Tài liệu Báo cáo khoa học: Lipopolysaccharide-evoked activation of p38 and JNK leads to an increase in ICAM-1 expression in Schwann cells of sciatic nerves ppt

Lipopolysaccharide-evoked activation of p38 and JNK
leads to an increase in ICAM-1 expression in Schwann
cells of sciatic nerves
Aiguo Shen
1,
*, Junling Yang
2,
*, Yangyang Gu
3
, Dan Zhou
4
, Linlin Sun
2
, Yongwei Qin
2
,
Jianping Chen
2
, Ping Wang
2
, Feng Xiao
2
, Li Zhang
2
and Chun Cheng
1,2
1 Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University, Jiangsu, China
2 Department of Microbiology and Immunology, Medical College, Nantong University, Jiangsu, China
3 Department of Surgery, RICH Hospital, Nantong, Jiangsu, China
4 Department of Biochemistry, Medical College of Nantong University, Jiangsu, China
Intercellular adhesion molecule-1 (ICAM-1, CD54) is a

cell-surface glycoprotein that belongs to the immuno-
globulin superfamily of adhesion molecules. Its struc-
ture comprises a cytoplasmic tail, a transmembranous
region, and five extracellular domains binding to the
b
2
-integrin counter-receptors lymphocyte function-
associated antigen-1 (LFA-1) and CD11b ⁄ CD18
(MAC-1) [1–4]. The ICAM-1 gene promoter ⁄ enhancer
Keywords
intercellular adhesion molecule-1;
lipopolysaccharide; mitogen-activated
protein kinase; peripheral nervous system;
Schwann cell
Correspondence
C. Cheng, Jiangsu Province Key Laboratory
of Neurodegeneration, Nantong University,
19 Qi-xiu Road, Nantong, Jiangsu 226001,
China
Fax: +86 513 85051999
Tel: +86 513 85051999
E-mail:
*These authors contributed equally to this
work
(Received 30 April 2008, revised 22 June
2008, accepted 27 June 2008)
doi:10.1111/j.1742-4658.2008.06577.x
Lipopolysaccharide is a major constituent of the outer membrane of
Gram-negative bacteria. It activates monocytes and macrophages to
produce cytokines such as tumor necrosis factor- a and interleukins IL-1b

and IL-6. These cytokines appear to be responsible for the neurotoxicity
observed in peripheral nervous system inflammatory disease. It has been
reported that, in the central nervous system, the expression level of inter-
cellular adhesion molecule-1 (ICAM-1) was dramatically upregulated in
response to LPS, as well as many inflammatory cytokines. ICAM-1 con-
tributes to multiple processes seen in central nervous system inflammatory
disease, for example migration of leukocytes to inflammatory sites, and
adhesion of polymorphonuclear cells and monocytes to central nervous sys-
tem cells. In the present study, we found that lipopolysacharide evoked
ICAM-1 mRNA and protein expression early at 1 h post-injection, and the
most significant increase was seen at 4 h. Immunofluorescence double-label-
ing suggested that most of the ICAM-1-positive staining was located in
Schwann cells. Using Schwann cell cultures, we demonstrated that ICAM-1
expression in Schwann cells is regulated by mitogen-activated protein
kinases, especially the p38 and stress-activated protein kinase ⁄ c-Jun
N-terminal kinase pathways. Thus, it is thought that upregulation of
ICAM-1 expression in Schwann cells may be important for host defenses
after peripheral nervous system injury, and reducing the biosynthesis of
ICAM-1 and other cytokines by blocking the cell signal pathway might
provide a new strategy against inflammatory and immune reaction after
peripheral nerve injury.
Abbreviations
CNS, central nervous system; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM-1,
intercellular adhesion molecule-1; IL, interleukin; LFA-1, lymphocyte function-associated antigen-1; LPS, lipopolysaccharide; MAPK, mitogen-
activated protein kinase; MHC, major histocompatibility complex; NF-jB, nuclear factor jB; PNS, peripheral nervous system; SAPK ⁄ JNK,
stress-activated protein kinase ⁄ c-Jun N-terminal kinase; SCs, Schwann cells; TNF, tumor necrosis factor.
FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS 4343
has binding sites for a number of transcription factors
[5–8]. During inflammation, ICAM-1 is dramatically
upregulated by bacterial lipopolysaccharide (LPS)

