2 pathways in murine peritoneal macrophages

Life Sciences 81 (2007) 362 – 371 www.elsevier.com/locate/lifescie

Dextran sulfate sodium enhances interleukin-1β release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages Ki Han Kwon, Hajime Ohigashi, Akira Murakami ⁎ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received 5 February 2007; accepted 21 May 2007

Abstract Interleukin (IL)-1β is a pro-inflammatory cytokine that has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD), however, the molecular mechanisms underlying the production of IL-1β in IBD are not fully understood. We investigated dextran sulfate sodium (DSS)-induced IL-1β production and caspase-1 activities in murine peritoneal macrophages (pM/). Further, the activation status of p38 mitogenactivated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun NH2-terminal kinase (JNK1/2), as well as their upstream target kinases, were examined by Western blotting. In addition, mRNA expression was assessed by RT-PCR and CXC chemokine ligand 16 (CXCL16) protein was detected by immunocytochemistry. DSS-treated pM/ released IL-1β protein in a time-dependent manner without affecting mRNA levels during 3–24 h, and caspase-1 activity peaked at 5 min (29-fold). IL-1β release and caspase-1 activity induced by DSS were significantly inhibited by a MAPK kinase 1/2 inhibitor, a p38 MAPK inhibitor, and NAC, however, not by JNK1/2 or a protein kinase C inhibitor. In addition, DSS strikingly induced the phosphorylation of p38 MAPK and ERK1/2 within 2 and 10 min, respectively. DSS also induced intracellular generation of reactive oxygen species (ROS). Pre-treatment with anti-CXCL16 for 24 h, but not anti-scavenger receptor-A, antiCD36, or anti-CD68 antibodies, significantly suppressed DSS-induced IL-1β production. Our results suggest that DSS triggers the release of IL-1β protein from murine pM/ at a post-translational level through binding with CXCL16, ROS generation, and resultant activation of both p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation. © 2007 Elsevier Inc. All rights reserved. Keywords: Interleukin-1β (IL-1β); Dextran sulfate sodium (DSS); p38 mitogen-activated protein kinase (MAPK); Extracellular signal-regulated kinase (ERK); Inflammatory bowel disease (IBD)

Introduction Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, relapsing, and remitting inflammatory condition of unknown origin that afflicts individuals of both sexes throughout life (Podolsky, 1991; Strober et al., 1998). The disease is characterized by a pronounced infiltration of neutrophils into colonic lesions, accompanied by epithelial cell necrosis and ulceration. Although infection, environmental factors, heredity, and immunologic abnormalities have been proposed as causes (Sartor, 1997; Shanahan, 2001), the precise pathogenesis of IBD is poorly understood. Several models of experimental colitis have been ⁎ Corresponding author. Tel.: +81 75 753 6282; fax: +81 75 753 6284. E-mail address: [email protected] (A. Murakami). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.05.022

developed to investigate the molecular and cellular mechanisms of inflammation and immunological disorders (Elson et al., 1995), with a dextran sulfate sodium (DSS)-induced colitis animal model the most widely used in studies of IBD. That DSS model exhibits many symptoms similar to those seen in human UC, i.e., diarrhea, bloody feces, body weight loss, mucosal ulceration, and shortening of the colorectum (Okayasu et al., 1990), therefore, it is considered to be reliable for studying the pathogenesis of UC and testing drugs for treatment (Dieleman et al., 1994; Elson et al., 1995; Murthy et al., 1993; Okayasu et al., 1990). However, the precise mechanism of colitis induction by DSS remains unclear. Increased levels of several pro-inflammatory cytokines during active states of IBD have been implicated (Brynskov et al., 1992). For example, tissue levels of interleukin (IL)-1β, IL-6, IL-8, and granulocyte macrophage-colony stimulating

