Syndecan-4 Regulates Early Neutrophil Migration
and Pulmonary Inflammation in Response
Yoshinori Tanino1,2, Mary Y. Chang3, Xintao Wang1, Sean E. Gill4,5, Shawn Skerrett5,
John K. McGuire5,4, Suguru Sato1, Takefumi Nikaido1, Tetsuhito Kojima6, Mitsuru Munakata1,
Steve Mongovin7, William C. Parks4,5, Thomas R. Martin5,7, Thomas N. Wight8, and
Charles W. Frevert3,4,5
1Department of Pulmonary Medicine, Fukushima Medical University School of Medicine, Fukushima, Japan;2Department of Pediatrics, University of
Washington School of Medicine, Seattle, Washington;3The Comparative Pathology Program in the Department of Comparative Medicine,
University of Washington School of Medicine, Seattle, Washington;4The Center of Lung Biology, University of Washington School of Medicine,
Seattle, Washington;5Department of Medicine, University of Washington School of Medicine, Seattle, Washington;6Nagoya University School of
Health Sciences, Department of Medical Technology, Nagoya, Japan;7VA Puget Sound Medical Center, Seattle, Washington; and8The Hope Heart
Matrix Biology Program at the Benaroya Research Institute at Virginia Mason, Seattle, Washington
Proteoglycans (PGs) and their associated glycosaminoglycan side
chains are effectors of inflammation, but little is known about
changes to the composition of PGs in response to lung infection or
injury. The goals of this study were to identify changes to heparan
sulfate PGs in a mouse model of gram-negative pneumonia, to
identify the Toll-like receptor adaptor molecules responsible for
these changes, and to determine the role of the heparan sulfate PG
in the innate immune response in the lungs. We treated mice with
intratracheal LPS, a component of the cell wall of gram-negative
bacteria, to model gram-negative pneumonia. Mice treated with
intratracheal LPS had a rapid and selective increase in syndecan-4
mRNAthat wasregulatedthrough MyD88-dependentmechanisms,
whereas expression of several other PGs was not affected. To
determine the role of syndecan-4 in the inflammatory response,
we exposed mice deficient in syndecan-4 to LPS and found a signif-
icant increase in neutrophil numbers and amounts of CXC-
chemokines and total protein in bronchoalveolar lavage fluid. In
studies performed in vitro, macrophages and epithelial cells treated
using BEAS-2B cells showed that pretreatment with heparin and
syndecan-4 decreased the expression of CXCL8 mRNA in response
to LPS and TNF-a. These findings indicate that the early inflamma-
tory response to LPS involves marked up-regulation of syndecan-
4, which functions to limit the extent of pulmonary inflammation
and lung injury.
Keywords: innate immunity; macrophages; neutrophils; proteoglycan;
Lung infections place a much higher burden on public health
than better recognized diseases such as HIV/AIDS, cancer,
heart attacks, and stroke (1). The innate immune system is the
body’s first line of defense against lung infection (2, 3). LPS, a
component of the cell wall of gram-negative bacteria, is used to
study lung infection and injury caused by gram-negative bacteria
because it results in a potent and reproducible activation of the
innate immune system (4, 5). Recognition of LPS by the innate
immune system occurs via the Toll-like receptor (TLR)4/MD-2/
CD14 receptor complex (2, 6). Upon binding LPS, TLR4 signals
through two adaptor molecules, MyD88 and TRIF, which are
required for the pulmonary inflammatory response and clearance
of gram-negative bacteria from lungs (7, 8). An essential step in
the clearance of most gram-negative bacteria from lungs is the
pulmonary recruitment of neutrophils, whose influx is mediated
by CXC chemokines, such as CXCL8 in humans and macrophage
inflammatory peptide (MIP)-2 and KC in rodents (9–11). Al-
though neutrophils are an important component of pulmonary
host defenses, in excess numbers they contribute to tissue dam-
age, leading to lung injury (12–14).
Once thought to be the molecular glue that provides struc-
tural support and imparts biomechanical properties to lung tis-
sue, it is now recognized that proteoglycans (PGs) are important
biological modifiers that regulate a number of processes such as
tissue inflammation, lung development, homeostasis, and wound
healing (15–17). Growing evidence supports important roles
for PGs in the innate immune response to lung infection (17).
