Am J Physiol Gastrointest Liver Physiol 305: G797–G806, 2013.
First published October 10, 2013; doi:10.1152/ajpgi.00058.2013.
Kupffer cells and activation of endothelial TLR4 coordinate neutrophil
adhesion within liver sinusoids during endotoxemia
Braedon McDonald,1 Craig N. Jenne,2 Lisheng Zhuo,3 Koji Kimata,3 and Paul Kubes1
1
Calvin, Phoebe, and Joan Snyder Institute for Chronic Disease, Department of Physiology and Pharmacology, University
of Calgary, Calgary, Alberta, Canada; 2Calvin, Phoebe, and Joan Snyder Institute for Chronic Disease, Department of
Microbiology, Immunology, and Infectious Disease, University of Calgary, Calgary, Alberta, Canada; and 3Institute for
Molecular Science of Medicine, Aichi Medical University, Aichi, Japan
Submitted 22 February 2013; accepted in final form 5 October 2013
inflammation; lipopolysaccharide; TLR4; leukocyte recruitment
SEVERE SEPSIS IS A SYNDROME of systemic inflammation and organ
dysfunction caused by infection (7). Despite intensive research,
there are no specific therapies to combat the inflammatory
pathology of severe sepsis, and as a result mortality from this
disease remains extremely high at ⬃20 –50% (12, 20, 28). In
gram-negative bacterial sepsis, shedding of bacterial lipopolysaccharide (LPS) into the blood can stimulate an exuberant
systemic innate immune response that results in accumulation
of neutrophils in the expansive capillary beds of organs such as
Address for reprint requests and other correspondence: P. Kubes, Director,
Snyder Institute for Chronic Disease, Dept. of Physiology and Pharmacology,
Univ. of Calgary, Alberta, Canada, 3330 Hospital Dr. NW, Calgary, AB, T2N
4N1, Canada (e-mail: [email protected]).
http://www.ajpgi.org
the liver and lungs, causing organ damage and dysfunction (1,
10, 36). Septic inflammation in the liver is of particular interest
as this organ is both a target of immune-mediated pathology as
well as a key site for the clearance of bacteria and bacterial
toxins from the blood (17, 38). In particular, neutrophils
recruited to sinusoids generate an intravascular defense system
against the spread of septic infections (31). However, this
antimicrobial response is mounted at the expense of significant
neutrophil-mediated injury and vascular dysfunction in the
liver (8, 9, 17, 31). Therefore, understanding the mechanisms
that initiate and regulate neutrophil recruitment to the liver
during sepsis/endotoxemia may reveal ways to therapeutically
manipulate the innate immune response to avoid organ damage
while optimizing antibacterial defenses.
During endotoxemia, LPS is detected by Toll-like receptor 4
(TLR4) on sentinel cells that initiate the subsequent inflammatory response, including neutrophil recruitment to the liver. It
is widely believed that stimulation of TLR4 on neutrophils
results in cellular hyperactivation, causing neutrophils to accumulate in the narrow capillaries of the liver (and lungs) (10,
14). However, TLR4 is expressed on many other cells, both
leukocytes and nonleukocytes, that may contribute to the
coordination of neutrophil recruitment in liver sinusoids. Tissue-resident leukocytes such as macrophages and mast cells are
hallmark sentinel cells and have well-described roles in the
initiation of inflammatory responses in various disease models
(37). In addition, there is increasing evidence that nonleukocytes also play key roles in detecting microbial products and
coordinating leukocyte recruitment to inflamed tissues. Notably, several recent studies have uncovered critical roles for the
vascular endothelium in initiating and regulating the systemic
inflammatory response to endotoxemia and bacterial sepsis (3,
4, 43, 44). Such evidence has led us to hypothesize that
vascular endothelial cells are a primary TLR4-expressing sentinel population for the detection of circulating LPS and, as
such, can coordinate the recruitment of neutrophils to the liver
during endotoxemia.
During the systemic inflammatory response of sepsis or
endotoxemia, neutrophils are sequestered within liver sinusoids by an adhesive interaction between neutrophil CD44 and
endothelial hyaluronan (HA) (30). Adhesion between CD44
and HA has been shown to support leukocyte-endothelial
interactions in a number of inflammatory models. The constitutive expression of CD44 on numerous leukocyte subsets as
well as HA on vascular endothelium suggests that their binding
is functionally regulated in response to inflammatory stimuli.
CD44 on lymphocytes and monocytes can be induced into an
HA-avid state by stimulation with cytokines (TNF-␣, IL-1␣,
0193-1857/13 Copyright © 2013 the American Physiological Society
G797
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McDonald B, Jenne CN, Zhuo L, Kimata K, Kubes P. Kupffer
cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia. Am J Physiol Gastrointest Liver Physiol 305: G797–G806, 2013. First published October 10, 2013; doi:10.1152/ajpgi.00058.2013.—A key pathological
feature of the systemic inflammatory response of sepsis/endotoxemia
is the accumulation of neutrophils within the microvasculature of
organs such as the liver, where they cause tissue damage and vascular
dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to
blood-borne microbes and microbial products in sepsis/endotoxemia.
In this study, we aimed to understand the role of endothelium, and
specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression
by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced
neutrophil adhesion in liver sinusoids was equivalent between wildtype mice and transgenic mice that express TLR4 only on endothelium (tlr4⫺/⫺Tie2tlr4), revealing that activation of endothelial TLR4
alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest
within sinusoids of endotoxemic mice requires adhesive interactions
between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serumderived hyaluronan-associated protein (SHAP) within sinusoids,
which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAPindependent mechanism. This study sheds new light on the control of
innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the
detection of intravascular infections and coordination of neutrophil
recruitment to the liver.
