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PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular
permeability changes during neutrophil transmigration in vivo
*Björn Petri,1 *Jaswinder Kaur,1 Elizabeth M. Long,2 Hang Li,3 Sean A. Parsons,1 Stefan Butz,3 Mia Phillipson,4
Dietmar Vestweber,3 Kamala D. Patel,1 Stephen M. Robbins,2 and Paul Kubes1
1Immunology Research Group, Department of Physiology and Pharmacology, the Calvin, Phoebe, and Joan Snyder Institute for Infection, Immunity &
Inflammation and 2Departments of Oncology and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB; 3Department of
Vascular Cell Biology, Max-Planck-Institute for Molecular Biomedicine, Münster, Germany; and 4Department of Medical Cell Biology, Division of Integrative
Physiology, Uppsala University, Uppsala, Sweden
The endothelium actively participates in
neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent
rearrangements leading to the formation
of transmigratory cups in vitro, and to
domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin–binding protein
recently shown to be in the endothelium,
is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial
LSP1 is expressed in the nucleus and
cytosol of resting endothelial cells and
associates with the cytoskeleton upon
endothelial activation. Two-photon microscopy revealed that endothelial LSP1
was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to
endogenously produced chemokines
stimulated by cytokines (tumor necrosis
factor ␣ [TNF␣] or interleukin-1␤ [IL-1␤]).
Endothelial domes were significantly reduced in Lsp1ⴚ/ⴚ compared with wildtype (WT) mice. Lsp1ⴚ/ⴚ animals not only
showed impaired neutrophil emigration
after KC and TNF␣ stimulation, but also
had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an
important role in forming endothelial
domes thereby regulating neutrophil
transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role
for LSP1 in endothelial barrier integrity.
(Blood. 2011;117(3):942-952)
Introduction
Recruitment of circulating neutrophils from the bloodstream to
sites of tissue injury and infection is the hallmark feature of the
inflammatory response. This process involves multiple, interdependent, regulated molecular interactions between the neutrophils and
the vascular endothelium.1,2 Initial tethering and rolling of leukocytes along the vessel wall is followed by firm adhesion to the
vascular endothelium caused by chemokine activation of leukocyte
integrins.3,4 The neutrophils then crawl to sites where they migrate
through the vascular endothelium,5 a process known as transendothelial migration or diapedesis. A growing body of literature
suggests that transendothelial migration is an interactive process
between leukocytes and endothelial cells, in which endothelial cells
are not passive bystanders but rather active participants that
regulate this process.6-9 There is also evidence that leukocyte
adhesion molecules binding to endothelial adhesion molecules2,10
induce intracellular signaling, stimulating the endothelial cytoskeleton and regulatory proteins to initiate transendothelial
migration.1,11,12
One such regulatory protein in endothelial cells is leukocytespecific protein 1 (LSP1), which was initially characterized in
lymphocytes and thymocytes.13,14 LSP1 has now been identified in
monocytes, macrophages, dendritic cells, Langerhans cells, and
neutrophils15-17 and in both murine and human endothelium.18
LSP1 is a cytoplasmic intracellular Ca2⫹- and F-actin–binding
protein that interacts with the cytoskeleton in leukocytes.19,20
Strategically positioned between the plasma membrane and the
cytoskeleton, neutrophil LSP1 may transmit signals that contribute
to cell polarization and cell motility.19 By contrast, endothelial
LSP1 but not leukocyte LSP1 is found primarily in the nucleus.18
LSP1 is a major substrate of the mitogen-activated protein kinase
(MAPK)–activated protein (MAPKAP) kinase 2 in the p38 MAPK
pathway as well as a substrate for protein kinase C (PKC21,22).
Intriguingly, MAPK-activated protein kinase 2 is also segregated to
the nucleus and transported to the cytoplasm on stimulation.23 Both
p38 and PKC pathways have been implicated in alterations of the
endothelial cytoskeleton.24,25
Numerous studies have documented the importance of the
endothelium and the endothelial cytoskeleton in the formation of
docking structures or transmigratory cups during transendothelial
migration in vitro.6,7,26,27 These endothelial structures are projections that move up the side of the leukocyte, suggesting a very
active, dynamic role of the endothelium in the leukocyte transmigration. Investigating neutrophil transmigration in vivo, we reported
that these transmigratory cups extended all the way to the top of
neutrophils, forming domes so that we could clearly see the
endothelium, both lumenally and ablumenally, with respect to the
Submitted February 16, 2010; accepted October 23, 2010. Prepublished online
as Blood First Edition paper, October 27, 2010; DOI 10.1182/blood-2010-02270561.
The online version of this article contains a data supplement.
*B.P. and J.K. contributed equally to this work.
© 2011 by The American Society of Hematology
942
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
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BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
neutrophil.8 This was not phagocytosis (also known as emperiopolesis) by the endothelium as the neutrophil never entered the
intracellular compartment of the endothelium; rather, it was a
covering and sealing of the neutrophil away from the mainstream
of blood. The seal would allow the neutrophil to penetrate the
ablumenal portion of the endothelium without creating a direct
conduit between the intravascular and extravascular space.
