORY AND HEMATOPOIETIC RECRUITMENT IN MICE
SREERAMKUMAR et al: ESL-1 IN INFLAMMATORY AND PROGENITOR TRAFFICKING
Vinatha Sreeramkumar1, Magdalena Leiva1, Anika Stadtmann2,3, Christophe
Pitaval1, Inés Ortega-Rodríguez1, Martin K. Wild3, Brendan Lee4, Alexander
Zarbock2,3 and Andrés Hidalgo1
1
Department of Epidemiology, Atherothrombosis and Imaging, Centro
Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández
Almagro 3, 28029 Madrid, Spain. 2 Department of Anesthesiology and Critical
Care Medicine, University of Münster,
Münster, Germany.
4
3
Max-Planck Institute Münster, 48151
Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, Texas, USA.
Corresponding author: Andrés Hidalgo ([email protected])
Department of Epidemiology, Atherothrombosis and Imaging, Centro Nacional
de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3,
28029 Madrid, Spain. Phone: +34 91 4531200 (Ext. 1504). Fax: +34 91
4531245
1
Copyright © 2013 American Society of Hematology
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KEY POINTS
•
ESL-1 and PSGL-1 cooperate to mediate E-selectin binding, myeloid
homeostasis and inflammatory cell recruitment
•
ESL-1 dominates E-selectin binding and homing of hematopoietic
progenitors
ABSTRACT
Beyond its well-established roles in mediating leukocyte rolling, E-selectin is
emerging as a multifunctional receptor capable of inducing integrin activation
in neutrophils, and of regulating various biological processes in hematopoietic
precursors. While these effects suggest important homeostatic contributions
of this selectin in the immune and hematological systems, the ligands
responsible for transducing these effects in different leukocyte lineages are
not well defined. We have characterized mice deficient in ESL-1, or in both
PSGL-1 and ESL-1, to explore and compare the contributions of these
glycoproteins in immune and hematopoietic cell trafficking. In the steady-state,
ESL-1 deficiency resulted in a moderate myeloid expansion that became
more
prominent
when
both
glycoproteins
were
eliminated.
During
inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation
and extravasation of mature neutrophils, but only the combined deficiency in
PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly,
we find that the levels of ESL-1 were strongly elevated in hematopoietic
progenitor cells (HPC). These elevations correlated with a prominent function
of ESL-1 for E-selectin binding and for migration of HPC into the bone marrow.
Our results uncover dominant roles for ESL-1 in the immature compartment,
and a functional shift towards PSGL-1-dependence in mature neutrophils.
2
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INTRODUCTION
In order to perform their immune functions, leukocytes traffic actively from the
blood into immune organs or inflamed tissues. In the particular case of
neutrophils, inflammatory recruitment critically relies on the expression of the
two endothelial selectins by the activated endothelium1. P- and E-selectins
recognize highly glycosylated proteins decorated with fucosylated and
sialylated moieties present on neutrophils, monocytes and some lymphocyte
subsets
2,3
. While P-selectin glycoprotein-1 (PSGL-1) was found in early
studies to almost exclusively mediate binding to P-selectin4,5, the identification
of leukocyte ligands for E-selectin has proven to be more challenging. This
has been in part due to the highly glycosylated structure and poor
immunogenicity of its ligands, which has prevented generation of functionblocking antibodies 6. Another reason has been that many different leukocyte
glyco-conjugates are capable of mediating binding to E-selectin, at least in
vitro.
Through the use of gene-deficient mice several surface glycoproteins,
including PSGL-1, CD44 and CD43, have been shown to function as
physiological ligands for E-selectin in different leukocyte subsets3. PSGL-1
and CD44 were shown in these mice to cooperate for E-selectin-mediated
recruitment of neutrophils and inflammatory T cells in vivo7,8, however the
significant selectin-binding activity remaining in double-deficient mice argued
for the presence of one or more additional ligands. Although the unavailability
of mice deficient in E-selectin-ligand-1 (ESL-1; encoded by Glg1) have
prevented analysis of the contribution of this glycoprotein to leukocyte
trafficking, studies that used shRNA-mediated silencing of Glg1 suggested
that this glycoprotein cooperated with PSGL-1 and CD44 for E-selectin
binding and recruitment of transduced neutrophils during inflammation 9.
However, the reduced fraction of transduced leukocytes in these studies
precluded analysis of global hematological or immune alterations due to the
absence of the glycoprotein during inflammation or in the steady-state. These
limitations also prevented dissection of the roles of ESL-1 in rare populations
of hematopoietic cells. Particularly relevant among these are hematopoietic
3
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progenitor cells (HPC), which rely on E-selectin for efficient migration to the
bone marrow (BM)10-12. Remarkably, however, the ligands that control Eselectin binding on HPC in vivo have not been defined.
Beyond its contributions to rolling, recent studies have uncovered important
additional functions for E-selectin. Engagement of E-selectin on rolling
neutrophils can initiate intracellular signals through the canonical ligands
PSGL-1 and CD44, and these signals in turn promote Gαi-independent
activation of the β2 integrin LFA-1, resulting in slow rolling, enhanced
adhesion and recruitment into inflamed tissues13,14. More recently, an elegant
study demonstrated that E-selectin expressed by vascular cells in the BM also
controls the quiescence, self-renewal and chemoresistance of hematopoietic
stem cells15. Notably, this study also revealed that E-selectin binding activity
in murine HPC is largely independent of PSGL-1 and CD44, implying that a
different ligand mediates E-selectin binding in the immature compartment.