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MATERIALS AND METHODS
Animals. C57BL6/J mice (referred to as WT), TLR4 knockout mice
(referred to as tlr4⫺/⫺), and B6.SJL-Ptprca Pep3b/BoyJ mice were
purchased from Jackson Laboratories. Bikunin knockout mice (referred to as Bik⫺/⫺) were generated in the laboratory of Dr. Kimata as
described previously (46). The tlr4⫺/⫺Tie2tlr4 transgenic mice were
generated in our laboratory as previously described (4). It is important
to note that this line of mice was lost as a result of a breeding error and
has not been regenerated to date. All animals were on a C57BL/6J
background. At the time of use, mice weighed between 20 and 35 g
and were 6 to 10 wk old. All animals were anesthetized with a mixture
of 200 mg/kg ketamine (Rogar/STB) and 10 mg/kg xylazine (MTC
Pharmaceuticals) administered via an intraperitoneal injection. Experimental animal protocols carried out in this study were approved by
the University of Calgary Animal Care Committee and met the
guidelines of the Canadian Council for Animal Care.
Generation of chimeric mice. Bone marrow chimeras between
C57BL/6 and tlr4⫺/⫺ mice, and C57BL/6 and B6.SJL-Ptprca Pep3b/
BoyJ mice were generated by following a standard protocol (3). Bone
marrow was isolated from donor mice euthanized by cervical spine
dislocation. Recipients were irradiated with two doses of 5 Gy
(Gammacell 40 137Cs ␥-radiation source), with a 3-h interval between
doses; 8 ⫻ 106 isolated donor bone morrow cells were injected via the
tail vein into irradiated recipient mice. Transplanted mice received
water with 0.2% neomycin and remained in germ-free microisolator
cages for 8 wk to allow full hematological reconstitution.
Intravital microscopy in the mouse liver. The procedure for transillumination intravital microscopy of the murine liver was performed
as outlined previously (41). Following general anesthesia, the right
jugular vein was cannulated for administration of additional anesthetic
and for injection of antibodies in some experiments. Throughout all
procedures the body temperature was maintained at 37°C with infrared heat lamps.
Leukocytes that remained stationary on the venular or sinusoidal
endothelium for ⱖ30 s were considered adherent cells. Adherent cells
were counted per 100 ␮m in postsinusoidal venules, or per field of
view in sinusoids. The percentage of sinusoids perfused was determined by dividing the number of perfused sinusoids by the total
number of sinusoids in a field of view.
Spinning disk confocal microscopy. Intravital microscopy was
performed as previously described, and the anterior surface of the
liver was viewed with a spinning disk multichannel confocal microscope. Images were acquired with an Olympus BX51 (Olympus)
inverted microscope using a ⫻10 or ⫻20/0.75 NA air objective. The
microscope was equipped with a confocal light path (WaveFx, Quorum) based on a modified Yokogawa CSU-10 head (Yokogawa
Electric). Livers were imaged with 488-, 561-, and 680-nm laser
excitation (Cobalt), and visualized with the appropriate long-pass
filters (Semrock). Typical exposure times for 488-, 561-, and 680-nm
laser excitation were 120, 240, and 500 ms, respectively. A 512 ⫻ 512
pixel back-thinned EMCCD camera (C9100-13, Hamamatsu, Bridgewater, NJ) was used for fluorescence detection. Volocity Acquisition
software (Improvision, Lexington, MA) was used to drive the confocal microscope. Images were acquired as 20 ␮m z-stacks (1-␮m
slices) through sinusoids and are presented in extended focus format.
Intravital immunofluorescence labeling was performed as previously
reported (19, 30, 31). HA expression in sinusoids was visualized with
use of 25 ␮g Alexa Fluor 488-labeled (Molecular Probes) HABP
(Calbiochem). SHAP was detected by use of Alexa Fluor 555-labeled
antibodies against SHAP [anti-inter-␣-trypsin inhibitor (I␣I) heavy
chains 1 and 2 mAbs, 1.6 ␮g each; Santa Cruz Biochemicals].
Neutrophils were visualized with 10 ␮g Alexa Fluor 647-labeled
anti-Gr-1 mAb (clone RB6-8C5, BD Pharmingen). Kupffer cell chimerism after bone marrow transplant was evaluated by visualization
of Kupffer cells (Alexa Fluor 660-labeled F4/80 mAb) and leukocyte
markers to differentiate donor from recipient (PE-labeled anti-CD45.1
and FITC-labeled anti-CD45.2, respectively). All antibodies and labeled proteins were injected intravenously through a jugular vein
cannula 30 min prior to imaging.
Experimental protocols. For each experiment, mice received 1
mg/kg ultrapure LPS from E. coli O111:B4 (List Biological Laboratory) administered intravenously (iv) 4 h prior to intravital microscopy. This concentration is sufficiently low that all animals survived
general anesthesia, and perfusion in the liver vasculature remained
such that leukocyte-endothelial interactions occurred under flow conditions. In some experiments, mice were injected with 20 ␮g of
purified anti-mouse CD44 mAb (clone KM81, Cedarlane) administered iv 30 min before LPS injection. Intravascular HA was depleted
by intraperitoneal administration of 20 U/g hyaluronidase type-IV
(Sigma-Aldrich), a procedure known to deplete HA from the vascular
endothelium (6, 23). The liver was then prepared for intravital microscopy and leukocyte kinetics were investigated as described above.