and inflammatory cytokines, such as tumor necrosis
factor-a (TNF-a), interleukin-1b (IL-1b) and inter-
feron-c (IFN-c) [9].
LPS is a major constituent of the outer membrane
of Gram-negative bacteria, and its recognition and
signal transmission are key events in the host defense
reaction towards Gram-negative bacteria. Generally,
LPS activates monocytes and macrophages to produce
cytokines such as TNF-a, IL-1b and IL-6, which, in
turn, serve as endogenous inflammatory mediators
[10,11], and are responsible for the neurotoxicity
observed in neurodegenerative diseases such as Guil-
lain–Barre
´
syndrome, amyotrophic lateral sclerosis
and multiple sclerosis in the peripheral nervous system
(PNS) inflammation [12].
In the central nervous system (CNS), ICAM-1
expression is frequently upregulated in inflammatory
diseases. In vitro, ICAM-1 expression can be upregu-
lated in astrocytes, the most common cell type in the
CNS, in response to an immune reaction [13]. It has
been reported that ICAM-1 is associated with multiple
steps of the CNS inflammation process, for example
migration of leukocytes to inflammatory sites [14,15]
and adhesion of polymorphonuclear cells and mono-
cytes to CNS cells [16,17].
Schwann cells (SCs) are glia cells found in the PNS.
In addition to their roles in myelination, trophic sup-
port and axon regeneration, SCs exhibit potential

immune functions, similar to the non-myelinating glia
of the CNS. SCs can be induced to produce cytokines
and chemokines, to express major histocompatibility
complex (MHC) class II molecules and adhesion mole-
cules, and to serve as antigen-presenting cells [18–20].
These chemokines and inflammatory proteins may
recruit macrophages from the blood vessels, leading to
local inflammation [21].
Nuclear factor jB (NF-jB), a critical participant
in cytokine-induced ICAM-1 upregulation [5,7,22,23],
mediates the rapid induction of cytokines and adhe-
sion molecules that are implicated in immune and
inflammatory responses [24,25]. Mitogen-activated
protein kinases (MAPKs) are important mediators
of cytokine expression; in particular, p38 and extra-
cellular signal-regulated kinase (ERK) play key roles
in LPS-induced signal transduction pathways. Numer-
ous studies have clearly demonstrated the essential
role of NF-jB in ICAM-1 expression [26,27], as well
in activation of the c-Jun N-terminal kinase (JNK),
but an unequivocal demonstration of ICAM-1 regula-
tion in SCs is currently lacking.
Thus, the goal of the present study was to determine
whether LPS upregulates ICAM-1 expression in vivo
and in vitro, and whether ERK, p38 or JNK, the
MAPK family members, mediate LPS-induced ICAM-
1 expression in SCs. We found that ICAM-1 expres-
sion in sciatic nerves is upregulated in response to LPS
injection, and that activation of MAPKs, especially
p38 and the stress-activated protein kinase

(SAPK) ⁄ JNK pathways, might contribute to this
process.
Results
LPS upregulates ICAM-1 mRNA and protein
expression in rat sciatic nerves
To examine ICAM-1 mRNA expression in rat sciatic
nerves, RT-PCR analysis was performed. The ICAM-1
mRNA content of the sciatic nerve increased over time
after intraperitoneal injection of LPS (Fig. 1A). In con-
trol rats, the ICAM-1 mRNA level was low but detect-
able. The peak level of ICMA-1 mRNA was found at
2–4 h after LPS administration peak (P = 0.01 versus
control) (Fig. 1A), and then decreased.
To determine whether ICAM-1 protein expression
increased in rat sciatic nerve after intraperitoneal injec-
tion of LPS, western blot analysis was performed. The
time course of ICAM-1 expression after LPS injection
is shown in Fig. 1B. The expression pattern for
ICAM-1 protein was similar to that for ICAM-1
mRNA. Compared with the control, expression of
ICAM-1 protein was elevated at 2 h after LPS admin-
istration, but this increase was not statistically signi-
ficant (P = 0.970) (Fig. 1B). The peak expression
occurred at 4 h (P = 0.001), and reduced gradually
but remained above initial levels until 48 h (Fig. 1B).
Expression of ICAM-1 in SCs of rat sciatic nerves
To identify the localization of ICAM-1 in sciatic
nerves after LPS administration, we performed double
immunostaining using ICAM-1 antibody with NF-200
(specific to neurofilaments), S100 (specific to Schwann