In vivo, endothelial but not neutrophil LSP1 was able to
regulate neutrophil transendothelial cell migration.18 Loss of LSP1
in the endothelium resulted in the most profound inhibition in
transmigration that we have ever reported.18 Because endothelial
LSP1 was so critical to the emigration process18 and could bind
F-actin, we tested the hypothesis that endothelial LSP1 left the
nucleus and associated with the cytoskeleton, allowing the formation of domes in vivo during transendothelial migration. Our data
show that in response to an inflammatory stimulus, endothelial
LSP1 was indeed recruited from the nucleus as well as cytosol to
the cytoskeleton. Moreover, endothelial domes were important
during transendothelial migration in vivo; Lsp1⫺/⫺ endothelium (in
LSP1 knockout and chimeric animals lacking LSP1 only in the
endothelium) often failed to construct these structures. Finally,
disproportionately increased permeability despite reduced numbers
of transmigrating neutrophils underscores the physiologic relevance of domes and the role for LSP1 in endothelial barrier
integrity.
ENDOTHELIAL LSP1 PROMOTES DOME FORMATION IN VIVO
943
Detection of endothelial domes by 2-photon microscopy
Our 2-photon system consisted of an FV300 laser scanning confocal unit
(Olympus) that was modified in house to allow for multiphoton imaging
(see Supplemental methods). For visualization the cremaster blood vessels
were labeled with anti–mouse PECAM-1 Ab coupled to Alexa Fluor 594 or
488 (each 70 ␮g/animal). In some instances the endothelium was labeled
with soybean agglutinin (SBA)–lectin coupled to Alexa Flour 555. Infiltrating neutrophils were labeled with anti–mouse Gr-1 coupled to Alexa 488 or
594 (each 50 ␮g/animal). To investigate endothelial domes under WT
conditions, lys–enhanced green fluorescent protein (EGFP) mice were used
and the vasculature was labeled with anti mouse PECAM-1 Ab coupled
to Alexa Fluor 594 (70 ␮g/animal). The cremaster was imaged using
800 nm excitation at approximately 50 mW total power. To quantify
endothelial domes, events were counted in a given vessel in the field of view
along 100 ␮m of the venule and expressed as percentage, per event, of
adherent cells.
Induction of neutrophil recruitment in cremaster muscle
To induce an inflammatory stimulus, the cremaster muscle was superfused
with 5nM KC (R&D Systems, MN,). In some experiments, recombinant
mouse TNF␣ (0.5 ␮g; R&D Systems) or IL-1␤ (50 ng; R&D Systems) in
200 ␮L of saline was injected intrascrotally before the experiment. The
neutrophil rolling flux, rolling velocity, adherence, and emigration were
measured in the cremasteric venule from 4 hours to 5.5 hours after the
TNF␣ injection at 10-minute intervals. In the case of KC, the above
parameters were measured every 10 minutes for 90 minutes after the start of
the superfusion.
Microvascular permeability measurement
Methods
Antibodies
Mouse anti–human LSP1 mAb (Clone 16), rat mAb RB6-8C5 against
mouse Ly-6G (Gr1), mAb RB6-8C5 against mouse Ly-6G conjugated to
fluorescein isothiocyanate (FITC) or phosphatidylethanolamine (PE) and
rat mAb against mouse PECAM-1 (Clone 390, which has been previously
reported not to interfere with leukocyte recruitment28,29) were purchased
from eBioscience. Other antibodies are described in supplemental Methods
(available on the Blood Web site; see the Supplemental Materials link at the
top of the online article).
Animals
WT 129/SvJ and WT C57BL/6 mice were purchased from The Jackson
Laboratory. Jongstra-Bilen and colleagues (Toronto General Hospital, ON)
kindly supplied the Lsp1⫺/⫺ mice, which were generated on the 129/SvJ
background by homologous recombination as previously described and
bred onto a C57BL/6 background.30 Both backgrounds behaved similarly in
experiments. Lys-EGFP mice in which the enhanced GFP gene was
knocked into the murine lysozyme M (lys) locus, were kindly provided by
Thomas Graf (Albert Einstein College of Medicine, Bronx, NY) and
generated as previously described.31 Bone marrow (BM)–chimeric mice
were generated following standard protocols in our laboratory.18 All mice
were bred at the University of Calgary animal center and used in
experiments when they were between 8 and 16 weeks of age for studies
involving neutrophil recruitment. All animal protocols and procedures were
approved by the University of Calgary Animal Care Committee and
conformed to Canadian Council for Animal Care guidelines.
Intravital microscopy
A mixture of 10 mg/kg xylazine (Bayer Inc Animal Health) and 200 mg/kg
ketamine hydrochloride (Rogar/STB Inc) was injected intraperitoneally to
anesthetize male mice. In all protocols, the left jugular vein was cannulated
to administer additional anesthetics or antibodies. The mouse cremaster
muscle was used to study neutrophil recruitment as previously described.18
To quantify the degree of vascular albumin leakage from cremasteric
venules of Lsp1⫺/⫺ and WT mice, microvascular permeability was measured as described previously32,33 and in the Supplemental methods.
Whole-mount staining of cremaster muscle tissue
WT animals were injected with either 0.5 ␮g TNF␣ or 50 ng IL-1␤ in
200 ␮L of saline intrascrotally 3 hours before tissue acquisition. Preparation and staining of the cremaster muscle was done as described in
Supplemental methods.
TEM
In some instances, after superfusion with 5nM KC, the cremaster preparation was separated and prepared for transmission electron microscopy
(TEM) analysis as described in Supplemental methods.