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IL-1␤, IL-2), chemokines (IL-8, MIP-1␤, RANTES), as well
as bacterial products such as LPS (reviewed in Ref. 35). CD44
binding activity can be modulated by numerous molecular
modifications including glycosylation, phosphorylation, sulfation, and expression of splice variant forms of CD44 (21, 27,
34). It was originally hypothesized that sequestration of neutrophils within the liver microvasculature in response to LPS
was the result of neutrophil activation causing enhanced CD44
adhesiveness for endothelial HA. Surprisingly, stimulation of
neutrophils with many different mediators (including LPS,
cytokines such as TNF-␣, or chemokines such as MIP-2) was
insufficient to induce neutrophil CD44 to bind to HA (30).
Such evidence suggests that neutrophil recruitment within liver
sinusoids may not be regulated at the level of the neutrophil but
may instead be regulated by functional activation of vascular
endothelium and its surface HA. Recent studies have demonstrated that HA can be functionally activated into a CD44-avid
state through the binding of a serum-derived hyaluronanassociated protein (SHAP, composed of the heavy chains H1
and H2 of inter-␣-trypsin inhibitor) (45). Deposition of SHAP
on the luminal surface of liver sinusoids during endotoxemia
has been reported, but its functional role in neutrophil adhesion
within these vessels has not been assessed (29, 30).
In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the
regulation of neutrophil recruitment to the liver microvasculature during endotoxemia. First, using bone marrow chimeras as
well as transgenic mice that express TLR4 only on selected cell
populations, we systematically identified vascular endothelial
cells as a key TLR4-expressing sentinel population regulating
neutrophil recruitment within the liver microvasculature in
response to endotoxemia. Activation of endothelial TLR4
alone was found to be sufficient to initiate CD44-HA dependent neutrophil adhesion in liver sinusoids. Furthermore, we
examined the contribution of SHAP to the regulation of
CD44-HA interactions, revealing that activation of endothelial
TLR4 leads to neutrophil adhesion through the induction of
SHAP-dependent adhesion of HA to neutrophil CD44. Finally,
we investigated the role of another key sentinel population,
Kupffer cells, and demonstrated that endothelium and Kupffer
cells provide unique and additive contributions to neutrophil
recruitment within liver sinusoids. Overall, this study provides
important new insight into the molecular mechanisms that
govern neutrophil recruitment to the liver during endotoxemia
and sepsis.
ENDOTHELIAL TLR4 AND NEUTROPHIL RECRUITMENT TO LIVER
G799
Cytokine measurement. Mice were anesthetized and blood was
collected by cardiac puncture. Blood was allowed to clot at room
temperature for 30 min after which samples were centrifuge at 5,000
rpm for 10 min for the retrieval of serum. The serum samples were
analyzed for inflammation-relevant cytokines and chemokines by
using a Luminex 200 apparatus (Applied Cytometry Systems) and
25-plex MILLIPLEX MAP mouse cytokine/chemokine magnetic
panel kit from Millipore according to manufacturer’s instructions. The
data was analyzed with the StarStation V.2.3 software from Applied
Cytometry Systems.
Statistical analysis. All data are presented as mean values ⫾ SE. A
Student’s t-test was used to determine the significance between
population means when two groups were compared. When more than
two groups were compared, a one-way analysis of variance with post
hoc Bonferroni test was used for multiple comparisons. Statistical
significance was set at P ⬍ 0.05.
LPS stimulation of TLR4 on non-bone marrow-derived cells
initiates neutrophil adhesion in the liver microvasculature. We
first aimed to define the role of TLR4 on bone marrow-derived
vs. non-bone marrow-derived cell populations in the induction
of neutrophil recruitment to the liver in response to iv LPS.
Bone marrow transplantation between Tlr4⫺/⫺ and wild-type
(WT, Tlr4⫹/⫹) mice generated chimeric animals that expressed
TLR4 only on bone marrow-derived cells (WT¡Tlr4⫺/⫺) or only
on non-bone marrow-derived cells (Tlr4⫺/⫺¡WT). Syngeneic
transplants between WT¡WT mice and Tlr4⫺/⫺¡Tlr4⫺/⫺ mice
served as controls. In untreated WT¡WT control mice, 3 ⫾ 0.3
neutrophils adhered per field of view in liver sinusoids, and 2 ⫾
0.2 adherent neutrophils were observed per 100 ␮m in postsinusoidal venules (Fig. 1, A and B). Four hours after iv administration of LPS, neutrophil adhesion increased significantly to 12 ⫾
0.5 cells/field in sinusoids and 10 ⫾ 0.5 cells/100 ␮m in venules
(Fig. 1, A and B). As anticipated, Tlr4⫺/⫺¡Tlr4⫺/⫺ control mice
showed no response to LPS (Fig. 1, A and B).
Within the sinusoids of Tlr4⫺/⫺ mice that received WT bone
marrow (WT¡Tlr4⫺/⫺), LPS administration failed to induce
neutrophil adhesion to sinusoidal endothelium, yielding an
equivalent response to that observed in the negative control
Tlr4⫺/⫺¡Tlr4⫺/⫺ group (Fig. 1A). Conversely, WT mice that
received Tlr4⫺/⫺ marrow (Tlr4⫺/⫺¡WT) responded to LPS
identically to the WT¡WT positive controls (Fig. 1, A and B).
