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

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)

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

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

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

(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

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

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

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

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

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

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;

adhesion ligand of LFA-1, is homologous to the neural
cell adhesion molecule NCAM. Nature 331, 624–627.
4 Staunton DE, Marlin SD, Stratowa C, Dustin ML &
Springer TA (1988) Primary structure of ICAM-1
demonstrates interaction between members of the
immunoglobulin and integrin supergene families. Cell
52, 925–933.
5 Chen CC & Manning AM (1995) Transcriptional regula-
tion of endothelial cell adhesion molecules: a dominant
role for NF-kappa B. Agents Actions Suppl 47, 135–141.
6 De Launoit Y, Audette M, Pelczar H, Plaza S & Baert
JL (1998) The transcription of the intercellular adhesion
molecule-1 is regulated by Ets transcription factors.
Oncogene 16, 2065–2073.
7 Hou J, Baichwal V & Cao Z (1994) Regulatory
elements and transcription factors controlling basal and
cytokine-induced expression of the gene encoding inter-
cellular adhesion molecule 1. Proc Natl Acad Sci USA
91, 11641–11645.
8 Roebuck KA, Rahman A, Lakshminarayanan V,
Janakidevi K & Malik AB (1995) H
2
O
2
and tumor
necrosis factor-alpha activate intercellular adhesion
molecule 1 (ICAM-1) gene transcription through
distinct cis-regulatory elements within the ICAM-1
promoter. J Biol Chem 270, 18966–18974.
9 Wahl SM, Feldman GM & McCarthy JB (1996) Regu-

109.
17 Sobel RA, Mitchell ME & Fondren G (1990) Intercellu-
lar adhesion molecule-1 (ICAM-1) in cellular immune
reactions in the human central nervous system. Am J
Pathol 136, 1309–1316.
18 Constantin G, Piccio L, Bussini S, Pizzuti A, Scarpini
E, Baron P, Conti G, Pizzul S & Scarlato G (1999)
Induction of adhesion molecules on human schwann
cells by proinflammatory cytokines, an immunofluores-
cence study. J Neurol Sci 170, 124–130.
19 Vougioukas VI, Roeske S & Bruck W (2000) Involve-
ment of intercellular adhesion molecule-1 in myelin
recognition by macrophages. Acta Neuropathol 99,
673–679.
20 Cheng C, Qin Y, Shao X, Wang H, Gao Y, Cheng M
& Shen A (2007) Induction of TNF-alpha by LPS in
Schwann cell is regulated by MAPK activation signals.
Cell Mol Neurobiol 27, 909–921.
21 Orlikowski D, Chazaud B, Plonquet A, Poron F, Shar-
shar T, Maison P, Raphael JC, Gherardi RK & Cre-
ange A (2003) Monocyte chemoattractant protein 1 and
chemokine receptor CCR2 productions in Guillain–
Barre
´
syndrome and experimental autoimmune neuritis.
J Neuroimmunol 134, 118–127.
22 Jobin C, Hellerbrand C, Licato LL, Brenner DA & Sar-
tor RB (1998) Mediation by NF-kappa B of cytokine
induced expression of intercellular adhesion molecule 1
(ICAM-1) in an intestinal epithelial cell line, a process

LFA-1 and Mac-1 with intercellular adhesion molecule-
1 in facilitating adherence and transendothelial migra-
tion of human neutrophils in vitro. J Clin Invest 83,
2008–2017.
29 Springer TA (1990) Adhesion receptors of the immune
system. Nature 346, 425–434.
30 Wawryk SO, Novotny JR, Wicks IP, Wilkinson D,
Maher D, Salvaris E, Welch K, Fecondo J & Boyd AW
(1989) The role of the LFA-1 ⁄ ICAM-1 interaction in
human leukocyte homing and adhesion. Immunol Rev
108, 135–161.
30a Patil C, Rossa C Jr & Kirkwood KL (2006) Action-
bacillus actinomycetemcomitans lipopolysaccharide
induces interleukin-6 expression through multiple
mitogen-activated protein kinase pathways in period-
ontal ligament fibroblasts. Oral Microbiol Immunol 21,
392–398.
30b Thorley AJ, Ford PA, Giembycz MA, Goldstraw P,
Young A & Tetley TD (2007) Differential regulation of
cytokine release and leukocyte migration by lipopoly-
saccharide-stimulated primary human lung alveolar type
II epithelial cells and macrophages. J Immunol 178,
463–473.
31 Gold R, Toyka KV & Hartung HP (1995) Synergistic
effect of IFN-gamma and TNF-alpha on expression of
immune molecules and antigen presentation by Schw-
ann cells. Cell Immunol 165, 65–70.
32 Kingston AE, Bergsteinsdottir K, Jessen KR, Van der
Meide PH, Colston MJ & Mirsky R (1989) Schwann
cells co-cultured with stimulated T cells and antigen

molecule-1 on human eosinophilic leukaemia EoL-1
cells is mediated by the activation of nuclear factor-jB
pathway. Clin Exp Allergy 33, 241–248.
40 Xia YF, Liu LP, Zhong CP & Geng JG (2001) NF-kap-
paB activation for constitutive expression of VCAM-1
and ICAM-1 on B lymphocytes and plasma cells.
Biochem Biophys Res Commun 289, 851–856.
41 Shibuya T, Takei Y, Hirose M, Ikejima K, Enomoto N,
Maruyama A & Sato N (2002) A double-strand
decoy DNA oligomer for NF-kappaB inhibits
TNFalpha-induced ICAM-1 expression in sinusoidal
endothelial cells. Biochem Biophys Res Commun 298,
10–16.
42 Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T,
Karin M & Davis RJ (1994) JNK1: a protein kinase
stimulated by UV light and Ha-Ras that binds and
phosphorylates the c-Jun activation domain. Cell 76,
1025–1037.
43 Gupta S, Campbell D, Derijard B & Davis RJ (1995)
Transcription factor ATF2 regulation by the JNK
signal transduction pathway. Science 267, 389–393.
44 Voraberger G, Schafer R & Stratowa C (1991) Cloning
of the human gene for intercellular adhesion molecule 1
and analysis of its 5’-regulatory region. Induction
by cytokines and phorbol ester. J Immunol 147,
2777–2786.
45 Kobuchi H, Roy S, Sen CK, Nguyen HG & Packer L
(1999) Quercetin inhibits inducible ICAM-1 expression
in human endothelial cells through the JNK pathway.
Am J Physiol 277, C403–C411.

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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