Human umbilical vein endothelial cell isolation
Human umbilical vein endothelial cells (HUVECs) were harvested and
cultured from fresh human cords as previously described.34
Subcellular fractionations
Subcellular fractionations were recovered using the Calbiochem ProteoExtract Subcellular Extraction Kit. Confluent HUVEC monolayers grown on
gelatin in T75 flasks were used. As per kit protocol, the confluent
monolayers were subjected to the buffers (I-IV) in sequential manner with
the required protease inhibitor cocktails and enzymes. Fraction I (cytosol)
Fraction II (membrane/organelle), Fraction III (nuclear material), and
Fraction IV (cytoskeleton) were collected. Seventy-five microliters of the
supernatant was removed, and 25 ␮L of 4⫻ sample buffer was added to the
samples. The samples were sonicated to dissolve the pellets and then loaded
onto a 10% sodium dodecyl sulfate (SDS) gel for Western blotting.
Western blotting
Whole-cell lysates and cellular fractionations prepared from confluent
HUVECs were subjected to electrophoresis on a 10% SDS–polyacrylamide
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944
PETRI et al
BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
Figure 1. Total cell lysates and subcellular proteome extraction of HUVECs stimulated with TNF␣. (A) Representative Western blot of total cell lysates stimulated with
TNF␣ and IL-8 and densitometry. HUVEC monolayers were stimulated for 30 minutes or 4 hours with IL-8, or for 4 hours with TNF␣. (B) Rrepresentative Western blot of
subcellular proteomic extraction. HUVEC monolayers were stimulated and treated with or without LMB (nuclear export inhibitor). Fractions were blotted for LSP1 (arrows),
pan-cadherin (membrane marker), ATF2 (nucleus), and actin (cytoskeleton). Densitometry of the Western blots in panel A (n ⫽ 3) is relative to total actin (as standardization).
Error bars indicate SEM. *P ⬍ .05.
gel, transferred to a nitrocellulose membrane, and blotted using mAbs and
polyclonal Abs against specific proteins. HUVEC fractionations from the
same day were tested on different gels. After washing, the membranes were
incubated with a secondary Ab conjugated to horseradish peroxidase and
developed using enhanced chemiluminescence reagents, with subsequent
exposure to X-ray film for the desired amount of time. As there was still
some variability of LSP1 expression in different HUVEC isolations, we
compensated for this variability with densitometry on each of the blots
using ImageJ software (v1.41o; National Institutes of Health; http://
rsb.info.nih. gov/ij/). LSP1 expression levels in total lysate were also
corrected against ␤-actin.
Statistical analysis
The data are expressed as means ⫾ SEM. For comparing differences within
2 groups, a Student t test with Bonferroni correction was used. Analysis of
variance was used for statistical analysis for the differences between more
than 2 groups. A P value of less than .05 was considered statistically
significant.
Results
Endothelial LSP1 associates with the cytoskeleton after
stimulation with inflammatory mediators
In previous studies we have shown that LSP1 is expressed not only
in leukocytes, but also in mouse and human endothelial cells.18
Using biochemical techniques, we further investigated in this study
the overall expression and subcellular localization of endothelial
LSP1 after stimulation. HUVEC cell lysates showed significantly
increased expression of total LSP1 after stimulation with TNF␣,
whereas treatment with IL-8 (the human analog of murine KC) for
either 30 minutes or 4 hours did not affect LSP1 (Figure 1A). Next,
we subjected cultured adherent HUVECs to proteome extraction
and isolated the different subcellular fractions, including the
cytosol, membrane/organelles, nuclear material, and cytoskeleton.
Under basal conditions, LSP1 signal was predominantly expressed
in the cytosol and nuclear fraction, and to a much lesser degree in
the organelle/membrane and cytoskeletal fractions (Figure 1B). On
TNF␣ stimulation, there was a very noticeable increase in LSP1 in
the cytoskeletal region, but not in the nuclear region. Treatment
with leptomycin B (LMB, a general nuclear export inhibitor)
increased the expression of LSP1 in the nuclear fraction, demonstrating that LSP1 was not able to shuttle out of the nucleus. Moreover,
the cytoskeletal-associated LSP1 was strongly inhibited with the
LMB inhibitor (Figure 1B). This clearly suggests that the cytoskeletal LSP1 was derived in a large part from the nucleus. Interestingly, LSP1 was also noted in the cytosol, but levels did not change
on treatment with TNF␣, suggesting a second pool of LSP1 outside
the nucleus that may serve another function (Figure 1B).
In leukocytes, LSP1 is a substrate of MK2,35 a signaling
molecule downstream of p38 MAPK. Under basal conditions
MK2 predominantly localizes to the nucleus, but on cell activation
it moves into the cytosol with bound p38 MAPK. To determine
whether endothelial LSP1 is also under the regulation of the
p38 MAPK pathway, we exposed the HUVEC monolayers to
SKF86002, an inhibitor of p38 MAPK, and examined LSP1
translocation in response to TNF␣. In the presence of SKF, there
was no difference in the expression of LSP1 in the nuclear fraction.
Expression of LSP1 in the cytoskeletal fraction was reduced
(supplemental Figure 1). To confirm the activation of the p38 MAPK
pathway, we examined phosphorylation of MK2. TNF␣ treatment
induced the phosphorylation of MK2, and pretreatment with the
SKF inhibitor blocked this phosphorylation of MK2 in the nucleus;
no MK2 could be found in the cytoskeleton. Total p38 did not
change under these conditions (data not shown).
