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

leads to an increase in ICAM-1 expression in Schwanncells of sciatic nerves Aiguo Shen1,*, Junling Yang2,*, Yangyang Gu3, Dan Zhou4, Linlin Sun2, Yongwei Qin2, Jianping Chen2, Ping Wang2

leads to an increase in ICAM-1 expression in Schwann

cells of sciatic nerves

Aiguo Shen1,*, Junling Yang2,*, Yangyang Gu3, Dan Zhou4, Linlin Sun2, Yongwei Qin2,

Jianping Chen2, Ping Wang2, Feng Xiao2, Li Zhang2and Chun Cheng1,2

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

b2-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: cheng_chun@yahoo.com.cn

*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 staindouble-label-ing 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.

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-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

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)1LPS appeared to induce ICAM-1 protein

to a lesser extent than treatment with 10 lgÆmL)1LPS; 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.

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

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

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.

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)1LPS 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)1LPS 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.

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 ± 1C 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.

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 l M ) or SP600125 (10, 20, 40 l M ) for 40 min, and then stimulated with 1 lgÆmL)1LPS 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 l M ) or SP600125 (20 l M ) 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.

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 l M ) or

SP600125 (20 l M ) for 40 min and then

stim-ulated with 1 lgÆmL)1LPS 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.

(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

94C 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 4C 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⁄ Pi 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 4C, washed with NaCl ⁄ Pi, and then blocked with NaCl⁄ Pi containing 1% BSA and 0.1 m glycine overnight at 4C The fixed monolayer was then incubated for 1 h at 37C with a monoclonal antibody to ICAM-1 (anti-mouse, 1 : 10 000; BD Pharmingen) in NaCl⁄ Pi containing 1% BSA After three washes with NaCl⁄ Picontaining 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⁄ Pi 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 lL of

1 m H2SO4, 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

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 4C 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

4C After washing in NaCl ⁄ Pithree 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 4C 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⁄ Pi for 5 min, and incubated with

NaCl⁄ Pi containing 3% normal goat serum blocking

solu-tion for 1 h The cells were incubated overnight at 4C

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⁄ Pi, 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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