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factor are elevated in active UC and CD, and correlate with the severity of inflammation (Ishiguro, 1999; Sartor, 1994). These cytokines may play some important roles in the induction and amplification of inflammatory responses, as well as the healing of intestinal tissue injury. Dieleman et al. have reported that chronic experimental colitis induced by DSS is characterized by Th1 and Th2 cytokines (Dieleman et al., 1998). A number of studies have strongly suggested that IL-1β is activated by IL-1β-converting enzyme (ICE, also known as caspase-1) at the early stage of the cascade of events, resulting in intestinal inflammation characteristic of experimental colitis (Cominelli and Dinarello, 1989). IL-1β is a pro-inflammatory cytokine that is produced primarily by activated monocytes and macrophages, as well as other cell types, including fibroblasts, smooth muscle cells, and endothelial cells (March et al., 1985). The cytokine possesses a wide spectrum of biological activities and has been demonstrated to be involved in the regulation of immune responses, as well as the pathogenesis of several acute and chronic inflammatory diseases (Dinarello, 1991). Enhanced production of IL-1β has been detected at both the mRNA and protein levels in human IBD (Cappello et al., 1992), and in DSSinduced colitis (Savendahl et al., 1997; Tsune et al., 2003). In our previous study, we found that IL-1β levels were profoundly increased in both colonic mucosa and murine peritoneal macrophages (pM/) in DSS-induced colitis (Kwon et al., 2005). Therefore, pM/-derived IL-1β may be closely and critically associated with the pathogenesis of the disease. Mitogen-activated protein kinases (MAPKs) are conserved among all eukaryotes and participate in multiple cellular processes (Widmann et al., 1999). Three groups of MAPKs have been identified in mammalian cells, extracellular signalregulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs) or stress-activated protein kinases, and p38 MAPKs (Chang and Karin, 2001; Robinson and Cobb, 1997), each of which are activated by specific upstream MAPK kinases, leading to phosphorylation of both their tyrosine and threonine residues (Kunnimalaiyaan and Chen, 2006). p38 MAPK and JNK are activated by inflammatory stimuli and environmental stress, while ERK is stimulated mainly by growth factors and tumor promoters (Robinson and Cobb, 1997; Takenobu et al., 2003). Each MAPK cascade cooperates in the orchestration of inflammatory responses, while extensive cross-talk with other inflammatory pathways, such as nuclear factor-κB and Janus kinase/STAT signaling, has been described (Kyriakis and Avruch, 2001; Tibbles and Woodgett, 1999). IL-1β is regulated by p38 MAPK and ERKs in human smooth muscle cells (Jung et al., 2001). Most recently, aggregated ursolic acid, a natural triterpenoid, induces IL-1β release from murine peritoneal macrophages via activation of p38 MAPK and ERK1/2 pathways (Ikeda et al., 2007). However, the molecular mechanism by which DSS induces IL-1β production in pM/ remains to be elucidated. In the present study, we investigated DSS-induced IL-1β production by examining DSS-induced caspase-1 activity, as well as the status of MAPKs and reactive oxygen species (ROS) generation, in pM/. Our results highlighted CXC chemokine ligand 16 (CXCL16) as a possible receptor for DSS.