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BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
ENDOTHELIAL LSP1 PROMOTES DOME FORMATION IN VIVO
945
Figure 2. In vivo endothelial dome formation in WT
mice visualized by whole-mount staining of the cremaster muscle and 2-photon microscopy. The cremaster of C57BL/6 WT mice was fixed in paraformaldehyde
and stained for platelet endothelial cell adhesion molecule-1 (PECAM-1; green, Alexa 488) and MRP-14 (red,
Alexa 568) 3 hours after application of IL-1␤ into the
scrotum. (A) Overview merge of a postcapillary venule
showing a migrating neutrophil covered by a dome
(arrows in box; scale bar: 10 ␮m). (B-D) Magnifications of
the area in panel A showing the single channels for
PECAM-1 (B), MRP-14 (C), and the merge (D). The
dome is highlighted by arrows. Presumably rolling and
adherent cells not covered by a dome are highlighted by
open arrows. Scale bar represents 5 ␮m. Image acquisition, panels A-D: Zeiss LSM510Meta confocal fluorescent
microscope on Axiovert 200M (Zeiss), 40⫻/1.2 NA water
C-Apochromat (Zeiss); DAKO fluorescent mounting medium; LSM510Meta photo-multiplier tubes (Zeiss); LSM
image Examiner Version 4.0.0.241 (Zeiss). (E) Percentage quantification of the whole-mount staining based on
the number of cells interacting with the vasculature/
vessel in the field of view in WT vs Lsp1⫺/⫺ animals.
Eleven to 16 vessels per whole mount and per background (n ⫽ 5) were analyzed. ***P ⬍ .001. Error bars
indicate SEM. For 2-photon microscopy, the cremaster of
lys-EGFP mice was superfused with 5nM KC and the
endothelium stained with anti–PECAM-1 Ab coupled to
Alexa 594. (F) PECAM-1 positive stained endothelium
demonstrating the formation of a dome (arrow).
(G) Neutrophils (green) migrate and are encapsulated by
endothelial domes (red) as highlighted by arrows. Insets
represent magnifications of the dome and the encapsulated neutrophil from panels G and F. The dome reaches
into the vessel lumen (L) and is highlighted by the arrow.
Image acquisition, panels F-G: Olympus FV300 laser
scanning confocal unit on Olympus BX61WI; Olympus
20⫻/0.95 NA water XLUMPLAN FI; bicarbonate superfusion buffer (132mM NaCI, 4.7mM KCI, 1.2mM MgSO4,
Olympus Fluoview (FV300 O3D V5.0). (H) Percentage
quantification of the 2-photon microscopy images in
WT vs Lsp1⫺/⫺ animals. Calculations were based on the
number of cells interacting with the vasculature/vessel in
the field of view (5- 8 vessels per mouse were observed;
each group contained at least 5 animals). ***P ⬍ .001.
Error bars indicate SEM. Scale bar represents 50 ␮m.
Endothelial LSP1 is necessary for endothelial domes during
neutrophil diapedesis
Three approaches were taken to analyze the role of LSP1 in dome
formation in vivo: first, whole-mount staining of cremaster muscle
tissue; second, 2-photon microscopy; third, transmission electron
microscopy. As seen in Figure 2, whole mounts 3 hours after TNF␣
or IL-1␤ stimulation revealed endothelial domes in WT mice (filled
arrows in Figure 2A, B, D; cremaster muscle treated with
cytokine). Endothelial PECAM-1 surrounded a single adherent
neutrophil (Figure 2B) and colocalized with the neutrophil marker
(anti–Myeloid-related protein [MRP]–14). Most neutrophils were
rounded, presumably rolling or free in circulation, and did not show
PECAM-1–positive domes (open arrows in Figure 2A-D). As the
formation of domes is a very dynamic and rapidly occurring
process, few neutrophils inside the vessel of the whole mount were
covered with domes at the time the tissue was fixed. Emigration
was an equally rare event. Quantification of domes in Lsp1⫺/⫺
animals showed fewer than half of these structures compared with
WT animals (Figure 2E).
To visualize the dynamic formation of domes in vivo, neutrophil interactions with the endothelium of the microvasculature
were visualized using 2-photon microscopy. Although both cytokines and chemokines induced dome-associated neutrophil adhesion and subsequent emigration, the chemokine KC induced more
concentrated emigration in a shorter period of time, allowing
observation of more frequent dome formations. Figure 2F shows
the formation of a dome (arrows) around a lys-EGFP neutrophil
(Figure 2G). Higher digital magnification reveals that the dome
(arrows) is forming around the neutrophil (Figure 2G insets). On
occasion, rolling neutrophils (open arrow in supplemental Figure
2B) could be seen rolling across a dome covering a neutrophil
(filled arrow in supplemental Figure 2A-B insets). The dynamic
process of a dome forming up the sides of an adhering neutrophil
over time is shown in supplemental Figure 3. At the end stage of
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PETRI et al
BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
Figure 3. Electron micrographs of migrating neutrophils in WT and LSP1ⴚ/ⴚ mice. (A) WT; (B) Lsp1⫺/⫺
mice. Sections (70 nm) were taken after KC superfusion
(5nM) of the cremaster. Images and corresponding cartoons demonstrate endothelial dome formations (arrows)
in WT mice (A) and the lack of such formations in LSP1
deficient mice (B arrow). Scale bars indicate 2 ␮m
(A) and 1 ␮m (B). Image acquisition, panels A-B: Hitachi
H-7000 transmission electron microscope; direct magnification: 3000⫻ (A), 4000⫻ (B); 16000 AMT camera, AMT
Capture Engine software (V.600.128); images generated
with Microsoft Office PowerPoint 2003 (SP3). Percentage
quantification of the electron micrograph sections based
on the adherent neutrophils per vessel that underwent
transendothelial migration in WT vs Lsp1⫺/⫺ animals (C).