cells) and CD31 (a marker of endothelial cells). In pre-
vious studies, ICAM-1–integrin interactions mediated
adhesion of leukocytes to the vascular endothelium,
revealing a key role in migration of leukocytes to
inflammation sites [28–30]. In the control rats, most of
the ICAM-1 staining co-localized with CD31, implying
expression of ICAM-1 in sciatic nerve blood vein
endothelial cells (Fig. 2A–C); only a few SCs were
ICAM-1-positive (Fig. 2D–F). Four hours after LPS
injection, co-localization of ICAM-1 with CD31 was
LPS increases ICAM-1 expression in Schwann cells of sciatic nerves A. Shen et al.
4344 FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS
still found in sciatic nerve blood vein endothelial
cells (Fig. 3A–C), but positive staining of ICAM-1 in
SCs was more apparent than that in the controls
(Fig. 3D–F). Rare co-localization of ICAM-1 and
NF-200 was found in the axons in both the control
group (Fig. 2G–I) and at 4 h after administration
(Fig. 3G–I).
Effects of LPS on expression of ICAM-1 in SCs
in vitro
In order to better explore the role of LPS-induced
ICAM-1 expression in SCs, a series of experiments
were performed in vitro. SCs were treated with various
concentrations of LPS for 2 h. Using western blot
analysis, we found that LPS induced ICAM-1 protein
expression in a concentration-dependent manner
(Fig. 4A). A significant increase was observed at
1 lgÆmL
)1

(P = 0.001) (Fig. 4A). Treatment with
100 lgÆmL
)1
LPS appeared to induce ICAM-1 protein
to a lesser extent than treatment with 10 lgÆmL
)1
LPS;
this might reflect a loss of cell viability or numbers at
the high LPS concentration. Time-course experiments
were performed at the concentration of 1 lgÆmL
)1
(Fig. 4B). Conspicuous ICAM-1 biosynthesis was
observed at 2 h (P = 0.05), and the maximum
response occurred at 4 h (P = 0.001) (Fig. 4B).
ELISA analysis showed that induction of ICAM-1
protein expression by LPS was dose- and time-depen-
dent (Fig. 4C,D). The expression pattern of ICAM-1
mRNA was similar to that of ICAM-1 protein
(Fig. S1).
LPS activates MAPKs in SCs
Activation of MAPKs has been proved to be impor-
tant in transmitting LPS-evoked cell signals in many
cell types [30a, 30b]. To investigate the role of these
signal transduction pathways in ICAM-1 expression,
we first examined the kinase activity of ERK, p38 and
SAPK ⁄ JNK, the three major members of the MAPK
family, in LPS-treated SCs. Briefly, as illustrated in
Fig. 5A, phosphorylation of p38 and SAPK ⁄ JNK
appeared at 30 min, and peaked at 2 h (P = 0.001)
and 1 h (P = 0.005), respectively (Fig. 5B). However

phosphorylation of ERK was not significant (Fig. 5).
Roles of MAPKs in LPS-induced ICAM-1 synthesis
Using U0126 (an MEK1⁄ 2 inhibitor), SB202190 (a
p38 MAPK inhibitor) and SP600125 (an SAPK ⁄ JNK
specific inhibitor), the roles of MAPKs in LPS-
induced ICAM-1 synthesis were examined. Pretreat-
ment of cells with SB202190 (1–20 lm) or SP600125
(10–40 lm) resulted in a significant attenuation of
ICAM-1 mRNA production in a concentration-
dependent manner, and the inhibition was nearly
complete when pretreated with SB202190 at 10 or
20 lm and SP600125 at 20 or 40 lm (Fig. 6A). In
contrast, U0126 had a minimal effect (Fig. 6A).
Expression of ICAM-1 protein detected by western
A
B
Fig. 1. Time course of ICAM-1 expression in rat sciatic nerves after
LPS injection. (A) Time course of ICAM-1 mRNA expression in LPS-
treated rats. Integrated band densities were obtained by densito-
metric scanning. The data are means ± SEM. *P = 0.01 (Student’s
t-test, n = 3) versus the corresponding control. (B) Time course of
ICAM-1 protein expression in LPS-treated rats. Immunoblots were
probed for ICAM-1 and b -actin, respectively. The bar chart shows
the ratio of ICAM-1 to b-actin at each time point. The data are
means ± SEM. **P = 0.001, *P = 0.014 (Student’s t-test, n =3)
versus the corresponding control.
A. Shen et al. LPS increases ICAM-1 expression in Schwann cells of sciatic nerves
FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS 4345
blot and ELISA revealed that induction of ICAM-1
was substantially inhibited by U0126 (20 lm), and