Heparan sulfate (HS) is the most abundant glycosaminoglycan
(GAG) in healthy lungs, and HS proteoglycans (HSPGs) play
a key role in modulating tissue inflammation (16, 18). The bind-
ing of the CXC chemokines CXCL8, KC, and MIP-2 to GAGs
such as HS in lungs provides fine-tune control of chemokine
gradient formation and neutrophil migration (19, 20). The bind-
ing of chemokines and cytokines to GAGs protects them from
proteolysis, thereby increasing their stability, which is thought
to promote the inflammatory response in patients with cystic
fibrosis (21–23). In addition, the shedding of syndecan-1,
a cell-surface HSPG, plays an important role in regulating neu-
trophil influx into lungs in animal models (24, 25).
Changes in the composition of GAGs and PGs in lungs have
beenreported in animal modelsand human lung disease (26–30).
After the treatment of lungs with LPS, the composition of
GAGs changes from the healthy lung where HS predominates
to inflamed lungs where chondroitin sulfate is the predominant
GAG (26, 27). The changes that occur to HSPGs during the
early stages of gram-negative pneumonia are not known. There-
fore, identifying these changes and characterizing how they alter
the inflammatory response could lead to a better understanding
of the mechanisms controlling the innate immune response and
the development of lung injury in patients with gram-negative
(Received in original form August 24, 2011 and in final form March 8, 2012)
This work was supported by National Institutes of Health grants HL098067,
GM37696, RR030249, HL082658, HL081764, and DK089507 and by the Med-
ical Research Service at the Department of Veterans Affairs and the Diffuse Lung
Diseases Research Group from the Ministry of Health, Labor and Welfare, Japan.
Correspondence and requests for reprints should be addressed to Yoshinori
Tanino, M.D., Ph.D., Fukushima Medical University School of Medicine, Depart-
ment of Pulmonary Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan. E-mail:
Am J Respir Cell Mol Biol
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2011-0294OC on March 15, 2012
Internet address: www.atsjournals.org
Vol 47, Iss. 2, pp 196–202, Aug 2012
The results provide evidence that expression of syndecan-4,
a cell surface HSPG, was rapidly and selectively increased in re-
sponse to LPS via MyD88-dependent signaling pathways.
Experiments performed with syndecan-4–null (Sdc4–/–) mice
showed that the lack of this HSPG resulted in a significant in-
crease in pulmonary inflammation and injury after treatment
with LPS. Finally, studies performed in vitro showed that mac-
rophages and epithelial cells increase the expression of
syndecan-4 in response to treatment with LPS and that pretreat-
ment with heparin and syndecan-4 decreased the expression of
CXCL8 mRNA in a human epithelial cell line treated with LPS
and TNF-a. These findings suggest that syndecan-4 has a critical
role in the early inflammatory response in lungs by limiting
tissue inflammation and injury.
MATERIALS AND METHODS
The reagents used in this study were Escherichia coli serotype 0111:B4
LPS (List Biological Laboratories, Campell, CA or Sigma-Aldrich, St.
Louis, MO), TaqMan primer-probes for quantitative PCR (Applied
Biosystems, Foster City, CA), human TNF-a (Sigma-Aldrich), BEAS-
2B cells (ATCC, Manassas, VA), human syndecan-4 (R&D, Minneapolis,
MN), and low-molecular-weight heparin (Fragmin Pfizer, Tokyo, Japan).
The Animal Research Committees of the Veterans Affairs Puget Sound
Health Care System and the Fukushima Medical University approved
all animal experiments. C57BL/6 and C57BL/6J-Ticam1Lps2/J (TRIF
mutant) mice (Jackson Laboratories, Bar Harbor, ME), MyD88 knock-
out (MyD882/2) (Dr. S. Akira at University of Osaka), and Sdc42/2
mice (Dr. T. Kojima) were housed in specific pathogen-free facilities.
The intratracheal injection of LPS (1 mg/g), animal death at specified
times, bronchoalveolar lavage (BAL), total and differential cell counts,
ELISA for murine KC and MIP-2 (R&D) and measurement of total
protein (Thermo Scientific, Rockford, IL) were performed as described
(20, 31, 32).
Isolation of RNA
RNA was isolated with Absolute RNA Miniprep Kit (Stratagene, La
TX), and RNA was reverse transcribed with a High Capacity cDNA
Archive Kit (Applied Biosystems).