Thirty-eight to 42 vessel sections per background were
analyzed. ***P ⬍ .001. Error bars indicate SEM. e1-e3
indicate endothelial cells; n, neutrophil; and L, vessel
lumen.
this dynamic process, the formed domes completely cover the
transmigrating neutrophil (as seen in other in vivo experiments;
arrows in supplemental Figure 4A-B insets). Quantification of the
in vivo images showed that endothelial domes in Lsp1⫺/⫺ animals
were found at 40% frequency compared with WT animals (Figure
2H). For completeness, we also performed experiments where we
injected TNF␣ into the scrotum to activate the cremaster vasculature. Whereas 4 hours after injection the majority of the neutrophils
could be found outside the vessel, at an earlier time point (2 hours)
neutrophils could be seen transmigrating. Similar to the results
obtained with the chemokine superfusion, endothelial domes could
be observed in WT animals, but were less frequently seen in
Lsp1⫺/⫺ animals (data not shown). From z-stacks we were also able
to create a 3-dimensional image and a video of a migrating
neutrophil, which enabled us to visualize the endothelial domes in
more detail in z-stacks over time (supplemental Figure 5 and
supplemental Video 1). The cells that appear blurry represent
crawling or rolling neutrophils.
Using transmission electron microscopy, a striking difference
was consistently observed between the WT and Lsp1⫺/⫺ mice.
Neutrophils in WT mice were completely covered by the endothelium, whereas in Lsp1⫺/⫺ mice the endothelium often only covered
the neutrophil pseudopod and did not climb up the sides of the
extravasating neutrophil. Figure 3 shows electron micrographs and
a cartoon representation of these endothelial domes (right panel).
The WT neutrophils are completely covered by the endothelium,
but endothelium was also below the neutrophil (Figure 3A arrows
e1); in Lsp1⫺/⫺ animals, the neutrophils have pushed very long
pseudopods displacing the endothelium, yet neither an endothelial
dome nor a clear docking structure can be seen (Figure 3B arrows).
WT neutrophils had much smaller pseudopod extensions before the
endothelium moved up the sides. Although we cannot absolutely
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BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
ENDOTHELIAL LSP1 PROMOTES DOME FORMATION IN VIVO
947
Figure 4. Serial sections from micrographs of migrating neutrophils in WT and Lsp1ⴚ/ⴚ mice. Sections
were taken after KC superfusion (5nM) of the cremaster.
Serial sections (each 70 nm) were cut 350 nm apart.
Filled arrows demonstrate endothelial dome formations in
WT mice (A-D), and open arrows point the lack of such
formations in LSP1-deficient mice (E-H). Scale bar indicates 500 nm. e indicates endothelial cell; n, neutrophil;
and L, vessel lumen. Image acquisition: Hitachi H-7000
transmission electron microscope; direct magnification:
6000⫻; 16000 AMT camera, AMT Capture Engine software (V.600.128); images generated with Microsoft
Office PowerPoint 2003 (SP3).
state that eventually a dome-like structure did not form in Lsp1⫺/⫺
mice, we never saw the huge pseudopods with no endothelium
moving up the sides of WT neutrophils. Serial sections of
transmigrating neutrophils demonstrated that the endothelium was
indeed covering the whole cell body of the neutrophil whenever a
serial section was taken (Figure 4A-D arrows; supplemental Figure
8) in WT animals, whereas in Lsp1⫺/⫺ animals no endothelial dome
structure could be observed (Figure 4E-H open arrows). Although
in some serial sections domes could be observed while neutrophils
were migrating in what appeared to be a transcellular fashion
(Figure 4A-D arrows), in most cases formation of domes in WT
animals were observed while the neutrophils were emigrating
between 2 endothelial cells (Figure 3A arrows e2 and e3). In those
situations, both endothelial cells contributed to the formation of the
dome structure. Quantification of the transmission electron microscopy sections revealed that endothelial dome formation was
reduced by more than half in Lsp1⫺/⫺ animals compared with
WT animals (Figure 3C). Whether the dome formed in LSP1⫺/⫺
mice only after a very significant proportion of the neutrophil had
invaginated the endothelium, or whether another protein can
replace LSP1, remains unknown.