In this study we have analyzed mice deficient in ESL-1 and PSGL-1, and
generated double-deficient mutants to dissect their physiological contributions
within the hematopoietic compartment. We report that PSGL-1 and ESL-1
cooperate to maintain myeloid homeostasis in the steady-state and allow
neutrophil recruitment during inflammation, but only PSGL-1 controls integrin
activation and slow rolling. In contrast, ESL-1 was the dominant E-selectin
ligand in, and controlled homing of, immature hematopoietic progenitors to the
BM. Our results thus reveal a functional shift of selectin ligand use, from ESL1 to PSGL-1, during myeloid maturation.
4
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MATERIALS AND METHODS
Mice
Six to twelve week-old C57BL/6 male and female mice were used for the
transplantation studies. DsRed-transgenic mice (under a β-actin promoter)
were used as donors in some transplantation studies. ESL-1-deficient
(Glg1−/−) mice were generated as reported16.
PSGL-1- (Selplg−/−) and E-
selectin-deficient (Sele−/−) deficient mice have already been described5,9,11.
Selplg−/− Glg1−/+ mice were bred to generate ESL-1/PSGL-1 double-deficient
mice. The genotypes were determined by PCR. Chow and water were
available ad libitum. All mice were in a pure C57BL/6 background. Mice were
housed in a specific pathogen-free facility at Centro Nacional de
Investigaciones Cardiovasculares. Experimental procedures were approved
by the Animal Care and Ethics Committee of the Centro Nacional de
Investigaciones Cardiovasculares.
Generation of BM chimeras by transplantation
We harvested donor BM cells from the appropriate genotype (wild-type, wildtype DsRed, PSGL-1−/−, ESL-1−/− or ESL-1/PSGL-1−/− DKO) which were
mixed with equal numbers of WT-Dsred BM cells for transplantation. For
some experiments only BM cells from the four groups without DsRed+
competitors were used. Recipient wild-type C57BL/6 mice were lethally
irradiated (two doses of 6.5 Gy 3 h apart; total 13 Gy) prior to receiving 2
million BM cells intravenously. We assessed engraftment of recipient animals
6 weeks after transplantation by flow cytometry.
Flow Cytometry and E-Selectin-Binding Assay
Primary blood leukocytes were incubated with DyLight650-conjugated antiLy6G antibody (clone 1A8, BioXcell) to detect the neutrophil population. Fluidphase binding of the E-selectin-IgM chimera to blood leukocytes was
performed as described previously7. Neutrophils were gated on the basis of
Ly6G+ expression and E-selectin binding was compared between DsRednegative and DsRed+ populations within the same sample. For hematopoietic
progenitors, 5 x 106 BM cells were first stained to exclude lineage (CD11b, Gr5
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1, CD3ε, B220 and TER119) positive cells and then stained for Sca-1 and cKit. LSK cells were defined as LineageNEG Sca1+ c-kitHI.cells and myeloid
progenitors as LineageNEG Sca1NEG c-kitHI. In other experiments blood or BM
cells were stained with DyLight649-conjugated anti-Gr1 antibody (Clone RB6,
eBioscience) and PE-conjugated or biotin-conjugated anti-CD115 antibody
(BioXcell) followed by Streptavidin-eFluor 450 (eBioscience) to identify
neutrophil and monocytes (see Figure S3A). These samples were additionally
stained with PerCP/Cy 5.5-conjugated anti-CXCR2 antibody (Biolegend), PEconjugated anti-CXCR4 antibody (eBioscience), or isotype controls.
For
Annexin V binding, blood leukocytes stained for Gr-1/CD115 were stained
with PE-conjugated Annexin V following the manufacturer instructions (BD
Biosciences). Samples were acquired using a FACS Canto flow cytometer
equipped with DIVA software (BD Biosciences). Data were analyzed with the
DIVA or FlowJo (TreeStar Inc.; Ashland, OR) software. All experiments were
conducted at the CNIC-Cellomics Unit.
Intravital Microscopy
Intravital microscopy (IVM) of the cremaster muscle after TNFα injection (0.5
μg, intrascrotal injection) was performed exactly as reported
17
. The IVM
system was built by 3i (Intelligent Imaging Innovations, Denver, CO) upon an
Axio Examiner Z.1 work station (Zeiss, Oberkochen, Germany) mounted on a
3-Dimensional Motorized Stage (Sutter Instrument, Novato, CA) allowing
precise computer-controlled lateral movement between XY positions and a Z
focusing drive to allow the focal plane to be rapidly changed. The microscope
is equipped with a CoolLED pE widefield fluorescence LED light source
system (CoolLED Ltd. UK), A quad pass filter cube was used with a Semrock
Di01-R405/488/561/635 dichroic and FF01-446/523/600/677 emitter. We used
a plan-APOCHROMAT 40x NA 1.0
∞/0
water-immersion objective (Zeiss).