Measurement of mRNA
Quantitative PCR was performed using TaqMan primer probes or
Power SYBR Green PCR master mix and an ABI PRISM 7000 (Ap-
plied Biosystems) (32). The threshold cycle (Ct) was calculated using
threshold cycles for the target genes and 18-S. Relative mRNA expres-
sion was expressed as fold increase over values obtained from RNA
from normal lungs, untreated cells, or human reference total RNA
Bone marrow–derived macrophages (BMDMs) were cultured in mac-
rophage medium (RPMI 1640, 10% FBS, 30% L929 cell supernatant,
2 mM L-glutamine, 100 IU/mL penicillin, and 100 mg/ml streptomycin)
as described (20). After being cultured in macrophage medium for 6
days, BMDMs were isolated, counted, and cultured in macrophage
media for 24 hours and then stimulated with LPS (10 or 100 ng/ml),
IL-4/IL-13 (10 ng/ml), IL-10 (10 ng/ml), or RPMI 1640 for up to
48 hours. Alveolar macrophages isolated with repeated BAL using
PBS were cultured for 24 hours in macrophage media and then stim-
ulated with LPS for 4 hours. BEAS-2B cells were cultured in RPMI
1640 supplemented with 10% BSA, penicillin, and streptomycin for
5 to 6 days until they reached 90% confluence. The culture medium
was then replaced, and low-molecular-weight heparin, syndecan-4, or
RPMI 1640 was added for 1 hour. Cells were washed with PBS and
incubated with LPS (1,000 ng/ml), TNF-a (1 ng/ml), or RPMI 1640
without heparin or syndecan-4 for 3 hours. Mouse tracheal air–liquid
interface cultures were performed as described and stimulated with
LPS or RPMI 1640 for 4 and 24 hours (33). Supernatants were removed
and RNA was harvested.
Flow Cytometric Analysis
BMDMs were detached, resuspended, incubated with Fc block (BD
Pharmingen, San Diego, CA), and centrifuged at 1,500 rpm for
5 minutes at 48C. The supernatant was removed, and BMDMs were
resuspended in 100 ml binding buffer (PBS and 1% BSA) containing
phycoerythrin-conjugated syndecan-4 antibody. After 1 hour, BMDMs
were washed twice and analyzed using the Guava System (Millipore,
Comparisons between multiple groups were performed using one-way
ANOVA with Bonferroni’s multiple comparison test or the Kruskal-
Wallis test and Dunn’s multiple comparison test. Student’s t test and
Figure 1. Changes in the relative amounts of mRNA for the heparan
sulfate proteoglycans syndecan-1, -2, and -4 and perlecan, were deter-
mined using mRNA collected from whole lung homogenates and quan-
titative real-time PCR. Comparison of mRNA recovered from lungs of
mice treated with PBS (open symbols) and LPS (closed symbols) were
made at 2, 6, and 24 hours (A). Expression of syndecan-4 in whole lung
homogenates collected from C57BL/6 (WT), MyD88 knockout (MyD88
KO), and Trif mutant mice (Trif mut) treated with PBS (open bars) or
LPS (closed bars) for 2 hours (B). Values are the mean 6 SEM with
a minimum n ¼ 3 for each group studied. The expression of mRNA
for each proteoglycan studied is expressed as a relative fold increase in
mRNA over the 0-hour control group. *Groups that are significantly
different (P < 0.05) when mice treated with PBS and LPS were com-
pared. The Mann-Whitney U-test was used for comparisons between
the two treatment groups (A) and the Kruskal-Wallis test with Dunn’s
multiple comparison test was performed when multiple comparisons
were made (B).
Tanino, Chang, Wang, et al.: Syndecan-4 Moderates Pulmonary Inflammation197
the Mann-Whitney U test where used for comparisons between two
groups. For all analyses, P < 0.05 was accepted as significant. The
values shown are means 6 SEM.
Measurement of mRNA for HSPGs in Whole
To characterize changes in the expression of mRNA for the
HSPGs perlecan, syndecan-1, syndecan-2, and syndecan-4, mice
were treated with LPS, and quantitative real-time PCR was per-
formed using mRNA collected from whole lung homogenates.
This work showed that, of the four HSPGs studied, only
LPS (11.4 6 1.7-fold and 10.4 6 1.7-fold at 2 and 6 h, respec-
tively) when compared with control mice (Figure 1A). These
findings show that among the HSPGs studied, syndecan-4 is
rapidly and selectively up-regulated in response to LPS.