completely abolished by SB202190 (10 lm) and
SP600125 (20 lm), respectively (Fig. 6B,C).
Immunofluorescent staining showed nuclear staining
of ICAM-1 in SCs after LPS stimulation. In unstimu-
lated cells, ICAM-1 was detected in the cytoplasm
(Fig. 7A, arrow), partly co-localized with S100
(Fig. 7C). Two hours after LPS stimulation, the inten-
sity of ICAM-1 staining was much greater and signifi-
cantly co-localized with S100 (Fig. 7D–F). Using
specific inhibitors of MAPKs resulted in a weakened
intensity of fluorescence in the cells (Fig. 7G–I). It
may be concluded that LPS-induced activation of the
p38 and SAPK ⁄ JNK MAPK cascades is responsible
for the synthesis of ICAM-1 in SCs.
Discussion
The present study demonstrated that LPS induces
ICAM-1 expression in SCs of sciatic nerves. We first
examined the ICAM-1 mRNA and protein levels in
A
B
C
D
EF
G
H
I
Fig. 2. Double immunofluorescence staining
for ICAM-1 and various phenotype-specific
markers in control sciatic nerves. Horizontal
sections were labeled with total ICAM-1

(green) and various phenotype-specific
markers (red), such as CD31 (endothelial
cells), S100 (Schwann cells), NF200 (neuro-
filaments). Yellow staining indicates co-local-
ization of ICAM-1 with the various
phenotype-specific markers. (A–C) The
majority of co-localization was seen in endo-
thelial cells. (D–F) A few SCs were ICAM-1-
positive. (G–I) Rare co-localization occurred
for ICAM-1 and NF-200. Scale bar = 20 lm.
A B
C
D
E
F
G
H
I
Fig. 3. Double immunofluorescence staining
for ICAM-1 and various phenotype-specific
markers in sciatic nerves at 4 h after LPS
injection. Horizontal sections were labeled
with total ICAM-1 (green) and various phe-
notype-specific markers (red), such as
CD31, S100 and NF200 (see Fig. 2). Yellow
staining indicates co-localization of ICAM-1
with the various phenotype-specific mark-
ers. (A–C) ICAM-1 and CD31 co-localized in
sciatic nerve blood vein endothelial cells.
(D–F) Co-localization of ICAM-1 and S100

was more frequent than that in controls,
and the intensity of staining was much
greater. (G–I) Rare co-localization occurred
for ICAM-1 and NF-200 was found. Scale
bar = 20 lm.
LPS increases ICAM-1 expression in Schwann cells of sciatic nerves A. Shen et al.
4346 FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS
the sciatic nerve at several time points after LPS injec-
tion and found that their levels had increased by 1 h
and were especially high at 4 h. This increase lasted
for 12 h. We conclude that ICAM-1 is expressed in rat
sciatic nerves at an early stage of inflammation. In our
experiments, SCs were found to produce ICAM-1
in vivo and in vitro (Figs 3 and 4). The results suggest
that this integral transmembrane protein can moderate
cell-to-cell communication and serve as a signal alter-
ing afferent neuronal function after inflammation.
Previous studies have already addressed the participa-
tion of SCs in immune responses in the PNS [31].
These cells may function as antigen-presenting cells
and activate T cells in vitro in an antigen-specific and
MHC-restricted manner [32], especially in the presence
of cytokines.
Natural ligands of ICAM-1and LFA-1 are expressed
on the surface of T and B lymphocytes, natural killer
cells, monocytes, macrophages and granulocytes [33],
and interaction between these two adhesion molecules
plays a pivotal role in cell-contact-mediated immune
mechanisms [30,34], including antigen-specific respon-
ses, binding of lymphocytes to the endothelium and

