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: 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 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. 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