We next investigated the signaling pathways responsible for
the increased expression of syndecan-4. The binding of LPS to
the TLR4/MD2/CD14 complex results in signaling through
two adaptor molecules, MyD88 and Trif (34). To evaluate the
roles of these pathways in regulating the expression of
syndecan-4 in lungs, mRNA was obtained from whole lung
homogenates collected from C57BL/6 (i.e., wild-type controls),
MyD88-null (MyD882/2) mice, and Trif mutant mice. Mice
were killed 2 hours after treatment with LPS to minimize the
chance that cytokines or growth factors produced in response to
the LPS would indirectly increase the expression of syndecan-4
mRNA. The results showed that syndecan-4 mRNA was signif-
icantly increased in the wild-type and Trif mutant mice treated
with LPS but not in the mice lacking MyD88 (Figure 1B). Thus,
the increased expression of syndecan-4 in response to LPS was
mediated via MyD88-dependent signaling pathways.
Measurement of the Innate Immune Response
in Mice Deficient in Syndecan-4
To identify the role of syndecan-4 in modulating the pulmonary
recruitment of neutrophils and the development of lung injury,
wild-type mice and mice deficient in syndecan-4 (Sdc42/2mice)
were studied 3 and 6 hours after treatment with intratracheal
LPS. We recovered significantly more neutrophils and greater
amounts of the neutrophil chemotactic factors KC and MIP-2
from the airspaces of the Sdc42/2lungs at 3 and 6 hours com-
pared with wild-type control mice (Figures 2 and 3). Consistent
with enhanced neutrophil influx leading to more injury, Sdc42/2
mice had a significant increase in total protein in the BAL fluid
(Figure 4). These findings indicate that syndecan-4 plays an
important role in the innate immune response to LPS by limit-
ing the inflammatory response and lung injury in mice treated
Measurement of Syndecan-4 mRNA in Macrophages
and Epithelial Cells Treated with LPS
We assessed which cells in the lungs were responsible for the
increased expression of syndecan-4. Immunohistochemistry of
Figure 2. The total number of neutrophils recovered in bronchoalveo-
lar lavage fluid collected from wild-type and syndecan-4 knockout
(Sdc42/2) mice treated with LPS (1 mg/g) and followed for 3 hours
(A) and 6 hours (B) was determined. Values are the mean 6 SEM with
n ¼ 8 to 10 mice per group for the 3-hour study and n ¼ 11 mice per
group for the 6-hour study. The y axis differs on the graphs displaying
the results for the 3- and 6-hour studies. *Significantly different using
the Mann-Whitney U test and a P < 0.05.
Figure 3. The amount of two neutrophil chemotactic factors, KC (A)
and MIP-2 (B), was measured in the bronchoalveolar lavage fluid collected
from wild-type and syndecan-4 knockout (Sdc42/2) mice treated with
LPS (1 mg/g) and followed for 6 hours. Values are the mean 6 SEM with
n ¼ 8 to 10 mice per group for the 3-hour study and n ¼ 11 mice per
group for the 6-hour study. *Significantly different using the Mann-
Whitney U test and (P < 0.05).
198 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 472012
tissues from Sdc42/2mice with commercial antibodies demon-
strated that none was specific for this antigen (data not shown).
Thus, we used cell culture models to identify the cell types in
lungs that might be responsible for the increased expression of
syndecan-4. Macrophages and lung epithelial cells were chosen
for these studies because they are the first cells exposed to an
airway challenge of LPS and because both cell types express the
TLR4/MD2/CD14 complex required to recognize and respond
to LPS (35–38). For studies where mRNA was collected to
measure alterations in syndecan-4 expression in macrophages,
BMDMs were used for most experiments because of the num-
bers of cells required to make direct comparisons among the
different treatments, which included two concentrations of
LPS (10 and 100 ng/ml). The combination of IL-4/IL-13 or
IL-10 alone was used for 4, 24, and 48 hours. LPS was used
to differentiate cells into classically activated macrophages
(M1), whereas IL-4/IL-13 in combination or IL-10 alone was
used to differentiate these cells into alternatively activated
macrophages (M2a and M2c, respectively) (39–41). The expres-
sion of syndecan-4 mRNA by macrophages was significantly
increased after treatment with LPS but not with IL-4/IL-13 or
IL-10 (Figure 5A), suggesting that this HSPG is a new marker
of M1 macrophages. Compared with 0-hour control macro-
phages, syndecan-4 mRNA increased 41 6 21-fold at 4 hours
with 10 ng/ml LPS and increased 45 6 28-fold with 100 ng/ml
LPS. By 24 hours, the amount of syndecan-4 mRNA expression
in macrophages treated with LPS had returned to control levels.