Images were collected with a CoolSnap HQ2 camera (6.45 x 6.45-µm pixels,
1392 x 1040 pixel format; Photometrics, Tucson, AZ). The SlideBook software
(Intelligent Imaging Innovations), run on a Dell Precision T7500 computer
system (Dell Inc., Round Rock, TX), coordinated image acquisition and
facilitated offline data analysis. Six to ten venules per mouse were analyzed
6
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150 to 210 min after TNF-α treatment by acquisition of fluorescence (Cy3
channel for dsRed) and bright-field images with 2×2 or 4x4 binning for 2 min.
For obtaining centerline blood velocities, a 7–10 s movie was acquired for
each venule at 40 Hz with the Cy3 channel, 4×4 binning, to allow velocity
measurement of free-flowing cells.
Thioglycollate-Induced Peritonitis
To assess recruitment efficiency of mutant neutrophils, we generated cohorts
of hematopoietic chimeras by transplantation of BM cells from WT-DsRed
mice mixed with BM cells from the four experimental genotypes (WT, PSGL1−/−, ESL-1−/− or DKO). After a 6-week recovery period, mice were injected
intraperitoneally with 1 ml of 3% thioglycollate. 8 h after thioglycollate injection,
venous blood and peritoneal exudates were collected and aliquots stained
with DyLight650-conjugated anti-Ly6G antibody. Samples were analyzed by
flow cytometry as indicated above. For estimating the efficiency of recruitment
into the peritoneum, the ratio of knockout versus WT-DsRed neutrophils was
determined in blood and peritoneal exudates.
Analysis of leukocyte rolling in autoperfused flow chambers
Autoperfused flow chamber experiments were performed as described
previously
14,18
. In brief, rectangular glass capillaries were coated with 2.5
µg/ml E-selectin alone or in combination with 2 µg/ml ICAM-1 (R&D Systems)
for 2 h and then blocked for 1 h using casein (Thermo Fisher Scientific). To
control the wall shear stress in the capillary, a PE-50 tubing (BD) was
connected to one side of the capillary. The other side of the chamber was
connected to a PE-10 tubing and inserted into a mouse carotid artery.
Leukocyte rolling was recorded for 1 min using an SW40/NA0.75 objective
and a digital camera (Sensicam QE; Cooke Corporation).
Progenitor homing assays
BM cells were extracted from WT, PSGL-1−/−, ESL-1−/− or ESL-1/PSGL-1−/−
DKO mice and mixed in a 1:1 ratio with BM cells from a WT-DsRed donor
mouse. 107 donor BM cells (5x106 from each donor) were injected into lethally
irradiated recipient mice. Irradiated mice that did not receive a transplant were
7
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used as controls. 3 hours after injection, we harvested femurs and 50% of the
total volume was subjected to 60% Percoll gradient purification. One half of
the mononuclear fraction was washed and used for colony-forming units in
culture (CFU-C) assay, which were scored on day 7 to 10 using an
epifluorescence inverted microscope. The presence or absence of DsRed
fluorescence was used to determine whether the colonies were from WT
(fluorescent) or mutant (non-fluorescent) donors, and the ratio of CFU-C from
experimental donors relative to control WT-DsRed CFU-C was evaluated. In
each experiment we plated 0.5% of the injected mix to assess the input
WT:mutant ratios and to calculate the homing efficiencies relative to the WTDsRed reference. The average mutant:WT-DsRed ratios for the WT, ESL-1−/−,
PSGL-1−/− and DKO groups were 1.03, 1.58, 1.15 and 1.03, respectively.
CFU-C counts in the BM of non-transplanted mice were 0 in all experiments.
Statistical analysis
Data are represented as mean ± standard error of the mean, and analyzed
using Prism software (Graph pad, Inc.). Data consisting of only two data sets
were analyzed using two-tailed Student´s t-test unless stated otherwise. For
data with more than two data sets we used one way- or two-way- ANOVA for
statistical analysis. Tukey’s multiple comparisons post-tests and Bonferroni’s
post-tests were used. We deemed P values below 0.05 as significant. * P <
0.05, ** P < 0.01, *** P < 0.001.
8
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RESULTS
ESL-1 cooperates with PSGL-1 to maintain myeloid homeostasis.
ESL-1 presents robust E-selectin binding activity in vitro
19
, but Glg1-silenced
leukocytes display only minor alterations in E-selectin binding in vitro and of
rolling dynamics in vivo 9. To understand how this glycoprotein may contribute
to global immune and hematological homeostasis we set out to analyze mice
deficient in ESL-1. These mice display major skeletal defects and reduced
body weight16,20, but are fertile and live for at least 40 weeks. The frequency
of live ESL-1-deficient mice born from heterozygous parents was however
very low (1.7%; 7 out of 412 live births), suggesting a high rate of embryonic
lethality. Hematological characterization of the mice at 7 to 12 weeks of age
revealed mild elevations in total blood leukocytes, monocytosis, neutrophilia
(Figure 1A) as well as splenomegaly (Figure 1B), but no difference in
lymphocyte counts. All other hematological values were normal and ESL-1
heterozygous littermates were indistinguishable from WT controls (Table S1).