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Materials and methods Mice Female specific pathogen-free ICR mice were purchased at 6 weeks of age from Japan SLC (Shizuoka, Japan) and quarantined for 1 week. They were housed 5 per cage and given fresh tap water ad libitum and commercial rodent chow (Oriental Yeast, Kyoto, Japan), which was freshly changed twice a week, and handled according to Guidelines for the Regulation of Animals, as provided by the Experimentation Committee of Kyoto University. The mice were maintained in a controlled environment of 24 ± 2 °C with a relative humidity of 60 ± 5% and a 12-hour light/dark cycle (lights on from 06:00 to 18:00). Chemicals Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). DSS, with a molecular weight of 40,000, was from ICN Biomedicals (Aurora, OH). Actinomycin D and cycloheximide were obtained from Funakoshi (Tokyo, Japan). PD98059, SB203580, SP600125, Negative control, GF109293X and caspase-1 inhibitor came from Calbiochem (La Jolla, CA). Antibodies were purchased from the following sources: rat anti-CXCL16 was from TECHNE® Inc. (Minneapolis, MN); rabbit anti-phospho-ERK1/2, rabbit antiERK1/2, rabbit anti-phospho-p38, rabbit anti-p38, rabbit antiactive-JNK1/2, rabbit anti-JNK1/2, rabbit anti-phospho-MAPK kinase (MEK)1/2, rabbit anti-MEK1/2, rabbit anti-phosphoMKK3/6, rabbit anti-MKK3, and anti-rabbit antibody horseradish peroxidase-linked immunoglobulin G (IgG) antibodies were from Cell Signaling Technology Inc. (Beverly, MA); goat anti-β-actin, goat anti-scavenger receptor (SR)-A, rabbit anti-CD36, and rabbit anti-CD68 antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); and horseradish peroxidase-conjugated antirabbit IgG, anti-rat IgG, and anti-goat IgG from Dako (Glostrup, Denmark). Oligonucleotide primers were synthesized and purified by Proligo (Kyoto, Japan). A Qiashredder™ kit and RNeasy Mini Kit® came from Qiagen (Hilden, Germany), and an RNA PCR Kit (AMV) Ver. 2.1 from TaKaRa Bio. (Shiga, Japan). A caspase-1 colorimetric assay kit was purchased from R&D Systems Inc. (Minneapolis, MN) and a mouse enzyme-linked immunosorbent assay (ELISA) kit for IL-1β measurement from Endogen Inc. (Woburn, MA). All other chemicals were from Wako Pure Chemicals (Osaka, Japan), unless otherwise specified. Preparation of pM/ For the preparation of macrophage monolayers, female ICR mice at 7 weeks of age were killed by cervical dislocation, after which each peritoneal cavity was washed twice with 5 ml of DMEM medium containing 10 U/ml heparin and 0.5 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethenesulfonic acid supplemented with 5% FBS. Peritoneal exudate cells were harvested and centrifuged at 3000 ×g for 5 min at 4 °C. pM/ were obtained by washing the plate twice with Hank's balanced buffer solution (Kwon et al., 2002).

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Cell viability Cell viability was measured using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) test (Murakami et al., 2000). The value for cell viability of the positive control cells, which were treated with 0.5% dimethylsulfoxide (DMSO) and DSS, was standardized as 100%. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from the pM/ (1 × 107 cells) using the Qiashredder™ kit and RNeasy Mini Kit®. Steadystate messenger RNA (mRNA) levels of IL-1β and CXCL16 were detected by RT-PCR, with a hypoxanthine guanine phosphoribosyltransferase (HPRT) transcript used as the internal control. The primer sequences used for PCR amplification and PCR product sizes are listed in Table 1. cDNA was synthesized using 1 μg of total RNA and an RNA PCR Kit (AMV). PCR amplification was then performed using a PTC100™ thermal cycler (MJ Research Inc., Watertown, MA). The PCR conditions consisted of 35 cycles, with 30 s of denaturation at 94 °C, 30 s of annealing at 60 °C, and 30 s of primer extension at 72 °C. Amplified cDNA was separated by electrophoresis on 2% agarose gels and stained with SYBR Gold® (Molecular Probes, Eugene, OR). DNA band sizes were confirmed using a Gene Ruler 100-bp DNA ladder (Invitrogen, Grand Island, NY). Image analyses were performed using NIH image software. The ratios of expression levels of each gene were determined by dividing the band intensity of the product of interest by that of the corresponding HPRT band. Western blotting For Western blot analysis, 1 × 107 cells were lysed in lysis buffer [protease inhibitor, phosphatase inhibitor (TaKaRa Bio., Shiga, Japan), 10 mM Tris, pH 7.4, 1% sodium dodecyl sulfate (SDS), 1 mM sodium vanadate (V)] and the lysates were boiled for 5 min. Denatured proteins (30 μg) were separated using SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel and then transferred onto Immobilon-P membranes (Millipore, MA). After blocking overnight at 4 °C in Block Ace® (Dainippon Pharmaceutical, Osaka, Japan), the membranes were first incubated with each antibody at dilutions of 1:1000 (goat anti-β-actin, rabbit anti-phospho-ERK1/2, rabbit

Table 1 List of primer sequences for RT-PCR Gene

Primer

Sequence (5′ to 3′)

Product size (bp)

IL-1β

Forward Reverse Forward Reverse Forward Reverse

ATggCAATgTTCCTgAACTCAACT CAggACAggTATAgATTCTTTCCTTT ACTACACgAggTTCCAgCTCC CTTTgtCCgAggACAgTgATC gTAATgATCAgTCAACgggggAC CCAgCAAgCTTgCATTAACCA