In a limited number of experiments, murine alveolar macro-
phages were studied to confirm that these macrophages also
increased the expression of syndecan-4 mRNA after treatment
with LPS. These studies show that alveolar macrophages trea-
ted with LPS (100 ng/ml) for 4 hours had a 96.8 6 6.8-fold
increase in syndecan-4 mRNA (Figure 5B).
We next used flow cytometry to measure the amount of
syndecan-4 present on the surface of BMDMs. Consistent with
the increased expression of syndecan-4 mRNA (Figure 5A),
macrophages treated with LPS had a significant increase in
the amount of syndecan-4 measured on their cell surface (Fig-
ures 5C and 5D). Control macrophages had a mean fluorescence
of 11.56 6 0.19, whereas cells treated with LPS had a mean
fluorescence of 129.2 6 6.38. Confirmatory experiments using
antibodies for syndecan-4 and the macrophage marker F4/80
showed that the syndecan-4–positive cells were macrophages
(data not shown).
We also found that syndecan-4 expression by epithelial cells
was increased by LPS (Figure 6) but not to the degree seen in
macrophages. Murine tracheal epithelial cells grown at an air–
liquid interface and treated with LPS at concentrations of 10
and 100 ng/ml for 4 hours had a 3.2 6 0.74-fold and a 5.8 6 0.85-
fold increase in syndecan-4 mRNA, respectively (Figure 6A). In
comparison when the human bronchial epithelial cell line
BEAS-2B cells were treated with LPS (1,000 ng/ml), there
was a 5.87 6 1.53-fold and 3.94 6 1.35-fold increase in
Figure 4. The amount of total protein in the bronchoalveolar lavage
fluid collected from wild-type and syndecan-4 knockout (Sdc42/2)
mice treated with LPS (1 mg/g) was measured at 6 hours. Values are
the mean 6 SEM with n ¼ 11 mice per group. *Significantly different
using the Mann-Whitney’s U test and (P < 0.05).
Figure 5. (A) Quantitative real-time PCR showing the rel-
ative expression for syndecan-4 mRNA collected from
bone marrow–derived macrophages (BMDMs) treated in
vitro with RPMI media (control), two concentrations of the
M1 agonist LPS (10 and 100 ng/ml), the M2a agonists
IL-4/IL-13 (10 ng/ml), or the M2c agonist IL-10 for up to
48 hours (n ¼ 4–13). (B) Quantitative real-time PCR show-
ing the relative expression for syndecan-4 mRNA collected
from alveolar macrophages treated with RPMI media (con-
trol) or LPS (100 ng/ml) for 4 hours (n ¼ 4). (C and D) A
representative histogram (C) and the geometric mean of
syndecan-4 expression (D) on BMDMs measured with flow
cytometry. Flow cytometry was performed 3 hours after
the treatment of mouse BMDMs with RPMI or LPS (10 ng/ml)
(n ¼ 3). Values are the mean 6 SEM. *Groups different
from the control mice (P < 0.05) using the Kruskal-Wallis
test with Dunn’s multiple comparison test (A) and the
Mann-Whitney U test (B and D).
Tanino, Chang, Wang, et al.: Syndecan-4 Moderates Pulmonary Inflammation 199
syndecan-4 mRNA after treatment for 3 and 6 hours, respec-
tively. Taken together, these findings show that LPS causes
a rapid up-regulation of syndecan-4 mRNA in macrophages
and epithelial cells. In addition, we show that the rapid increase
in the expression of syndecan-4 is a characteristic of classically
Effect of Low-Molecular-Weight Heparin or Syndecan-4
on CXCL8 Expression in BEAS-2B Cells
We assessed if soluble forms of heparin or syndecan-4 alter the
inflammatory response of BEAS-2B cells treated with LPS or
TNF-a, a heparin-binding proinflammatory cytokine. Heparin
was used for these studies because it is readily available and
because it has a similar, though more sulfated, GAG backbone
to heparan sulfate (18). BEAS-2B cells treated with LPS alone
had significantly increased CXCL8 mRNA production com-
pared with control cells (no LPS) (8.5 6 0.45-fold increase
versus control cells). However, when BEAS-2B cells were pre-
treated with low-molecular-weight heparin (LMWH) and then
treated with LPS, these cells expressed significantly less CXCL8
mRNA (2.8 6 0.67 for cells) as compared with cells treated with
LPS alone (Figure 7A).