These results suggest a more prominent role for ESL-1 in hematological
homeostasis than anticipated.
PSGL-1 is a prominent ligand for E-selectin and the main ligand for P-selectin
in myeloid leukocytes
21
, and mice deficient in PSGL-1 consequently display
elevations in neutrophil counts (Figure 1A and
5,21
). To determine whether
ESL-1 cooperates with PSGL-1 to allow myeloid cell trafficking, we generated
mice doubly deficient in both glycoproteins (herein referred to as DKO mice).
About 2% of double-deficient mice were born from PSGL-1–/– ESL-1+/– parents
(5 out of 251 live births), and displayed morphological defects and
splenomegaly (Figure 1B) similar to single ESL-1-deficient mice (Table S1).
Compared to the PSGL-1–/– or ESL-1–/– parental lines, however, DKO mice
had further elevations in blood neutrophil and monocyte counts, but not in
lymphocyte numbers (Figure 1A). Analysis of BM chimeras generated by
transplantation of BM cells from the different mutant mice into WT recipients
(Figure S1A) confirmed that the myeloid expansion in the mutant mice was
intrinsic to the hematopoietic compartment. This peripheral expansion in DKO
mice correlated with mild elevations in G-CSF levels in plasma (Figure S2A),
9
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while no changes were observed in the absolute number of total cells or
myeloid subsets in the BM of any group (Figure S2B). Since extravasation
and clearance of neutrophils is required for the homeostatic control of
granulopoiesis through the production of G-CSF22, these data support a role
for both glycoproteins in myeloid trafficking in the steady-state. We also
measured the levels of CXCR2 and CXCR4, two receptors that are modulated
during neutrophil aging23,24. While the levels of CXCR2 were not altered in
mutant mice, those of CXCR4 were moderately elevated in both neutrophils
and monocytes that lacked ESL-1 (Figure S3). In addition, the low frequency
of Annexin V-binding cells in all groups ruled out major effects in cell viability
in the absence of ESL-1 or PSGL-1 (Figure S3). Altogether, these findings
provide strong evidence for an important combined role for ESL-1 and PSGL1 during the homeostatic trafficking and clearance of myeloid leukocytes.
Contributions of PSGL-1 and ESL-1 to neutrophil rolling and recruitment
during inflammation
Since the phenotypes of mice deficient in ESL-1 were consistent with a role
for this glycoprotein in neutrophil trafficking, we analyzed the adhesive
behavior of ESL-1-deficient leukocytes in vitro and in vivo. Due to the limited
availability of ESL-1–/– and DKO mice, we generated mixed hematopoietic
chimeras by BM transplantation for our analyses. We transplanted lethally
irradiated recipient mice with a 1:1 mixture of BM cells obtained from WTDsRed transgenic mice together with BM from non-fluorescent WT, ESL-1–/–,
PSGL-1–/– or DKO mice. This approach afforded two additional advantages:
first, it provided an internal WT reference within each mouse; and second, it
eliminated potential environmental alterations originating from ESL-1deficiency in non-hematopoietic cells. Interestingly, the frequency of ESL-1
and DKO neutrophils was elevated relative to WT-DsRed control cells (not
shown), further confirming that both glycoproteins are important for neutrophil
homeostasis.
We next assessed the capacity of blood Ly6GHI neutrophils from the different
chimeric mice to bind an E-selectin/IgM soluble protein by flow cytometry.
Binding was mildly but reproducibly reduced in the absence of ESL-1
10
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compared to co-circulating WT neutrophils, and the reduction was stronger in
the absence of PSGL-1 (Figure 2A-B and
9,25
). Notably, binding was reduced
by ~80% in the absence of both glycoproteins (Figure 2A-B). These data
confirmed that ESL-1 displays high-affinity E-selectin ligand activity, and
together with PSGL-1 accounts for the majority of E-selectin binding on
neutrophils. To simultaneously track the intravascular behavior of neutrophils
from each mutant relative to WT-DsRed controls, we next used high-speed
multichannel intravital microscopy of the cremasteric microcirculation after
TNFα treatment (Figure 2C, Table S2 and Supplementary Video), at times
during which neutrophils constitute the majority of recruited cells26. Compared
to the marked reductions in rolling flux fractions seen in the PSGL-1–/– group
(69% reduction), we found no alterations in the absence of ESL-1 alone
(Figure 2C). In contrast, and consistent with the soluble E-selectin binding
experiments, the rolling fractions were reduced by 88% in the DKO group,
although the differences did not reach significance compared with the PSGL1–/– group (Figure 2C). We observed a similar trend for the fraction of
neutrophils that adhered to the endothelium, with a marked decrease in the
PSGL-1–/– group that were further reduced in the absence of both ligands
(Figure 2D). We next used a model of thioglycollate-induced peritonitis in our
chimeric mice to assess the roles of ESL-1 and PSGL-1 during neutrophil
recruitment to inflamed sites. In keeping with the intravital imaging analyses,
ESL-1–/– neutrophils were recruited with efficiencies similar to those of WTDsRed control cells, whereas the number of recruited PSGL-1–/– neutrophils
was reduced by 60% (Figure 2E-F). Notably, DKO leukocytes were almost
undetectable in the inflamed peritoneum, with 95% reduction compared to
control WT cells (Figure 2E-F), indicating that the presence of both
glycoproteins is essential for the recruitment of neutrophils to inflamed tissues.