586

CXCL16 HPRT

304 196

anti-ERK1/2, rabbit anti-phospho-p38, rabbit anti-p38, rabbit anti-active JNK1/2, rabbit anti-JNK1/2, rabbit anti-phosphoMEK1/2, rabbit anti-MEK1/2, rabbit anti-phospho-MKK3/6, rabbit anti-MKK3, and rat anti-CXCL16 antibodies). The second incubation was performed with horseradish peroxidase-conjugated secondary anti-rabbit IgG, anti-rat IgG, or anti-goat IgG antibody (1:1000 dilution each). The blots were developed using an ECL Advance Western blotting detection reagent (Amersham Biosciences, Buckinghamshire, UK). Image analyses were performed using NIH image software. The ratios of expression levels of each protein were determined by dividing the band intensity of the product of interest by that of the corresponding β-actin band. Time course of IL-1β production Cells from peritoneal exudates were seeded onto a 96-well plate at a density of 1 × 106 cells/well in DMEM medium with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml), and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, pM/ were treated with the vehicle (PBS) or DSS (10 μg/ml) for 1, 3, 6, 12, or 24 h. The negative control cells were treated only with PBS. IL-1β protein was examined by ELISA, as described below. Effects of caspase-1 inhibitor Cells from peritoneal exudates were seeded onto a 96-well plate at a density of 1 × 106 cells/well in DMEM medium with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml), and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, pM/ were treated with the vehicle (0.5% DMSO, v/v) or various concentrations of caspase-1 inhibitor (0.001–100 μM) dissolved in DMSO. The negative control cells were treated only with 0.5% DMSO. After incubating at 37 °C for 30 min, pM/ were then treated with DSS (10 μg/ml) for 24 h for ELISA, as described below. Effects of specific inhibitors Cells from peritoneal exudates were seeded onto a 96-well plate (IL-1β) or a 35-mm dish (caspase-1) at a density of 1 × 106 cells/well or 2 × 106 cells/dish, respectively, in DMEM medium with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml), and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, pM/ were treated with the vehicle (0.5% DMSO, v/v) alone, or 50 μM of PD98059 (PD), 50 μM of SB203580 (SB), 10 μM of SP600125 (SP), 50 μM of Negative control (NEG), 10 μM of GF109293X (GF), or 20 mM of NAC dissolved in DMSO. The negative control cells were treated only with 0.5% DMSO. After incubating at 37 °C for 30 min, pM/ were then treated with DSS (10 μg/ml) for 24 h (IL-1β) or 5 min (caspase-1). IL-1β production and caspase-1 activity were examined by ELISA and a colorimetric assay kit, respectively, as described below.

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ELISA IL-1β concentrations in the media supernatants (50 μl each) without dilution were determined using an ELISA kit, according to the protocol of the manufacturer.

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Tokyo, Japan) (original magnification 1:1000) and pictures taken with a digital camera system. As a negative control, the primary antibody was replaced with rabbit normal IgG (1:1000), which corresponded to the species of the primary antibody. Protein determination

Caspase-1 activity Caspase-1 activity was determined using a Caspase-1 colorimetric assay kit (R&D Systems Inc., Minneapolis, MN). Triplicate samples of untreated and DSS-treated pM/ (2 × 106 cells) were washed in cold phosphate-buffered saline (PBS) at specified times, then re-suspended in 50 μl of cold lysis buffer and incubated on ice for 10 min. The cell lysates were pelleted, followed by transfer of the supernatants to microcentrifuge tubes. Fifty microliters of 2× reaction buffer with 1 M DTT and 5 μl of 4 mM WEHD-pNA substrate were added to each well, followed by a 1-hour incubation in a water bath at 37 °C. A control reaction of treated cells without WEHD-pNA was included. The optical density for each specimen was determined at 405 nm using an MPR-A4i microplate reader (TOSHO, Japan). The results are expressed as fold increase, with caspase-1 activity seen in nonstimulated cells (the control) standardized as 1-fold.