These results demonstrate that LPS-induced neutrophil recruitment to the liver is dependent on TLR4 signaling in non-bone
marrow-derived cells, with minimal input from TLR4 activation on leukocytes.
Notably, there was a small increase in neutrophil adhesion
within the postsinusoidal venules of Tlr4⫺/⫺ mice that received
WT marrow (WT¡Tlr4⫺/⫺), although this small response was
significantly less than that observed in LPS-treated WT¡WT
control group (Fig. 1B). This small increase is likely accounted
for by the integrin-dependent mechanism of adhesion that
neutrophils utilize to bind to endothelium in venules (15, 30).
As such, LPS stimulation of TLR4-expressing neutrophils
likely generated sufficient upregulation of integrin activity to
allow for some increased adhesiveness toward ligands on the
venular endothelium.
Although irradiation and bone marrow transplantation result
in ⬎95% reconstitution of circulating leukocytes by donor
cells, liver-resident Kupffer cells have previously been found
to be somewhat radioresistant, resulting in substantial chime-
Fig. 1. LPS-induced neutrophil adhesion within liver sinusoids requires TLR4
on non-bone marrow-derived cells. The number of adherent leukocytes in
sinusoids per field of view (A) and the number of adherent leukocyte per 100
␮m in postsinusoidal venules (B) were measured via intravital microscopy in
untreated (UT, open bars) and LPS-treated (solid bars) bone marrow chimeric
mice, generated in wild-type (WT) or Tlr4⫺/⫺ recipients transplanted with
either WT or Tlr4⫺/⫺ donor marrow. C: serum TNF-␣ concentrations at 90 min
after LPS administration in the indicated groups of mice (or untreated). Data
are presented as means ⫾ SE of 3–5 animals per group. *P ⬍ 0.05 and ***P
⬍ 0.001 relative to respective untreated group; #P ⬍ 0.05, ##P ⬍ 0.01 relative
to LPS-treated WT¡WT control group; N.S., not significant.
rism following bone marrow transplant (24). The origin of
Kupffer cells (donor vs. recipient) was investigated by transplantation of C57Bl/6 bone marrow (expressing the leukocyte
antigen CD45.2) into irradiated congenic mice expressing
CD45.1 (B6.SJL-Ptprca Pep3b/BoyJ mice), enabling quantification of donor (CD45.2⫹F4/80⫹) vs. recipient (CD45.1⫹F4/
80⫹) Kupffer cells within the liver. Indeed, we found that, 8 wk
after irradiation and transplantation, 69.2% (⫾7.1, SE) of
Kupffer cells were of donor origin, whereas 30.8% (⫾7.1, SE)
remained of recipient origin. Kupffer cells are key producers of
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RESULTS
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ENDOTHELIAL TLR4 AND NEUTROPHIL RECRUITMENT TO LIVER
Fig. 2. Endothelial TLR4 stimulation activates neutrophil adhesion in liver
sinusoids through CD44-HA interactions. WT mice or Tlr4⫺/⫺Tie2Tlr4 mice
were left untreated (open bars) or treated with intravenous LPS (solid bars).
Groups of WT and Tlr4⫺/⫺Tie2Tlr4 mice were pretreated with anti-CD44 mAb
(shaded bars) prior to LPS administration. Intravital microscopy was used to
quantify neutrophil adhesion within liver sinusoids (A) and postsinusoidal
venules (B). Data are presented as means ⫾ SE of at least 5 animals per group.
*P ⬍ 0.05 relative to untreated controls (open bars), #P ⬍ 0.05 relative to
LPS-treated positive control animals (solid bars).
Neutrophil adhesion in liver sinusoids after endothelial
TLR4 stimulation is dependent on SHAP binding to endothelial
HA. In 2006, Kimata and colleagues (45) reported that SHAP,
composed of the heavy chains of inter-␣-inhibitor, binds covalently to HA and greatly enhances its avidity for leukocyte
CD44. We therefore tested the hypothesis that endothelial HA
may be modified during endotoxemia by the formation of
SHAP-HA complexes and that these SHAP-HA complexes
were necessary to allow for adhesive interactions between
CD44 and HA in the liver sinusoids. Spinning disk confocal
intravital microscopy (SD-IVM) was used to visualize the
localization of HA [using Alexa Fluor 488-labeled hyaluronan
binding protein (HABP)], SHAP (using Alexa 555-labeled
anti-I␣I H1 and H2 Abs), and neutrophils (using Alexa Fluor
647-labeled anti-GR-1 mAb) within the liver sinusoids in vivo.
In the absence of LPS, HA was observed lining the sinusoidal
endothelium, but very little SHAP and very few neutrophils
were observed within these vessels (Fig. 3A). In stark contrast,
4 h after LPS injection both adherent neutrophils and SHAP
were observed in abundance within liver sinusoids, most often
colocalized with areas of HA expression (Fig. 3B). Similarly,
Tlr4⫺/⫺Tie2Tlr4 mice demonstrated identical HA expression,
SHAP localization, and neutrophil adhesion in sinusoids following LPS administration (Fig. 3C). To investigate whether
SHAP was in fact binding to endothelial HA, a group of
animals was depleted of intravascular HA by treatment with
HNase, and 4 h after LPS injection the absence of endothelial
HA correlated with an absence of SHAP (and fewer adherent
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TNF-␣ in response to LPS, and TNF-␣ can serve as a strong
stimulus for neutrophil recruitment in liver sinusoids (15).