Consistent with these results, we found that the absolute number of DKO
neutrophils and monocytes remained strongly elevated in the blood of mice
treated with thioglycollate (Figure S1B).
Because the differences between the PSGL-1–/– and DKO groups did not
reach significance for several parameters and this was important to
11
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conclusively define a role for ESL-1 in neutrophil recruitment, we also
compared the behavior of PSGL-1–/–and DKO neutrophils within the same
animals. To this end, we generated PSGL-1-DsRed+ mice whose BM was cotransplanted together with non-fluorescent DKO BM into lethally irradiated WT
recipient mice. Cytometric, intravital microscopy and migration analyses in
these mice revealed significant reductions in E-selectin binding, rolling,
adhesion and recruitment of neutrophils that lacked both glycoproteins
compared to those deficient only in PSGL-1 (Figure 3A-D). Together, these
results indicate that ESL-1 cooperates with PSGL-1 in all aspects of Eselectin-mediated recruitment during inflammation, from tethering and rolling
to extravasation; the results also showed that the absence of ESL-1 on
neutrophils is almost completely compensated by PSGL-1.
ESL-1 does not participate in E-selectin-induced slow rolling
Neutrophil rolling velocities are controlled both by direct engagement of
selectins as well as by partial activation of the integrin LFA-1, which can in
turn be initiated by signaling events following the engagement of E-selectin
13,14,18
. To test whether ESL-1 was capable of transducing LFA-1-activating
signals in neutrophils that promoted slow rolling, we next performed in vivo
and ex vivo experiments. Intravital microscopy experiments in chimeric mice
revealed that ESL-1–/– leukocytes rolled at velocities that were comparable to
those of WT controls in the same vessels, whereas PSGL-1–/– leukocytes
rolled significantly faster (Figure 3E). These data suggested that ESL-1 is not
involved in controlling rolling velocities and does not trigger LFA-1-activating
signals in circulating neutrophils. However, these findings contradicted our
previous results using silencing of the gene encoding ESL-1 9, possibly
because off-target effects of the shRNA or biased lentiviral transduction of
progenitor subsets caused experimental artifacts. We therefore decided to
confirm this observation in a more controlled setting in which we measured
leukocyte rolling velocities on E-selecin co-immobilized with the β2-integrin
ligand ICAM-1 in an autoperfused flow chamber system 18. Consistent with the
data in the microcirculation, PSGL-1–/– neutrophils rolled faster than WT cells,
whereas the absence of ESL-1 did not alter rolling velocities (Figure 3F).
12
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These data indicate that, although ESL-1 is a bona fide ligand for E-selectin
on mature neutrophils, it is not capable of inducing LFA-1 activation and slow
rolling on ICAM-1.
ESL-1 dominates E-selectin binding and homing of hematopoietic
progenitors to the bone marrow
Because ESL-1–/– mice displayed hematological abnormalities in the steadystate, we speculated that ESL-1 might also be a functional E-selectin ligand in
non-inflammatory leukocytes. Indeed, we found that the levels of HPC were
moderately elevated in the blood of ESL-1–/– mice in the steady-state (Figure
4A), which suggested a role for this glycoprotein in controlling HPC trafficking.
Because the functional E-selectin ligands that participate in homing to the BM
are not well characterized11,15, we decided to study the functions of ESL-1 in
hematopoietic precursors. We first analyzed the binding of soluble E-selectin
to myeloid progenitors (LineageNEG Sca-1NEG cKitHI) and to the more primitive
population of LineageNEG Sca-1+ cKitHI (LSK) precursor cells, together with
immature Ly6G+ neutrophils located within the same BM. To increase the
sensitivity of this assay, we again used chimeric mice reconstituted with WTDsRed+ BM together with the non-fluorescent experimental groups, and
compared binding relative to the internal WT competitor cells.
–/–
selectin binding to Ly6G+ BM-neutrophils in the PSGL-1
While E-
group was strongly
reduced (Figure 4B-C), binding to myeloid progenitors and LSK cells lacking
PSGL-1 was only partially reduced compared to control WT cells (Figure 4BC). In contrast, P-selectin binding was completely abrogated in PSGL-1–/– LSK
cells (Figure 4D), indicating that PSGL-1 was otherwise functional in HPC.
Interestingly, when we tested myeloid progenitors and LSK cells from ESL-1–/–
mice, we noted that binding of soluble E-selectin was reduced by 41% and
56%, respectively, and binding was not further reduced in LSK cells from DKO
mice (Figure 4B-C). While the reduced ligand activity of PSGL-1 in HPC
agrees with a recent report 15, the marked contribution of ESL-1 represents, to
our knowledge, the first identification of a glycoprotein with prominent Eselectin binding activity on hematopoietic progenitors. Thus, in contrast to the
dominant ligand activity of PSGL-1 in mature neutrophils, ESL-1 dominates Eselectin binding in immature progenitors.