migration of lymphocytes towards inflammatory
sites [35,36]. SCs have been implicated in human
inflammatory demyelinating neuropathies such as
Guillain–Barre
´
syndrome and chronic inflammatory
demyelinating polyneuropathy [31]. In experimental
autoimmune neuritis, an animal model of demyelinat-
ing disease of the PNS [37,38], Archelos et al. showed
that, by inhibiting early interactions between immuno-
competent cells after exposure to foreign antigen and
migration of primed T cells into the peripheral nerve,
ICAM-1 ⁄ LFA-1 adhesion molecules act on both the
induction and effect phases of the immune response
[38]. These observations, together with our data
A
B
C D
Fig. 4. LPS induced the expression of ICAM-1 protein in cultured SCs. (A) LPS induced ICAM-1 protein expression in a concentration-depen-
dent manner. Cultures were treated with various concentrations of LPS for 2 h. Data are means ± SEM of the maximum response
observed. *P = 0.001 (Student’s t test, n = 3) versus the corresponding control. (B) LPS induced ICAM-1 protein expression in a time-depen-
dent manner. Cultures were treated with 1 lgÆmL
)1
LPS for various durations (0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h). Data are means ± SEM of
the maximum response observed. *P = 0.001 (Student’s t-test, n = 3) versus the corresponding control. (C) ELISA showed that expression
of ICAM-1 protein in response to LPS stimulation was dose-dependent. SCs were cultured to form confluent monolayers. Cells were treated
with various concentrations of LPS for 2 h. Data are means ± SEM of the maximum response observed. *P = 0.001, (Student’s t-test,
n = 3) versus the corresponding control. (D) ELISA showed that LPS induces ICAM-1 protein expression in a time-dependent manner.
Cultures were treated with 1 lgÆmL
)1

LPS for various durations (0, 0.5, 1, 2, 4, 6, 8 and 12 h). Data are means ± SEM of the maximum
response observed. *P = 0.001 (Student’s t-test, n = 3) versus the corresponding control.
A. Shen et al. LPS increases ICAM-1 expression in Schwann cells of sciatic nerves
FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS 4347
indicating that LPS induces ICAM-1 expression in
SCs, suggest that ICAM-1 may play a role in the focal
accumulation and antigen-induced activation of T cells
in inflammatory demyelinating diseases of the PNS.
As mentioned above, MAPKs were implicated in the
activation of NF-jB in SCs in response to LPS stimu-
lation. Numerous studies have shown that NF-jB
serves as a transcriptional regulator of ICAM-1 in
various cell types [26,39–41], but the mechanisms that
regulate ICAM-1 expression in SCs are not well under-
stood. The present study showed no significant effect
of U0126 on ICAM-1 upregulation, while notable inhi-
bition was observed with SB202190 and SP600125,
indicated that MEK might not contribute to the acti-
vation of NF-jB by LPS.
Our results confirm the important role of SAPK ⁄ JNK
in mediating LPS-induced ICAM-1 expression in SCs.
JNK phosphorylates c-Jun and ATF-2 and increases
their ability to activate transcription, leading to c-jun
induction and subsequent activator protein-1 activation
[42,43]. ICAM-1 gene expression is also modulated by
multiple cis-acting elements, binding sites for activator
protein-1, NF-jB and the transcription factor specificity
protein-1 [44]. Consistent with the results reported by
Kobuchi et al., which showed that phorbol ester and
TNF-a induced ICAM-1 expression via activation of

the JNK pathway and activator protein-1 [45], the pres-
ent research suggests that the JNK pathway also plays a
significant role in the signaling cascade leading to induc-
tion of ICAM-1 expression [46].
In summary, upregulation of ICAM-1 expression in
SCs after direct stimulation with LPS occurred via
activation of MAPKs, especially the p38 and
SAPK ⁄ JNK pathways. Activation of MAPK pathways
might be a precondition for induction of ICAM-1
expression. Reducing the biosynthesis of ICAM-1 and
other cytokines by blocking the cell signal pathway
might provide a new strategy against inflammatory
and immune reactions after peripheral nerve injury.
However, our investigation involved the use of cell
cultures in vitro; in vivo experiments are still needed to
confirm the role of MAPKs. In addition, it is necessary
to clarify whether ICAM-1 expression in SCs is accom-
panied by infiltration of blood-borne monocytes and
contributes to the development of PNS neuropathy.
Experimental procedures
Experimental animals and treatments
Male Sprague–Dawley (SD) rats (Department of Animal
Center, Medical College of Nantong University, China)
were housed in plastic cages at 24 ± 1 °C under a 12 h
light ⁄ dark cycle and given free access to laboratory chow
and water. Rats in the LPS group were intraperitoneally
injected with 5 mgÆkg
)1
LPS (Sigma, St Louis, MO, USA).
All animal experiments were carried out in accordance with