To determine if pretreatment with heparin would affect the
activation of epithelial cells stimulated with the heparin-binding
cytokine TNF-a (42, 43), BEAS-2B cells were pretreated with
RPMI or LMWH and then treated with TNF-a. These studies
showed that the preincubation of epithelial cells with LMWH
significantly decreased the TNF-a induced up-regulation of
CXCL8 mRNA expression (Figure 7B). Finally, to determine
if syndecan-4 would affect the activation of epithelial cells stim-
ulated with TNF-a, BEAS-2B cells were preincubated with hu-
man recombinant syndecan-4 (2.5 mg/ml) before treatment
with TNF-a. The treatment of epithelial cells with recombi-
nant syndecan-4 significantly inhibited the TNF-a induced up-
regulation of CXCL8 mRNA in BEAS-2B cells (Figure 7C).
As was observed with the LMWH, syndecan-4 did not com-
pletely abolish the TNF-a activation of BEAS-2B cells. These
studies showed that pretreatment with heparin and HS are
able to decrease the inflammatory response of epithelial cells
subsequently treated with LPS and TNF-a in vitro.
The purpose of this work was to identify changes in the compo-
sition of HSPGs in mice exposed to gram-negative bacterial
products and to determine how these changes alter pulmonary
inflammation. To reduce animal-to-animal variability resulting
Figure 6. Quantitative real time PCR showing the relative expression
for syndecan-4 mRNA collected from murine tracheal air–liquid inter-
face cell cultures treated with LPS at 10 and 100 ng/ml (A) and BEAS-2B
cells treated with LPS at 1,000 ng/ml (B) for the specified times. For all
cultures, n ¼ 3; for BEAS-2B cells, n ¼ 4. Values are the mean 6 SEM.
*Groups different from the controls using the Kruskal-Wallis test with
Dunn’s multiple comparison test with P < 0.05.
Figure 7. (A and B) Pre-
treatment of BEAS-2B cells
with RPMI media or low-
(LMWH) (10 mg/ml) for 1
hour followed by treat-
ment with media, LPS (A),
or TNF-a (B) for 3 hours.
(C) Pretreatment of BEAS-
2B cells with RPMI media
or syndecan-4 (2.5 mg/ml)
for 1 hour followed by
treatment with media or
TNF-a for 3 hours. Values
are the mean 6 SEM with
n ¼ 6. An (a) shows groups
that are different from the
control group, and a (b)
shows differences between
LPS or TNF-a treatment
groups using the Kruskal-
multiple comparison test
with P < 0.05.
200AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 472012
from live bacteria, mice were treated with intratracheal LPS,
a component of the cell wall of gram-negative bacteria. The
treatment of mice with intratracheal LPS resulted in a rapid in-
crease in syndecan-4 mRNA in lungs that was regulated through
MyD88-dependent signaling. Work performed using Sdc4–/–
mice showed that the lack of this HSPG resulted in a significant
increase in pulmonary inflammation and injury after treatment
with LPS. Finally, experiments performed in vitro showed that
macrophages and epithelial cells increase the expression of
syndecan-4 in response to treatment with LPS and that pretreat-
ment with heparin and syndecan-4 decreased the expression of
CXCL8 mRNA in a human epithelial cell line treated with LPS
and TNF-a. These studies show that syndecan-4 plays a key
role in regulating the early innate immune response to lung
Little is known about how the composition of HSPG changes
during gram-negative pneumonia or about the signaling path-
only the expression of syndecan-4 increased in mice treated with
LPS. The rapid increase in syndecan-4 mRNA in lungs, which
occurs in a similar time frame to the increased expression of
proinflammatory cytokines and chemokines (44), suggests that
the expression of this HSPG in mice exposed to intratracheal
LPS is regulated directly through TLR4. To better characterize
the signaling pathways responsible for the increased expression
of syndecan-4, studies were performed in mice deficient in
MyD88 or Triff. The inability of the MyD882/2mice to increase
syndecan-4 mRNA in response to LPS shows that the recruit-
ment of MyD88 to TLR4 mediates signal transduction to down-
stream signaling components, such as NF-kB, which modulates
syndecan-4 production (Figure 1B). This extends the previous
work of Smith and colleagues who found that signaling through
NF-kB was responsible for the increased expression of syndecan-
4 in a gastric epithelial cell line treated with TLR2 and TLR4
agonists in vitro (45).