13
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To functionally confirm these findings, we next assessed the differential
requirement for each ligand during HPC homing to the BM. Lethally irradiated
recipient mice were injected with a mixture of BM cells obtained from WT,
ESL-1–/–, PSGL-1–/– or DKO mice, together with cells from WT-DsRed+ mice,
and the homing of fluorescent and non-fluorescent colonies in the BM was
scored 3h later using semisolid clonogenic cultures (Figure 5A). In agreement
with the in vitro E-selectin binding data, the homing of ESL-1–/– and DKO
progenitors (measured as colony-forming units in culture, or CFU-C) was
reduced by about 60%, while that of PSGL-1–/– CFU-C was reduced by ~45%
relative to the internal WT-DsRed controls (Figure 5B). The reduced homing
seen for PSGL-1–/– progenitors is comparable to that reported previously
using genetically deficient mice or using an antibody that exclusively interferes
with P-selectin binding
11
, suggesting that most of the impaired homing of
PSGL-1–/– progenitors may be due to the inability to bind P-selectin. Together,
these data demonstrate that ESL-1 is a major ligand for E-selectin on HPC
that contributes to their efficient homing to the BM.
Since these data revealed that ESL-1 was the dominant E-selectin ligand in
HPC, we searched for possible mechanisms underlying this lineage-restricted
function. Transcriptional regulation of fucosyltransferase genes (Fut) is the
critical regulator of selectin ligand synthesis in leukocytes. Because Fut4 and
Fut7 differentially fucosylate and mediate maturation of ESL-1 and PSGL-1,
respectively27, we explored whether expression of Fut4 was elevated in HPC.
We found, however, that the expression of both Fut genes was higher in
neutrophils that in any of the hematopoietic precursors analyzed (LSK,
myeloid progenitors, and granulocyte-monocyte progenitors; Figure 5C), thus
ruling out that differential expression of Fut genes was responsible for the
preferential use of ESL-1 in HPC. In contrast, we found that the levels of ESL1 protein in HPC were 10.1- times higher than those present in circulating
neutrophils (Figure 5D), whereas the levels of PSGL-1 were similar in the two
cell types (Figure S4). Therefore, the higher levels of ESL-1 on immature
progenitors may account for its predominant function in these cells.
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DISCUSSION
In this study we have examined the functions of ESL-1 in the hematopoietic
system, which allowed us to uncover distinct signaling and migratory functions
for this glycoprotein in the mature and immature hematopoietic compartments
(Figure 6). We report that, in contrast to the preferred use of PSGL-1 on
mature neutrophils, ESL-1 dominates E-selectin binding and migration of
immature hematopoietic progenitors. In neutrophils, our results reveal a
complex specialization of each ligand in specific signaling events; whereas
PSGL-1 and CD44 regulate activation of the β2 integrin LFA-1 and promote
slow rolling upon E-selectin engagement13,14, ESL-1 is devoid of this function.
Interestingly, ESL-1 has been reported to regulate activation of the other
major myeloid β2-integrin, Mac-1
17
, suggesting that E-selectin can deliver
different signals depending on the ligand involved even within the same
leukocyte.
By analyzing mice deficient in PSGL-1 and ESL-1 we demonstrate that
neutrophil recruitment is severely impaired in the absence of both receptors.
This impairment correlates with global alterations in myeloid homeostasis both
during inflammation and in the steady-state, which partially recapitulate those
reported in mice deficient in P- and E-selectins
22,28
. Although our results
show that the ligands are important for neutrophil clearance from the
circulation, at present we cannot rule out a role in myeloid retention or release
from the BM.
An important conclusion of our study is that each ligand dominates E-selectin
binding in different hematopoietic compartments, with a dominant role of
PSGL-1 in mature neutrophils, and an unexpected prominent function of ESL1 in immature progenitors. Besides neutrophils, monocytes and inflammatory
T lymphocytes have been shown to rely heavily on PSGL-1 for E-selectin
binding and migration29,30, suggesting a general use of this glycoprotein in
fully differentiated leukocytes. It is puzzling that HPC are largely independent
of PSGL-1 for binding to E-selectin, and we speculate that this may be related
to the additional roles that E-selectin plays in the hematopoietic niche;
15
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functions that may require a more specialized ligand endowed with additional
signaling properties15. ESL-1, which shares functional and structural
homology with receptors for the fibroblast growth factor19,31, may thus
represent a more suitable ligand in the immature compartment.
Interestingly, we find that deficiency in ESL-1 results in major HPC homing
defects not previously seen in mice deficient in E-selectin11. This is likely due
to the competitive nature of the assays used in this study, which yields more
robust quantitative results compared to those used to investigate E-selectindeficient mice. It is also possible that the stronger effect of ESL-1 deletion
arises from its capacity to activate additional homing receptors though both
selectin-dependent (as shown for β2-integrins in neutrophils3) and independent mechanisms. Although in the present study we have focused on
the migratory functions of ESL-1, its identification as the major glycoprotein
ligand for E-selectin in HPC raises the possibility that it mediates other
important functions of this selectin in the immature compartment, including the
recently reported regulation of HSPC dormancy and chemoresistance15. Our
current findings thus pave the way for future work on the control of HPC
biology by ESL-1.