the United States National Institutes of Health Guidelines
for the Care and Use of Laboratory Animals.
SC cultures
Rat primary Schwann cells were isolated and cultured using
a modified method based on that described by Brockes
et al. [47,48]. Briefly, Schwann cells were taken from excised
dorsal root ganglion, brachial plexus and sciatic nerves
from Sprague–Dawley rats and cultured in Dulbecco’s
modified Eagle’s medium containing 10% fetal bovine
serum. The next day, 10 lm cytarabine (AraC) (Sigma) was
added to the medium to eliminate contaminating fibro-
blasts. After 48 h, the medium was replaced by Dulbecco’s
modified Eagle’s medium containing 3% fetal bovine serum
with 3 lm forskolin (Sigma) and 20 ngÆmL
)1
neuregulin
A
B
Fig. 5. Activation of MAPKs in LPS-stimulated SCs. (A) Immuno-
blots were probed for phosphorylated ERK, p38 and JNK (p-ERK,
p-p38 and p-JNK) and total ERK, p38 and JNK (tERK, tp38 and
tJNK). (B) The ratio of phosphorylated to total ERK (p44 ⁄ 42), p38
and JNK at each time point. The data are means ± SEM.
**P = 0.001, *P = 0.029 (Student’s t-test, n = 3) versus the corre-
sponding control.
LPS increases ICAM-1 expression in Schwann cells of sciatic nerves A. Shen et al.
4348 FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS
A B
C
Fig. 6. Effects of U0126, SB202190 and SP600125 on ICAM-1 synthesis induced by LPS. (A) Cells were pretreated with various concentrations

of U0126 (10, 20, 40 l
M), SB202190 (1, 10, 20 lM) or SP600125 (10, 20, 40 lM) for 40 min, and then stimulated with 1 lgÆmL
)1
LPS for 4 h. Cells
were harvested for semi-quantitative RT-PCR analysis, and representative blots are shown. Data were normalized against GAPDH and are plotted
as means ± SEM. **P = 0.01 (Student’s t-test, n = 3) versus the corresponding control. (B) Effects of MAPK inhibitors on ICAM-1 protein syn-
thesis in SCs. Cells were pretreated with U0126 (20 l
M), SB202190 (10 lM) or SP600125 (20 lM) for 40 min, and then stimulated with 1 lgÆmL
)1
LPS for 4 h. Cells were harvested for western blot analysis. The bar chart shows the ratio of ICAM-1 to b-actin for each sample. **P = 0.001,
*P = 0.029 (Student’s t-test, n = 3) versus cultures with only treatment of LPS. (C) ELISA showed the effects of MAPK inhibitors on ICAM-1
protein synthesis in SCs. The data are means ± SEM. *P = 0.01 (Student’s t-test, n = 3) versus the cultures with only treatment of LPS.
A
B
C
D
E
F
G
H
I
Fig. 7. Immunofluorescence analysis of
ICAM-1 expression in SCs. (A–C) In non-
stimulated cells, ICAM-1 (green) was
detected at the cytoplasm (arrow). (D–F)
Two hours after stimulation with LPS in the
absence of inhibitors, the intensity of stain-
ing was much greater than for the control
(without LPS). (G–I) Cells were pretreated
with U0126 (20 l

M), SB202190 (20 lM)or
SP600125 (20 l
M) for 40 min and then stim-
ulated with 1 lgÆmL
)1
LPS for 2 h, and
weaker intensity of ICAM-1 (green) fluores-
cence was detected that when LPS was
used without the inhibitors. Double
immunofluorescence revealed that ICAM-1
co-localizes with S100 (red) (A–F). Scale bar
¼ 20 lm.
A. Shen et al. LPS increases ICAM-1 expression in Schwann cells of sciatic nerves
FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS 4349
(Sigma) to expand the cells. Cells were then detached from
the dishes by 0.25% trypsin treatment and subcultured by
replanting onto poly-l-lysine-coated plastic dishes at a 1 : 4
ratio before confluence. We obtained a Schwann cell culture
of > 99% purity by these procedures. Cells between pas-
sage 3 and 7 were used in all experiments.
RNA isolation and RT-PCR
Total RNA of sciatic nerves and SCs was extracted using a
Trizol extraction kit (Life Technologies, Rockville, MD,
USA) according to the manufacturer’s protocol. Total
RNA was reverse-transcribed using a ThermoScript
RT-PCR system (Invitrogen, Carlsbad, CA, USA). The pri-
mer pairs used for amplification of ICAM-1 (GenBank
accession number NM-012967) were 5¢-TCCAATGGCTT
CAACCCGTG-3¢ (sense) and 5¢-CTTCTGTGGGATGG
ATGGATACC-3¢ (antisense). The cycling parameters were