MyD88 is an adaptor molecular common to all TLRs (except
for TLR3- and for IL-1R1) (3). Therefore, it is likely that
syndecan-4 will be increased in a number of infectious and non-
infectious lung diseases, with the common link being activation
of MyD88 signaling pathways. For example, IL-1R1/MyD88
signaling and the inflammasome are essential in the develop-
ment of pulmonary inflammation and fibrosis in bleomycin- in-
duced lung injury (46), suggesting that IL-1R1/MyD88 signaling
may be responsible for the increased expression of syndecan-4
reported in mice treated with bleomycin (47). These findings
suggest an important role for syndecan-4 in regulating the in-
flammatory response in lungs after the activation of MyD88
To determine the role of syndecan-4 in the innate immune
response to LPS, we studied mice lacking syndecan-4. This work
showed that Sdc42/2mice had increased pulmonary inflamma-
tion and lung injury after treatment with LPS. These results are
consistent with previous reports, such as the work of Ishiguro
and colleagues, who found that the injection of LPS into the
peritoneum of Sdc42/2mice resulted in increased mortality
(48). In a more recent study, Jiang and colleagues showed that
Sdc42/2mice treated with bleomycin had increased pulmonary
fibrosis (47). In addition, studies performed in vitro with BEAS-
2B cells showed that pretreatment with heparin or syndecan-4
decreased the expression of CXCL8 mRNA when the BEAS-
2B cells were subsequently treated with LPS or TNF-a (Figure
7). Taken together, this body of work shows that syndecan-4
regulates the inflammatory response and suggests that syndecan-
4 may play a key role in preventing the adverse consequences of
tissue inflammation, such as lung injury, pulmonary fibrosis, and
the systemic inflammatory response.
sion of syndecan-4, murine macrophages and a human bronchial
epithelial cell line (BEAS-2B cells) were treated with LPS for up
to 24 hours. Macrophages and epithelial cells were evaluated be-
cause these cells are exposed to the highest concentrations of
LPS after an airway challenge. In response to treatment with
LPS in vitro, macrophages and epithelial cells showed a rapid
and significant increase in the expression of syndecan-4, suggest-
ing that they are in part responsible for the increased expression
of syndecan-4 mRNA. Whereas the in vitro studies were per-
formed using macrophages and epithelial cells, other cells, such
as fibroblasts and endothelial cells, are potential sources of the
increased recovery of syndecan-4 mRNA obtained from whole
lungs of mice treated with LPS.
Macrophages are broadly classified into two groups: M1 or
classically activated macrophages and M2 or alternatively acti-
vated macrophages. To characterize whether the early increase
of syndecan-4 was specific for M1 macrophages, cells were trea-
13 and IL-10 to differentiate them to a M2 phenotype (39, 40).
This work showed that the rapid increase in syndecan-4 expres-
sion in macrophages occurs only with LPS treatment, which
suggests that syndecan-4 is an early marker of M1 macrophages.
In summary, the results of this study show that syndecan-4 is
rapidly increased in response to the intratracheal instillation
of LPS with expression kinetics similar to that observed for
cytokines and chemokines. These studies also show an increased
pulmonary inflammatory response and lung injury in syndecan-
4–deficient mice treated with intratracheal LPS. Finally, pre-
treatment of lung epithelial cells with heparin or syndecan-4
inhibits the proinflammatory properties of LPS and TNF-a.
We conclude that the increased expression of syndecan-4 in lungs
of mice exposed to intratracheal LPS is an important mechanism
that regulates neutrophil recruitment and limits the extent of
pulmonary inflammation and lung injury. This suggests that the
development of therapeutic strategies to capitalize on the protec-
tive effects of syndecan-4 may provide treatments to minimize
the adverse effects of tissue inflammation.
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgments: The authorsthank Timothy Birkland,Ph.D., Kathleen R.Braun,
Gina Kiske. and Vivian Lee for excellent technical expertise and assistance.
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