Endothelial LSP1 is important to maintaining physiologic
barrier function in inflammation
The cremaster muscle of WT and LSP1-deficient mice was
superfused with 5nM of KC. There was no difference in the rolling
behavior of the neutrophils (Figure 5A) and slightly fewer adherent
cells (Figure 5B), but a large decrease in emigration (Figure 5C) in
LSP1⫺/⫺ compared with WT mice. Unexpectedly, the increase in
permeability was comparable in both WT and LSP1-deficient
animals (Figure 5D) although LSP1-deficient mice had fewer
neutrophils crossing the endothelial barrier (Figure 5C). The
increased permeability could be reduced to baseline in Lsp1⫺/⫺ and
WT mice if neutrophils were depleted, suggesting that the neutrophils were causing the increase in permeability in both strains of
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PETRI et al
BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
Figure 5. Microvascular permeability changes in cremasteric venules of WT and Lsp1ⴚ/ⴚ mice. Measurements were taken after KC superfusion of the cremaster muscle
preparation. Besides equal rolling flux (A) and decreased adhesion (B), LSP1-deficient animals show impaired neutrophil transmigration (C), but equal microvascular leakiness
(D) compared with WT animals. Depletion of neutrophils with anti Gr-1 Ab injection 24 hours before experiments shows a return of permeability to baseline levels (E). The
amount of permeability normalized for number of emigrated neutrophils, as shown 60 minutes after KC superfusion (F). Each group contained at least 3 animals. *P ⬍ .05;
***P ⬍ .001. Error bars indicate SEM.
mice (Figure 5E). Clearly, the increase in permeability per migrating neutrophil was significantly lower in WT than in LSP1deficient mice after 60 minutes of neutrophil emigration (Figure
5F). Injection of TNF␣ 4 hours before the cremaster preparation
showed similar results in neutrophil behavior and permeability
measurements (supplemental Figure 6).
It is possible that the LSP1-adherent neutrophils are more
activated and increase the release of mediators that induce permeability increases. We tested responses to KC in chimeric mice
where WT neutrophils were induced to adhere to Lsp1⫺/⫺ endothelium and Lsp1⫺/⫺ neutrophils were induced to adhere to WT endothelium. Both sets of chimeric mice demonstrated identical leukocyte rolling flux (Figure 6A) and adhesion (Figure 6B) on
KC superfusion of the muscle microvasculature. Chimeras that
lacked LSP1 only in their leukocytes emigrated as effectively
across the vasculature as WT mice (Figure 6C), and normal
permeability changes occurred in response to KC administration,
suggesting that adhesion per se of Lsp1⫺/⫺ neutrophils was not
sufficient to induce exaggerated increases in permeability. In
contrast, WT leukocytes reconstituted in Lsp1⫺/⫺ mice (that is,
lacking LSP1 in endothelium) displayed much more subtle transendothelial migration (Figure 6C), but the permeability in animals
lacking LSP1 only in the endothelium was significantly higher
(Figure 6D). The permeability per migrating neutrophil was also
only increased in the Lsp1⫺/⫺ endothelium (Figure 6F). Obviously,
the WT neutrophils attempting to cross the LSP1-deficient endothelium did so poorly and with increased permeability. The lack of
LSP1 in the endothelium is clearly responsible for the permeability
changes, which occur through the neutrophils. Depletion of the
neutrophils eliminated increases in permeability in both strains of
mice (Figure 6E).
Differential effects of endothelial LSP1 in different inflamed
vascular beds
The emigration of neutrophils in the peritoneum is quite different
from other organs using opaque sites. Indeed, we previously found
that neutrophils emigrated more in Lsp1⫺/⫺ mice than in WT mice.18
We therefore checked whether there were more domes in WT than
in Lsp1⫺/⫺ animals in the peritoneum. Although electron microscopy revealed (Figures 7A and B) endothelial domes (arrows)
during diapedesis after 4 hours of peritoneal cytokine treatment,
endothelial dome formation was less frequent in the peritoneal
endothelium compared with cremaster endothelium. Importantly,
dome formation in LSP1-deficient animals was not different from
WT controls in peritoneum (Figure 7C). Domes could be seen in
the peritoneum of Lsp1⫺/⫺ animals surrounding neutrophils, but
this was a rare event (Supplemental Figure 7). Vascular leakage in
the peritoneum based on Evan blue dye injection showed no
difference between LSP1-deficient and WT animals at an early
time point (2 hours) after saline or cytokine injection (Figure 7D).
At a later time point (4 hours), LSP1-deficient animals showed a
slight increased permeability into the peritoneal cavity compared
with WT mice, which was consistent with the greater number of
emigrating neutrophils. Surprisingly, saline-treated LSP1-deficient
animals showed an increased Evan blue leakage into the peritoneal
cavity at 4 hours.
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ENDOTHELIAL LSP1 PROMOTES DOME FORMATION IN VIVO
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Figure 6. Microvascular permeability changes in cremasteric venules of chimeric mice (Lsp1ⴚ/ⴚ into WT and WT into Lsp1ⴚ/ⴚ). Measurements were taken after
KC superfusion of the cremaster muscle preparation. Besides equal rolling flux (A) and adhesion (B), WT into LSP-1 deficient animals (lacking LSP1 only in the endothelium)
show impaired neutrophil transmigration (C) and increased microvascular leakiness (D) compared with Lsp1⫺/⫺ into WT animals (lacking LSP1 only in the neutrophils).
Depletion of neutrophils with anti Gr-1 Ab injection 24 hours before experiments shows a return of permeability to baseline levels (E). The amount of permeability normalized
for number of emigrated neutrophils as shown 60 minutes after KC superfusion (F). Each group contained at least 3 animals. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001. Error bars
indicate SEM.