94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. The
number of amplification cycles used was that necessary to
achieve exponential amplification where product formation
was proportional to starting cDNA, and was established
empirically [49]. The glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) was used as an internal control and was
detected using the following primers: sense, 5¢-TGATGA
CATCAAGAAGGTGGTGAAG-3¢; antisense, 5¢-TCCTT
GGAGGCCATGTGGGCCAT-3¢. Cycling parameters for
were as described previously [49]. The signal intensities
of RT-PCR products were quantified by calculating the
integrated volume of the band using Molecular Dynamics
densitometer (Scion, Frederick, MD, USA), and data are
expressed as the ratio of ICAM-1 ⁄ GAPDH.
Western blot analysis
Rats were killed at 0, 2, 4, 6, 8, 10, 12, 24 and 48 h after
intraperitoneal injection of LPS (n = 3 per time point). Sci-
atic nerves were removed by cutting the nerve shortly after.
The nerves were excised and snap frozen at )70 ° C until
use. To prepare lysates, frozen nerve samples were minced
with opthalmic scissors in ice. The samples were then homo-
genized in lysis buffer [1% NP-40 (Sigma), 50 mmolÆL
)1
Tris pH 7.5, 5 mmolÆL
)1
EDTA, 1% SDS, 1% sodium
deoxycholate, 1% Triton X-100 (Sigma), 1 mmolÆL
)1
phen-
ylmethanesulfonyl fluoride, 10 lgÆmL

)1
aprotinin and
1 lgÆmL
)1
leupeptin], and clarified by centrifuging at
12 000 g for 20 min in a microcentrifuge at 4 °C. The
protein concentration of the resulting supernatant by the
Bradford assay (Bio-Rad, Hercules, CA, USA), and
the supernatant was divided into aliquots containing 50 lg
of protein.
After appropriate stimulation, cells were washed twice
with ice-cold NaCl ⁄ P
i
and extracted in lysis buffer for
45 min on ice. Equal amounts of protein were subjected to
SDS–PAGE. The separated proteins were transferred to a
polyvinylidine difluoride membrane (Millipore, Bedford,
MA, USA) using a transfer apparatus at 0.35 mA for
2.5 h. The membrane was then blocked with 5% nonfat
milk and incubated with primary antibody against ICAM-1
(anti-mouse, 1 : 500; BD Pharmingen, San Diego, CA,
USA), ERK (anti-rabbit, 1 : 500; Cell Signalling, Danvers,
MA, USA), phosphorylated ERK (anti-rabbit, 1 : 500; Cell
Signal), p38 (anti-rabbit, 1 : 500; Cell Signal), phosphory-
lated p38 (anti-rabbit, 1 : 500; Cell Signal), SAPK ⁄ JNK
(anti-rabbit, 1 : 500; Cell Signal), phosphorylated
SAPK ⁄ JNK (anti-rabbit, 1 : 500; Cell Signal) or b-actin
(anti-mouse, 1 : 2000; Sigma). After incubating with goat
horseradish peroxidase-conjugated secondary antibody
against rabbit or mouse, protein was visualized using an

enhanced chemiluminescence system (Pierce, Rockford, IL,
USA).
After the chemiluminescence was exposed to Kodak
X-OMAT film (Eastman Kodak, Rochester, NY, USA),
the films were scanned using a Molecular Dynamics densit-
ometer. Relative amounts of proteins were quantified by
absorbance analysis. The level was normalized to b-actin, a
domestic loading control.
Cell surface ICAM-1 expression assays
The quantitative expression of ICAM-1 on the surface of
the SC monolayers was determined by modified ELISA in
96-well plates as described previously [50]. In brief, follow-
ing incubation with antagonists and agonists, SCs were
fixed with 3.7% formaldehyde (pH 7.4) containing 0.1 m
l-lysine monohydrochloride and 0.01 m sodium m-perio-
date for 20 min at 4 °C, washed with NaCl ⁄ P
i
, and then
blocked with NaCl ⁄ P
i
containing 1% BSA and 0.1 m
glycine overnight at 4 °C. The fixed monolayer was then
incubated for 1 h at 37 °C with a monoclonal antibody to
ICAM-1 (anti-mouse, 1 : 10 000; BD Pharmingen) in
NaCl ⁄ P
i
containing 1% BSA. After three washes with
NaCl ⁄ P
i
containing 0.1% BSA, the cells were incubated for