We present evidence that the preferential use of ESL-1 may be caused by the
elevated levels of the glycoprotein in progenitors, rather than by the
expression of glycosyltransferases involved in its functional maturation. It is
interesting that the increased levels of ESL-1 are not transcriptionally
regulated (not shown), suggesting that complex post-translational processing
of this glycoprotein regulates its cellular functions. It will be important to
understand in more detail how ESL-1 is processed in different hematopoietic
lineages, or in tumor cells that display tropism to the BM32.
In summary, the pleiotropic and specialized functions of ESL-1 in the
hematopoietic system identify this poorly characterized glycoprotein as a
potential target for both inflammatory and hematopoietic disorders. The
differential use of selectin ligands on mature and immature hematopoietic
16
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cells suggests the feasibility of targeting the migration of specific leukocyte
subsets for therapeutic purposes.
17
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ACKNOWLEDGEMENTS
We thank G. Crainiciuc, M. Nácher, V. Zorita, J.M. Ligos and the Cellomics
and Comparative Medicine Units at CNIC for technical support; L. Weiss and
D. Lucas for reviewing the manuscript. This study was supported by NIH P01HD070394 to B.L.; German Research Foundation ZA428/3-1, ZA428/6-1 and
INST211/604-1 to A.Z.; Ramón y Cajal Fellowship (RYC-2007-00697) and
SAF2009-11037 from MINECO, S2010/BMD-2314 from Comunidad de
Madrid, and FP7-People-IRG Program (246655) to A.H.; M.K.W. is supported
by the Max Planck Society. The Centro Nacional de Investigaciones
Cardiovasculares is supported by the Spanish Ministry of Economy and
Competitivity and the Pro-CNIC Foundation.
Contribution: V.S. performed experiments and wrote the manuscript; M.L.,
A.S. and C.P. performed experiments. I.O-R. maintained the animal colonies.
B.L. and M.W. contributed reagents. A.Z. designed experiments and edited
the manuscript; A.H. conceived the study and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial
interest.
18
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FIGURE LEGENDS
Figure 1. ESL-1-deficiency is characterized by a myeloid expansion in
the steady-state. Hematological characterization of WT, ESL-1−/−, PSGL-1−/−
and DKO mice. (A) Lymphocyte, neutrophil and monocyte counts in the blood
of WT and mutant mice. (B) Spleen to body weight (BW) ratios in WT and
mutant mice. n=3-9 mice per group. Data is shown as mean ± SEM, and was
analyzed by unpaired Student’s t-test.
Figure 2. ESL-1 cooperates with PSGL-1 in all stages of neutrophil
recruitment during inflammation. Analyses were performed in mice
transplanted with BM from WT, ESL-1−/−, PSGL-1−/− or DKO mice together
with competing WT-DsRed BM cells. (A) Flow-cytometric analyses of soluble
E-selectin binding to Ly6GHI blood neutrophils. Histograms show overlays of
E-selectin binding to experimental neutrophils (empty histograms) and WTDsRed competitors (dark grey histograms). Binding in the presence of EDTA
was used as a negative control (light grey histograms). (B) Quantification of
E-selectin binding, as measured by the mean fluorescence intensities, in all
groups. Values are represented as ratios relative to internal WT-DsRed
competitor cells. n=7-9 mice per group from 3 independent experiments. (C)
Intravital microscopy analysis of neutrophil rolling within inflamed cremastric
venules. Near-simultaneous acquisition in two channels discriminates
experimental (brightfield) and WT-DsRed (red) neutrophils. The bar graph
shows rolling flux fractions represented as ratios relative to internal WTDsRed competitor cells. n=22-58 venules in 5-7 mice per group. (D)
Representative micrographs of adherent neutrophils from the same groups
shown in (C). White dotted lines demarcate adherent non-fluorescent cells.
Non-fluorescent structures in the bottom panel are erythrocytes bound to WTDsRed cells. Bar graph shows ratios of adherent fractions relative to WTDsRed competitors. n=26-58 venules in 5-8 mice per group. (E) Neutrophil
extravasation after 8h of thioglycollate-induced peritonitis. Histograms show
percentages of mutant (grey histograms) and WT-DsRed (red histograms)
neutrophils in blood prior to extravasation (top panels), and in peritoneal
exudates (bottom panels). (F) Ratios of mutant relative to WT-DsRed
22
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neutrophils
in
blood
versus
the
peritoneum,
thereby
representing
extravasation efficiencies. n=5-14 mice per group from 2 independent
experiments. Data is shown as mean ± SEM, and was analyzed by One-way
ANOVA and Tukey´s multiple comparison test.