Discussion
Recent work has demonstrated that during emigration, the endothelium plays a critical role in forming docking structures by
Figure 7. Endothelial dome formation and microvascular permeability changes in peritoneal venules of
WT and Lsp1ⴚ/ⴚ mice. Measurements were taken 2 or
4 hours after IL-1␤ injection into the peritoneum. Image
and corresponding image demonstrate endothelial dome
formation (filled arrows) and the overlap of endothelial
cell borders (open arrow) in Lsp1⫺/⫺ mice (A-B)
4 hours after IL-1␤ injection. Scale bar indicates 500 nm.
Image acquisition, panels A-B: Hitachi H-7000 transmission electron microscope; direct magnification: 4000⫻;
16000 AMT camera, AMT Capture Engine software
(V.600.128); images generated with Microsoft Office
PowerPoint 2003 (SP3). Percentage quantification of
the electron micrograph sections based on the adherent
neutrophils per vessel that underwent transendothelial
migration with domes in WT vs Lsp1⫺/⫺ animals in the
cremaster and the peritoneum (C). Twenty to 25 vessel
sections per background were analyzed. (D) WT or
Lsp1⫺/⫺ mice were injected with Evan blue and injected
with saline or IL-1␤ as indicated; 2 or 4 hours after
injection, the dye that leaked out into the peritoneum
was quantified. *P ⬍ .05; ***P ⬍ .001. Error bars indicate SEM. e indicates endothelial cell; n, neutrophil; p,
pericyte; and L, vessel lumen.
extending projections up the side of the neutrophil, presumably
facilitating the emigration across the vessel wall.7,26,27,36 Herein, we
observed that one side of the docking structure extended all the way
over the neutrophil and covered the cell in vivo. Numerous other in
vivo studies have seen these domes,8,37-39 but this has not been
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BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
PETRI et al
noted in vitro despite detailed presentation of emigrating neutrophils across endothelium7,9,36,40 This raises, however, the possibility
that in vitro, the endothelium is restricted to making only docking
structures, perhaps because of the absence of a critical factor found
in vivo (such as shear or hydrostatic pressure), or that the dome
formation fails to form because of the absence of basement
membrane and other key components. It is probable that a source of
additional membrane for the formation of docking structures and
domes would be required. Indeed, Mamdouh and colleagues
reported membrane compartments in the endothelium adjacent to
junctions that were mobilized during paracellular and transcellular
emigration processes.41-43 Perhaps there are more of these storage
compartments in vivo than in vitro, limiting the degree of dome
formation in the latter setting.
As the formation of the domes as well as the docking structures
is very dynamic, the endothelium would require significant cytoskeletal involvement. Indeed, in this study, the endothelial F-actin
binding protein, LSP1, which moves from the nucleus to the
cytosol and is associated with the cytoskeleton on stimulation, was
important for the formation of the dome structures. Although the
reason for storage of proteins in the nucleus is unclear, such storage
is not unprecedented. Indeed, numerous proteins, including MK2, a
protein upstream of LSP1, have been found in the nucleus and are
mobilized to the cytosol during activation.23 This could be a
strategy to compartmentalize actin-binding proteins away from the
target site until they are absolutely needed. Overproduction of
LSP1 in patients with neutrophil actin dysfunction (NAD47/89) or
forced overexpression in highly motile cells led to hair-like
F-actin–rich projections with severe crawling defects.44,45 Clearly,
fine regulation of LSP1 is required for proper cytoskeletal dependent effects. We report that lack of LSP1 also impairs motility in
endothelium. Although in some cases LSP1-deficient endothelium
still formed domes, this may be more active penetration by
neutrophils then active covering by Lsp1⫺/⫺ endothelium. In most
cases the Lsp1⫺/⫺ endothelium remained in the same plane along
the entire vessel, and as such did not move up the sides of adhering
neutrophils.
The less frequent dome formation in the Lsp1⫺/⫺ endothelium
was associated with very few neutrophils emigrating out of the
vasculature. Although there was a decrease in adhesion of approximately 30%, the emigration was inhibited by more than 90%,
suggesting that a simple reduction in adhesion could not explain the
reduced emigration. Moreover, when chimeric mice were made,
identical levels of adhesion were noted but emigration was only
impaired in the Lsp1⫺/⫺ endothelium despite the presence of
WT neutrophils. Although LSP1 is also found in neutrophils,
LSP1-deficient neutrophils emigrated across WT endothelium
unperturbed but WT neutrophils could not cross LSP1-deficient
endothelium as shown in this study and elsewhere.18 This clearly
suggests that impairment in the endothelium was mediating the
reduced emigration. In this study, LSP1-deficient endothelium
failed to project up the sides of the neutrophils and cover the
neutrophils with domes. In fact, we found unusually large pseudopods penetrating the endothelium in the absence of any change in
endothelial structure (see Figures 3B and 4E-H). Previous work by
Carman et al reported that inhibition of SNARE protein–containing
membrane fusion complexes were required for the permissive
structures created by endothelium to permit emigration.36 Inhibition of these structures in the study by Carman and colleagues,36
much like inhibition of LSP1 in our paper, resulted in impaired
emigration, highlighting the key contribution by the endothelium.