1 h at room temperature with horseradish peroxidase-
conjugated goat anti-mouse IgG, washed three more times
with NaCl ⁄ P
i
containing 0.1% BSA, and incubated for
20 min in the dark with 100 lL tetramethyl benzidine solu-
tion. The reaction was stopped by the addition of 50 lLof
1 m H
2
SO
4
, and the absorbance of each well was measured
at 450 nm using a microplate reader. ICAM-1 expression
was calculated relative to the control value.
Immunohistochemistry
Four hours post-injection control and LPS-injected rats
were killed and perfused through the ascending aorta with
saline, followed by 4% paraformaldehyde. After perfusion,
normal and inflamed sciatic nerves were removed and post-
fixed in the same fixative for 3 h, which was then replaced
by 20% sucrose for 2–3 days, then 30% sucrose for
LPS increases ICAM-1 expression in Schwann cells of sciatic nerves A. Shen et al.
4350 FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS
2–3 days. Serial transverse sections (14 lm) were cut
through the tissues. For double labeling, sections were first
blocked with blocking solution, containing 10% normal
goat serum, 3% w ⁄ v BSA, 0.1% Triton X-100 and 0.05%
Tween-20 overnight at 4 °C to avoid non-specific staining.
Then the sections were incubated with antibody specific for
ICAM-1 (1 : 100; BD Pharmingen) and antibody for vari-

ous markers as follows: S100 (Schwann cell marker,
1 : 100; Sigma), NF-200 (neurofilament marker, 1 : 200;
Sigma) or CD31 (endothelial cell marker, 1 : 50; Santa
Cruz Biotechnology, Santa Cruz, CA, USA), overnight at
4 °C. After washing in NaCl ⁄ P
i
three times for 10 min, sec-
ondary antibodies [fluorescein isothiocyanate-labeled goat
anti-mouse, 1 : 100 (Jackson, Bar Harbor, ME, USA) and
tetramethyl rhodamine isothiocyanate-labeled donkey anti-
rabbit, 1 : 100 (Jackson)] were added in the dark and incu-
bated for 2–3 h at 4 °C. Images were captured using a
Leica fluorescence microscope (Wetzlar, Germany).
For immunocytochemistry, the cells were fixed with 4%
formaldehyde for 30 min, then treated with 0.1%
Triton X-100 in NaCl ⁄ P
i
for 5 min, and incubated with
NaCl ⁄ P
i
containing 3% normal goat serum blocking solu-
tion for 1 h. The cells were incubated overnight at 4 °C
with monoclonal mouse antibody against ICAM-1 (1 : 100;
BD Pharmingen) and polyclonal rabbit anti-S100 (1 : 100;
Sigma). After rinsing the cells with NaCl ⁄ P
i
, they were
incubated with fluorescein isothiocyanate-conjugated
anti-mouse (ICAM-1) in blocking solution and tetramethyl
rhodamine isothiocyanate-labeled anti-rabbit IgG (1 : 100;

Jackson) to visualize polyclonal antibody (S100). The cells
were rinsed and mounted onto slides, which were then ana-
lyzed and imaged using a Leica fluorescence microscope.
Statistical analysis
All data were analyzed using stata 7.0 statistical software
(Systat Software Inc., San Jose, CA, USA). The OD of the
immunoreactivity is represented as means ± SEM. One-
way ANOVA followed by Tukey’s post-hoc multiple com-
parison tests were used for statistical analysis. P values
< 0.05 were considered statistically significant. Each exper-
iment consisted of at least three replicates per condition.
Acknowledgements
This work was supported by the National Natural
Scientific Foundation of China (grants 30300099 and
30770488), the Natural Scientific Foundation of Jiangsu
Province (grants BK2003035 and BK2006547), the Col-
lege and University Natural Scientific Research Pro-
gramme of Jiangsu Province (grants 03KJB180109 and
04KJB320114), the Technology Guidance Plan for
Social Development of Jiangsu Province (grant
BS2004526), the Health Project of Jiangsu Province
(grant H200632), and the Foundation of Talented Per-
sons at the Summit of Six Fields of Jiang Su Province.
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Supporting information
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Fig. S1. LPS induces the expression of ICAM-1
mRNA in cultured Schwann cells.
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A. Shen et al. LPS increases ICAM-1 expression in Schwann cells of sciatic nerves
FEBS Journal 275 (2008) 4343–4353 ª 2008 The Authors Journal compilation ª 2008 FEBS 4353