Therefore, given the substantial chimerism in the Kupffer cell
population, we investigated serum levels of TNF-␣ following
LPS administration in the above WT↔Tlr4⫺/⫺ bone marrowtransplanted mice. No significant differences in levels of serum
TNF-␣ were found between WT¡Tlr4⫺/⫺ and Tlr4⫺/⫺¡WT
groups (Fig. 1C), confirming that the differences in neutrophil
recruitment observed between these groups of mice are not the
result of differing levels of TNF-␣ production in the setting of
incomplete repopulation of Kupffer cells.
Stimulation of TLR4 on endothelium alone is sufficient to
initiate neutrophil adhesion in liver sinusoids. Given that
neutrophil recruitment to the liver during endotoxemia was
dependent on TLR4 from non-bone marrow-derived cells, we
hypothesized that the primary LPS-responsive sentinel within
this population was the vascular endothelium. To test this
hypothesis, our laboratory generated a transgenic mouse that
expresses TLR4 exclusively on endothelial cells (4). Briefly, a
linearized transgene containing the Tlr4 coding sequence under
the control of the Tie2 promoter and enhancer was incorporated
into the genome of Tlr4⫺/⫺ mice, yielding Tlr4⫺/⫺Tie2Tlr4
animals. In adult Tlr4⫺/⫺Tie2Tlr4 mice, the only cells that
express TLR4 are those that also express Tie2 [exclusively
endothelial cells; see previous report (4) for full characterization of TLR4 expression]. Therefore, using these animals that
express TLR4 exclusively on endothelial cells, we tested the
hypothesis that TLR4 signaling within endothelium alone is
sufficient to initiate neutrophil recruitment to the liver during
endotoxemia.
In the absence of LPS, Tlr4⫺/⫺Tie2Tlr4 mice and WT mice
had equivalent hematocrits, total white blood cell and differential counts (data not shown), and very few neutrophils within
the liver sinusoids (Fig. 2A). Four hours after LPS administration, neutrophil adhesion in liver sinusoids and postsinusoidal
venules of Tlr4⫺/⫺Tie2Tlr4 mice increased significantly to levels that were not different from WT Tlr4⫹/⫹ mice (Fig. 2, A
and B). Importantly, Tlr4⫺/⫺Tie2Tlr4 mice fail to produce
significant amounts of serum TNF-␣ (or other proinflammatory
cytokines such as IL-1␤) in response to endotoxemia (4, 44),
indicating that neutrophil recruitment in the liver of these mice
is not the result of cytokine production, but rather a direct
response to LPS/TLR4 signaling in endothelial cells. These
findings reveal that neutrophil adhesion in the liver microvasculature can be initiated entirely by TLR4 signaling within
vascular endothelial cells.
LPS activation of endothelial TLR4 initiates CD44-dependent adhesion in liver sinusoids. We have previously reported
that neutrophil adhesion in murine liver sinusoids in response
to LPS is largely dependent on adhesive interactions between
neutrophil CD44 and endothelial HA, whereas recruitment
within postsinusoidal venules requires classical integrin-mediated adhesion (30). To determine whether stimulation of endothelial TLR4 initiated neutrophil adhesion in liver sinusoids
via the CD44/HA axis, WT and Tlr4⫺/⫺Tie2Tlr4 mice were
treated with a blocking antibody against CD44 prior to LPS
administration. Indeed, neutralization of CD44 reduced adhesion within sinusoids of Tlr4⫺/⫺Tie2Tlr4 mice to the same
extent as WT animals but had no effect on adhesion within
postsinusoidal venules (Fig. 2, A and B).
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Fig. 3. Endothelial TLR4 stimulation initiates serum-derived hyaluronan-associated protein (SHAP) deposition on endothelial hyaluronan. Spinning disk confocal
intravital microscopy was used to visualize hyaluronan expression (green, AF488-HABP) and detect the presence of SHAP [red, AF555 anti-inter-␣-trypsin
inhibitor (I␣I) H1 and H2 Abs] within liver sinusoids of untreated WT mice (A), LPS-treated WT mice (B), LPS-treated Tlr4⫺/⫺Tie2Tlr4 mice (C), and mice
pretreated with HNase [to depleted intravascular hyaluronan (HA)] prior to LPS (D). Neutrophils are displayed in blue (AF647 anti-Gr1 Ab). Representative of
3 experiments. Scale bars denote 50 ␮m.
neutrophils) within the liver sinusoids of these animals (Fig.
3D). This evidence suggests that LPS stimulation of endothelial TLR4 results in profound SHAP deposition on endothelial
HA within the liver sinusoids.
Finally, the role of SHAP deposition on endothelial HA was
probed for its contribution to neutrophil adhesion in the liver
sinusoids. Inter-␣-inhibitor, the circulating SHAP precursor, is
composed of a central protein called bikunin attached to heavy
chain proteins (H1 and H2), which are covalently transferred to
HA as “SHAP” at sites of inflammation (45). Therefore,
Bik⫺/⫺ mice are deficient of I␣I, and are thereby deficient of
SHAP generation in response to LPS (Fig. 4A) and other
inflammatory stimuli (46). Quantitative assessment of neutrophil adhesion in liver sinusoids using intravital microscopy
revealed that in SHAP-deficient Bik⫺/⫺ mice, neutrophil adhesion was significantly reduced by over 50% (Fig. 4B). The
reduction in sinusoidal neutrophil adhesion in Bik⫺/⫺ mice was
equivalent to that seen in anti-CD44 aAb treated WT animals
(Fig. 2A above). Furthermore, antibody blockade of CD44 or
depletion of HA in Bik⫺/⫺ mice yielded no further decrease in
the levels of neutrophil adhesion in liver sinusoids, indicating
that SHAP, HA, and CD44 function as a single interdependent
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ENDOTHELIAL TLR4 AND NEUTROPHIL RECRUITMENT TO LIVER
and overlapping mechanism rather than separate additive contributors (Fig. 4B).