Our data suggest that the neutrophil is dependent on the
endothelium for emigration. Interestingly, it has been reported that
beads approximately the size of neutrophils could be enveloped by
endothelium and would be released on the other side of the barrier,
consistent with the view that the endothelium can move even inert
objects from the lumenal to the ablumenal side.46 Indeed, once the
dome is formed over the top of the neutrophil, the endothelium
beneath the neutrophil could then retract, allowing easy passage for
the neutrophil. By extension, the neutrophil may not be permitted
to move across the ablumenal endothelium unless the dome
formation is complete. Although the concept of one cell taking up
another cell by invasion has been demonstrated during epithelial
cell cancer (entosis or emperiopolesis),47 it is important to note that
at no time did we see neutrophils inside the cytosol of the
endothelium, that is, no emperiopolesis. We always saw a double
membrane with gaps around the neutrophil. One reason endothelial
domes may occur is to prevent the neutrophil from creating
significant breaches in the endothelial barrier, thereby preventing
excess fluid and protein from leaking into the interstitium. Indeed,
the net positive hydrostatic pressure inside the postcapillary venule
would force intravascular fluid, proteins, and particles to move out
of the vasculature. One could imagine that the dome might function
like an air-lock type seal (such as in a submarine), which prevents
major leakage by first closing the top portal and then opening the
bottom portal, allowing only a minimal amount of protein and fluid
leakage out of the vasculature. Although there is growing acceptance of the view that a neutrophil could migrate either at
endothelial junctions or directly through the endothelium,1,2,48 one
might imagine significant disruption of the endothelial barrier if the
neutrophil forced its way straight through the body of the endothelium. However, with the formation of a dome, subsequent transcellular or paracellular emigration could occur with limited vascular
disruption. Domes could serve to limit vascular leakage regardless
of the route of emigration. Indeed, we observed endothelial dome
formation during transcellular and paracellular diapedesis events.
Study limitations
Although it is tempting to conclude that the lack of domes in
Lsp1⫺/⫺ mice explains the disproportional increase in permeability,
it is plausible that excessive soluble factors released by Lsp1⫺/⫺
adherent and emigrating neurophils could contribute to increase the
permeability.49 However, when Lsp1⫺/⫺ neutrophils were stimulated to cross the WT endothelium, excessive permeability increases were not seen. Nevertheless, it is also plausible that
regardless of the neutrophil genotype, the Lsp1⫺/⫺ endothelium
delays or prevents emigration, causing the neutrophil to become
overly activated and thereby increasing permeability. Previous
results have shown that histamine, a mediator that increases
permeability by retracting the endothelium independent of neutrophils,
caused lesser permeability increase in LSP1-deficient animals.18 Clearly,
LSP1 may also affect retraction of the endothelium by the cytoskeleton,
further supporting a role for LSP1 regulating cytoskeletal events in
endothelium. Our data support LSP1 as a new player during regulation
of fluid and protein leakage out of the inflamed blood vessel.
Serial sections were performed in an attempt to show that the
dome covered the entire neutrophil. Although we could never
obtain serial sections of the entire neutrophil, none of the serial
sections ever revealed that that the covering was discontinuous at any
point over the neutrophil, suggesting that a true dome was formed by the
endothelium, and that this dome was always followed by the
neutrophil emigrating across the underlying endothelium, as we
were able to observe using our 2-photon microscopy.
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BLOOD, 20 JANUARY 2011 䡠 VOLUME 117, NUMBER 3
ENDOTHELIAL LSP1 PROMOTES DOME FORMATION IN VIVO
951
Acknowledgments
Authorship
We thank Carla Badick and Lori Zbytnuik for their expert
assistance in animal care, and Pina Colarusso for the management
of the imaging facility and her help in the development of the
imaging assays.
This work was supported by the Canadian Heart and Stroke
Foundation and by an equipment and infrastructure grant from the
Canadian Foundation for Innovation and the Alberta Science and
Research Authority. B.P. is supported by a postdoctoral fellowship
from the Alberta Heritage Foundation for Medical Research
(AHFMR; CA#2997) and by the Immunology Training Program of
the Canadian Institutes of Health and Research (CIHR) at the
University of Calgary. P.K. is a Canada Research Chair and an
Alberta Heritage Foundation for Medical Research Scientist and
the Calvin, Phoebe and Joan Snyder Chair in Critical Care
Medicine. H.L., S.B., and D.V. are supported by SFB 492 of the
Deutsche Forschungsgemeinschaft and by the Max-Planck-Society.
Contribution: B.P., J.K., E.M.L., H. L., S.A.P., and K.D.P.,
performed, designed, and analyzed experiments; P.K. initiated the
study, designed, and supervised the research project. All experiments were done in the laboratory of P.K. or connected core
facilities (except whole-mount staining of the cremaster, done by
H.L. and S.B. in the laboratory of D.V.); S.B., D.V., and S.M.R.
provided valuable reagents; M.P. and K.D.P. contributed to data
interpretation; and B.P. and P.K. contributed to data analysis and
interpretation and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Paul Kubes, University of Calgary, Immunology Research Group, Department of Physiology and Pharmacology, The Calvin, Phoebe, and Joan Snyder Institute for Infection,
Immunity & Inflammation, 3280 Hospital Dr NW, Calgary,
AB T2N 4N1, Canada; e-mail: [email protected].
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2011 117: 942-952
doi:10.1182/blood-2010-02-270561 originally published
online October 28, 2010
Endothelial LSP1 is involved in endothelial dome formation, minimizing
vascular permeability changes during neutrophil transmigration in vivo
Björn Petri, Jaswinder Kaur, Elizabeth M. Long, Hang Li, Sean A. Parsons, Stefan Butz, Mia
Phillipson, Dietmar Vestweber, Kamala D. Patel, Stephen M. Robbins and Paul Kubes
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