Figure 3. ESL-1 mediates neutrophil recruitment in the absence of
PSGL-1, but is dispensable for integrin-mediated slow rolling. Analyses
in panels A to D were performed in WT mice transplanted with BM cells from
PSGL-1−/− DsRed+ and DKO donor mice. (A) Flow-cytometric analyses of
soluble E-selectin binding to Ly6GHI blood neutrophils. Histograms show
overlays of E-selectin binding to DKO vs. PSGL-1−/− DsRed+ neutrophils
present in the blood of the same mice. Binding in the presence of EDTA was
used as a negative control. E-selectin binding of WT neutrophils is included as
reference. Bar graphs at right show quantification of E-selectin-binding
intensities of DKO and PSGL-1−/− DsRed+ neutrophils. Data is from 4 mice,
and was analyzed using paired t-test. (B) Rolling-flux fractions of DKO and
PSGL-1−/− DsRed+ neutrophils in inflamed cremastric venules. n=27 venules
from 5 mice. (C) Representative micrograph of adherent neutrophils in an
inflamed venule during the adhesion phase. Dotted lines demarcate a nonfluorescent DKO cell. Bar graph at right shows quantification of the adherent
fractions. n=27 venules from 5 mice. (D) Neutrophil extravasation in
thioglycollate-induced
peritonitis.
Bar-graph
represents
the
relative
frequencies of DKO and PSGL-1−/− DsRed+ neutrophils in blood versus the
peritoneum, thereby representing extravasation efficiencies. n=4 mice from 2
independent experiments. Data was compared using a paired t-test. (E)
Cumulative frequency histograms of rolling velocities of mutant neutrophils
obtained from analyses of chimeric mice reconstituted with BM cells from WT,
ESL-1−/−, PSGL-1−/− or DKO mice. Dotted lines indicate velocities of the
median. The bar graph represents mean rolling velocities. n=37-82 cells per
group from 5-7 mice per group, from 2 independent sets of experiments. Data
was analyzed using a One-way ANOVA with Tukey’s multiple comparison test.
(F) Rolling velocities of leukocytes in autoperfused flow chambers coated with
E-selectin alone or in combination with ICAM-1. Data is from 3-4 individual
23
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mice per group, and was analyzed using two-tailed Student’s t-test. Data is
shown as mean ± SEM.
Figure 4. ESL-1 dominates E-selectin binding in hematopoietic
progenitors. (A) Number of progenitors in the blood of mice transplanted with
WT or ESL-1−/− BM cells, measured as CFU-C. (B) Representative dot plot at
left shows the gates used for myeloid progenitors and LSK cells among
LineageNEG BM cells. Panels at right show representative histograms of Eselectin binding to experimental (grey) and WT-DsRed (red) BM-derived
neutrophils (top panel), myeloid progenitors (middle) and LSK cells (bottom).
Dashed lines show levels of binding in the presence of EDTA. (C)
Quantification of E-selectin-binding to BM-neutrophils, myeloid progenitors
and LSK cells. Values represent fluorescence intensity ratios relative to
internal WT-DsRed competitor cells. n=4-6 mice per group from 3
independent experiments. (D) Quantification of P-selectin-binding in LSK cells
from all groups represented as in (C). n=4-5 mice per group from 3
independent experiments. Bars represent mean ± SEM. Data was analyzed
by One-way ANOVA using a Tukey’s multiple comparison test.
Figure 5. ESL-1 mediates progenitor homing to the bone marrow. (A)
Design of the BM homing assays. Lethally irradiated mice were injected with
experimental and DsRed+ competitor BM cells, which were allowed to home
for 3 hours. Homed CFU-C were scored 7-10 days later, and differentiated on
the basis of red fluorescence (WT-DsRed+) or no fluorescence, as illustrated
in the micrographs. (B) Homing efficiencies of progenitors from each group,
calculated as ratios of homed CFU-C from each mutant donor relative to
competing WT-DsRed+ progenitors, and corrected by the ratio of CFU-C
injected. n=6-8 mice per group from 3 independent experiments. Bars
represent mean ± SEM. Data was analyzed by One-way ANOVA using a
Tukey’s multiple comparison test. (C) Relative expression of Fut4 and Fut7 in
purified LineageNEG Sca-1+ cKitHI cells (LSK), myeloid progenitors (MP),
Granulocyte-monocyte progenitors (GMP) and circulating neutrophils. Data
shows the mean ± SEM from 3 independent samples, and was analyzed by
an unpaired Student’s t-test. (D) Western blot analysis of total ESL-1 protein
24
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and β-actin (load control) present in sorted LineageNEG cKitHI progenitors
(HPC) and blood neutrophils (PMN). Data is representative of two
independent experiments, with increases of 10.0 and 10.3-fold in ESL-1
protein levels in HPC relative to PMN.
Figure 6. Shared and unique functions of ESL-1 during mature and
immature leukocyte trafficking. Schematic representation of the E-selectin
ligands preferentially used by immature (HPC) and mature (neutrophils)
hematopoietic cells to migrate. The elevated levels of ESL-1 in HPC correlate
with its predominant function for E-selectin binding and migration in these
cells. In neutrophils, both functions rely predominantly on PSGL-1 despite
similar expression of the glycoprotein in mature and immature leukocytes.
25
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Prepublished online October 8, 2013;
doi:10.1182/blood-2013-07-514497
Coordinated and unique functions of the E-selectin ligand ESL-1 during
inflammatory and hematopoietic recruitment in mice
Vinatha Sreeramkumar, Magdalena Leiva, Anika Stadtmann, Christophe Pitaval, Inés Ortega-Rodriguez,
Martin K. Wild, Brendan Lee, Alexander Zarbock and Andrés Hidalgo
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