Resident macrophages in the liver augment neutrophil adhesion in a SHAP/HA/CD44-independent manner. The results
presented above demonstrate that stimulation of endothelial
TLR4 is sufficient to initiate neutrophil adhesion within liver
sinusoids during endotoxemia. However, the liver contains a
large population of intravascular macrophages (Kupffer cells)
that are intimately involved in many inflammatory and immunological responses in the liver (11) and may also affect
neutrophil recruitment during endotoxemia. In fact, an important role for Kupffer cells in the development of sepsis-induced
liver inflammation and pathology has previously been reported
(13, 40). To determine whether Kupffer cells contribute to
LPS-induced neutrophil recruitment in liver sinusoids, a group
of mice was depleted of Kupffer cells by iv administration of
clodronate liposomes (CLL) 48 h prior to LPS injection. This
treatment has been shown by us and others to deplete ⬎99% of
Kupffer cells from the liver (16, 26). SD-IVM of the liver with
staining for Kupffer cells with the pan-macrophage marker
F4/80 confirmed the depletion of Kupffer cells by CLL in our
experiments (Fig. 5A). Importantly, injection of CLL alone did
not induce any neutrophil recruitment in sinusoids or venules
(not shown). Interestingly, neutrophil adhesion in sinusoids was
reduced by ⬃40% in endotoxemic CLL-treated mice compared
with positive control animals that received LPS alone (Fig. 5, A
and B).
Surprisingly, SHAP deposition in liver sinusoids was unchanged in the absence of Kupffer cells, yet neutrophil adhesion
was decreased (Fig. 5A). This suggested the possibility that
Kupffer cells may contribute to neutrophil adhesion in sinusoids
independently of the CD44-HA/SHAP axis. To investigate this
possibility, mice lacking Kupffer cells (CLL treated) were treated
with anti-CD44 mAb prior to LPS administration, which resulted
in further reduction of neutrophil adhesion in sinusoids beyond
CLL-treatment alone (Fig. 5B). The additive effects of Kupffer
cell depletion and CD44 blockade suggest that these pathways are
independent contributors to neutrophil adhesion. Given that
Kupffer cells are an important source of serum cytokines during
endotoxemia, we investigated the early cytokine response in
Kupffer cell-depleted mice vs. controls following LPS administration. Interestingly, few differences were seen for notable cytokines and chemokines that have been implicated in promoting
neutrophil recruitment within the liver (TNF-␣, KC, MIP-2),
whereas reduced levels were seen for cytokines such as IL-1␤ and
IL-12 (Table 1).
DISCUSSION
In this study, we have identified resident non-bone marrowderived cells as the sentinels responsible for LPS detection
(and the induction of neutrophil recruitment) in the liver during
endotoxemia. Using transgenic animals that expressed TLR4
only on endothelium, we further specified that endothelial cells
are the primary sentinels responsible for LPS detection and
initiation of neutrophil adhesion in sinusoids. Stimulation of
endothelial TLR4 was found to initiate neutrophil adhesion
through a mechanism involving functional activation of HA on
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Fig. 4. Impaired neutrophil adhesion in the liver sinusoids of
SHAP-deficient mice. A: representative spinning-disk intravital microscopy images of liver sinusoids in LPS-treated WT
(left) and SHAP-deficient bikunin knockout mice (Bik⫺/⫺,
right). SHAP is shown in red (AF555 anti-I␣I H1 and H2
Abs), and neutrophils are shown in green (AF488 anti-Gr-1
Ab). Scale bar denotes 50 ␮m. B: intravital microscopy was
used to quantify the number of adherent neutrophils in liver
sinusoids of WT and Bik⫺/⫺) mice left untreated (open bar) or
treated with LPS (solid bars). In some experiments, bikunin
knockout mice were pretreated with anti-CD44 mAb, or
HNase prior to LPS administration. Data are presented as the
arithmetic means ⫾ SE of at least 5 animals per group. *P ⬍
0.05 relative to untreated controls (open bars), #P ⬍ 0.05
relative to LPS-treated WT (positive control) animals.
ENDOTHELIAL TLR4 AND NEUTROPHIL RECRUITMENT TO LIVER
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the luminal surface of the sinusoidal endothelium through
binding of SHAP, enabling firm adhesion to neutrophil CD44.
Sequestration of neutrophils within the microcirculation of
highly vascular organs such as the liver and lungs is a hallmark
of the systemic inflammatory response of endotoxemia and
sepsis (8). For many years, the prevailing dogma has been that
blood-borne bacterial products (LPS) cause hyperactivation of
circulating neutrophils, resulting in their accumulation in narrow capillaries of the liver and lungs (5, 14, 18, 22, 32, 42).
Our data contradict this view, instead revealing that TLR4
signaling in neutrophils (and other marrow-derived cells) is not
required for their recruitment to the liver. Interestingly, similar
observations have been reported for neutrophil sequestration in
the pulmonary microvasculature during endotoxemia/sepsis,
wherein TLR4 on bone marrow-derived cells (leukocytes) was
shown to be dispensable for recruitment to the lungs (3).
Therefore, contrary to the notion that neutrophil sequestration
in the liver (and lungs) is the result of cellular hyperactivation
by LPS, our data suggest that recruitment is coordinated by
non-leukocyte-tissue-resident sentinels.
There is emerging evidence that the vascular endothelium is
critical to the orchestration of inflammatory responses, especially those involving blood-borne microbes or microbial products. Although tissue-resident leukocytes including macrophages, mast cells, and others have long been considered the
primary sentinels for the detection of pathogens and orchestration of acute inflammation, vascular endothelial cells express many pattern recognition receptors (including TLRs,
NLRs, RLRs, and CLRs) and are capable of expressing cytokines, chemokines, and adhesion molecules (33). Furthermore,
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Fig. 5. Kupffer cells contribute to LPS-induced neutrophil adhesion in sinusoids via a CD44-independent pathway. A: spinning disk confocal intravital
microscopy of the liver was used to visualize Kupffer cells (blue, AF660 anti-F4/80 Ab), neutrophil adhesion (green, AF488 anti-Gr-1 Ab), and SHAP deposition
(red, AF555 anti-I␣I H1 and H2 Abs) in sinusoids of LPS-treated control mice and LPS-treated mice depleted of Kupffer cells (CLL ⫹ LPS). Scale bar denotes
50 ␮m. B: the number of adherent neutrophils in sinusoids was assessed by intravital microscopy in untreated control mice, LPS-treated control mice, LPS-treated
mice depleted of Kupffer cells, as well as Kupffer cell-depleted mice that were administered anti-CD44 mAb prior to LPS. Data are presented as means ⫾ SE
of 3–5 animals per group. *P ⬍ 0.05 and ***P ⬍ 0.001 relative to untreated control group.
G804
ENDOTHELIAL TLR4 AND NEUTROPHIL RECRUITMENT TO LIVER
Table 1. Serum cytokine levels following Kupffer cell depletion
LPS Control
CLL ⫹ LPS
0.78 ⫾ 0.34
3.52 ⫾ 1.78
254.2 ⫾ 78.9
8.9 ⫾ 8.9
4.49 ⫾ 1.74
20.8 ⫾ 5.7
28.0 ⫾ 10.5
27.6 ⫾ 26.1
304.2 ⫾ 59.3
60.0 ⫾ 31.0
6.38 ⫾ 3.8
82.8 ⫾ 13.1
38.1 ⫾ 9.8
19.6 ⫾ 6.4
58.8 ⫾ 36.1
1,023 ⫾ 48.9
32,418 ⫾ 721.3
33,014 ⫾ 1,603
23,245 ⫾ 199
228.7 ⫾ 32.53
299.3 ⫾ 40.9
25,026 ⫾ 1,449
2,422 ⫾ 464
61,734 ⫾ 413.9
125.2 ⫾ 23.9
20.0 ⫾ 2.5
17856 ⫾ 1095
686.4 ⫾ 112.5
207.9 ⫾ 12.9
18,553 ⫾ 3,975
977.8 ⫾ 222.7
30,083 ⫾ 650.5*
30,985 ⫾ 1,808
23,140 ⫾ 238
76.6 ⫾ 14.6†
171.6 ⫾ 21.7*
24,834 ⫾ 2478
821.2 ⫾ 185.2†
60,171 ⫾ 881
199.5 ⫾ 50.9
101.0 ⫾ 27.9*
16,781 ⫾ 1,082
686.1 ⫾ 108.2
148.3 ⫾ 13.2†
15,664 ⫾ 3,088
Concentrations (pg/ml) of various cytokines and chemokines in serum 90
min following administration of LPS (1 mg/kg) in the indicated groups of
mice. Data are presented as means ⫾ SE. UT, untreated; CLL, clodronate
liposomes. *P ⬍ 0.05 (LPS control vs. CLL⫹LPS). †P ⬍ 0.01 (LPS control
vs. CLL⫹LPS).
endothelial cells are the first cell type to contact circulating
bacteria or bacterial products, and they substantially outnumber leukocytes in the liver and lungs (2). In sepsis and endotoxemia, the central role of endothelial cells has recently been
highlighted in a number of studies. Ye et al. (43) demonstrated
using models of endotoxemia and polymicrobial sepsis that
endothelium-specific inhibition of NF-␬B reduced neutrophil
accumulation in multiple organs (including the liver), reduced
endothelial permeability, and attenuated hypotension and multiple organ dysfunction. In addition, the generation of Tlr4⫺/
Tlr4
⫺Tie2
mice that express TLR4 exclusively on endothelial
cells has allowed our laboratory to perform focused investigations of the role of endothelial TLR4 in a number of organs
during endotoxemia and bacterial sepsis. In response to systemic LPS administration, endothelial TLR4 is entirely sufficient to induce neutrophil recruitment to the pulmonary microvasculature (4), central nervous system (44), and liver (present
study). More recently, the importance of endothelium as a sentinel
system was expanded to antiviral responses, in which it was found
that cytokine production and leukocyte recruitment to the lungs
following influenza virus infection was coordinated entirely at the
level of the vascular endothelium (39).
We have previously reported using the same model of
endotoxemia that the majority of neutrophil adhesion in liver
sinusoids is mediated by interactions between neutrophil CD44
and endothelial HA (30). Importantly, it was found that binding
between neutrophil CD44 and endothelial HA was not induced
by neutrophil activation, since stimulation with LPS, various
cytokines, or chemokines did not increase neutrophil CD44
binding to HA under either static or flow conditions. Instead, it
was hypothesized that functional activation of endothelial HA
in response to LPS may regulate neutrophil adhesion in liver
sinusoids. A number of previous reports have provided in vitro
evidence that covalent binding of SHAP to HA in response to
inflammatory stimuli augments the adhesiveness of HA for
CD44 (11a, 45). Using an in vitro flow-chamber assay, Zhuo
and colleagues (45) demonstrated that CD44-expressing Tcells failed to bind to native HA under flow conditions but
would adhere in abundance to HA following SHAP binding.
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TNF-␣
IL-6
KC
MIP-2
IL-1␤
IL-12
MCP-1
IL-10
G-CSF
GM-CSF
IFN-␥
IP-10
RANTES
IL-1␣
MIP-1␤
UT Control
Our study represents the first in vivo evidence that functional
modification of HA by SHAP is important for leukocyte
adhesion during inflammation. Although the precise mechanism underlying activation of HA by SHAP has not been fully
elucidated, there is evidence that covalent binding between HA
and SHAP may yield structural changes in HA polymers that
generate increased avidity for CD44 on leukocytes (11a).
Evidence presented in this study and others demonstrates
that endothelial cells are important sentinels in the detection of
circulating microbial products and coordination of neutrophil
recruitment, yet other important sentinel cell populations exist
that also contribute to the recruitment of neutrophils. Within
the liver, Kupffer cells are optimally positioned within the
vasculature to encounter circulating bacteria and bacterial
products and are critical for their clearance from the bloodstream during steady state and during sepsis (11, 13, 26).
Furthermore, Kupffer cells have well-defined roles in the
coordination of hepatic inflammation in a number of diseases
(11, 26, 40). Importantly, although our bone marrow transplant
experiments suggest that TLR4 signaling in Kupffer cells was
dispensable, depletion of Kupffer cells with CLL resulted in an
⬃40% reduction in neutrophil adhesion in sinusoids of endotoxemic mice. These data indicate that Kupffer cells have an
important role in neutrophil recruitment, albeit downstream of
TLR4 signaling in other sentinels (including endothelium),
possibly through the production of cytokines or chemokines
that influence neutrophil trafficking. Surprisingly, data presented in Table 1 demonstrate that many cytokines and chemokines known to affect neutrophil recruitment were not different
between controls and Kupffer cell-depleted mice during the
early response to LPS. However, it is possible that Kupffer cell
TLR4 and cytokine/chemokine response may contribute to the
later stages of LPS-induced hepatic inflammation.
Interestingly, the reduction of neutrophil adhesion by ⬃40%
following Kupffer cell depletion may in fact be seen as paradoxical, since it could have been predicted to enhanced neutrophil adhesion. Kupffer cells occupy a great deal of endothelial surface area within the sinusoids, and therefore the removal
of these cells may have been predicted to expose more binding
sites for neutrophils to adhere to endothelium. However, removal of Kupffer cells did not result in enhanced neutrophilendothelial interactions and instead reduced adhesion significantly through an unknown mechanism. The additive inhibition provided by CD44 blockade in Kupffer cell-depleted mice
suggests that CD44-dependent adhesion and Kupffer celldependent adhesion are unique and independent pathways. The
precise mechanism by which Kupffer cells augment neutrophil
adhesion in sinusoids is unknown, but it is possible that some
neutrophils adhere directly on Kupffer cells using adhesion
molecules other than CD44, or that other signaling pathways in
Kupffer cells (including HMGB-1 or PPAR␥ as has been
proposed by others) lead to other unique mechanisms of
adhesion. Further studies are needed to fully characterize the
molecular mechanism that enables Kupffer cells to contribute
to neutrophil recruitment in endotoxemic liver.
Overall, this study sheds new light on the control of innate
immune activation within the vasculature of the liver during
endotoxemia and sepsis. Neutrophil recruitment to the liver
represents an important contributor to the intravascular immune response mounted during sepsis that protects the host
against dissemination of blood-borne bacterial infections. Our
ENDOTHELIAL TLR4 AND NEUTROPHIL RECRUITMENT TO LIVER
present study has furthered our understanding of the coordination of this important intravascular immune response, revealing
a key role for vascular endothelium as sentinels in the detection
of intravascular infections, and revealing novel roles for SHAP
and HA activation in the molecular mechanism supporting
neutrophil adhesion within the inflamed liver sinusoids.
ACKNOWLEDGMENTS
The authors thank Lori Zbytnuik, Mandy Tse, and the late Carla Badick for
excellent technical assistance.
GRANTS
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
B.M., L.Z., K.K., and P.K. conception and design of research; B.M. and
C.N.J. performed experiments; B.M., C.N.J., and P.K. analyzed data; B.M.,
C.N.J., and P.K. interpreted results of experiments; B.M. and P.K. prepared
figures; B.M. and P.K. drafted manuscript; B.M., C.N.J., and P.K. edited and
revised manuscript; B.M., C.N.J., L.Z., K.K., and P.K. approved final version
of manuscript.
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This work was supported by grants from the Canadian Institutes for Health
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