Total protein concentrations in pM/ were determined using a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA) according to the protocol of the manufacturer (dilution factor= 50), with γ-globulin employed as the standard. Statistical analysis Each experiment was performed at least 3 times and the data are shown as mean ± standard deviation (SD). Statistically significant differences between groups in each assay were determined using a non-parametric post-hoc test (Kruskal–Wallis test with post-hoc test). Results DSS promotes IL-1β protein release at post-transcriptional level

Determination of intracellular ROS generation pM/ (1 × 106 cells) were plated directly onto one-chamber slides (GLASS/PS, 9 × 9, IWAKI, Osaka, Japan). After washing, pM/ were suspended in 200 μl of PBS and then treated with 10 μM of 2,7′-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR) for 15 min. Thereafter, the medium was discarded and the pM/ was washed twice with PBS, followed by exposure to PBS or DSS at a concentration of 10 μg/ml for 5 or 15 min. The negative control cells were treated only with PBS. DSS-induced intracellular ROS was detected by fluorescence microscopic (OLYMPUS, Tokyo, Japan) examinations (original magnification 1:200).

IL-1β mRNA is expressed in a constitutive manner in nontreated pM/ and the levels did not change after treatment with

Immunocytochemistry Immunocytochemical staining was performed using the procedure of Itota et al. (Itota et al., 2004), with some modifications. Briefly, pM/ (1 × 106 cells) were plated directly onto onechamber slides. After 24 h, control and DSS-treated pM/ were washed with cold PBS twice, then fixed with Zamboni's fixative [10% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4)] for 7 h at 4 °C. After extensive washing with PBS, the cells were permeabilized by incubation with 0.1% Triton X-100 in 0.1 M PBS, then washed 3 times in PBS and blocked with Block Ace® for 12 h at 4 °C. The cells were then incubated with a specific primary antibody (rat anti-CXCL16, 1:100 dilution) for 12 h at 4 °C. After extensive washing with PBS, the cells were stained with fluorescein isothiocyanate-conjugated secondary antibody (anti-rat IgG; Dako, Glostrup, Denmark, 1:100 dilution) and propidium iodide (PI; Wako Pure Chemicals, Osaka, Japan, 1:2000 dilution). Stained cells were viewed using a confocal laser scanning microscope (Fluoro-View; Olympus Optical Co., Ltd.,

Fig. 1. Levels of IL-1β mRNA expression (A) and protein production (B) in DSSstimulated pM/. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 35-mm dish (mRNA) or a 96-well plate (protein) at a density of 1 × 107 cells/dish or 1 × 106 cells/well, respectively, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (PBS) or DSS (10 μg/ml) for 1, 3, 6, 12, or 24 h. The negative control cells were treated only with PBS. IL-1β mRNA and its protein were examined by RT-PCR (A) and ELISA (B), respectively, as described in the Materials and methods section. Each experiment was performed at least 3 times and the data are shown as mean ± SD. HPRT served as the internal control. Statistical analysis was performed using a non-parametric post-hoc test (Kruskal–Wallis test): ⁎P b 0.05, ⁎⁎P b 0.01 versus Control. ―○― Control, ―●― DSS (10 μg/ml).

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Effects of kinase inhibitors and antioxidant on caspase-1 activity and IL-1β production

Fig. 2. Involvement of caspase-1 activation in DSS-induced IL-1β production. (A) Effects of caspase-1 inhibitor on DSS-induced IL-1β production. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 96-well plate at a density of 1 × 106 cells/well, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (0.5% DMSO, v/v) or various concentrations of caspase-1 inhibitor (0.001–100 μM). The negative control cells were treated only with 0.5% DMSO. After incubating at 37 °C for 30 min, the pM/ were then treated with DSS (10 μg/ml) for 24 h. The concentration of IL-1β in the supernatant of the medium (50 μl) without dilution was determined using a mouse IL-1β ELISA kit, as described in the Materials and methods section. Cell viability was determined with an MTT test. Each value is shown as mean ± SD of 3 replicated experiments. Statistical analysis was performed using a non-parametric post-hoc test (Kruskal–Wallis test): ⁎P b 0.01 versus Control, ⁎⁎P b 0.05, ⁎⁎⁎P b 0.01 versus DSS. (B) Time course for caspase-1 activity in DSS-stimulated pM/. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 35-mm dish at a density of 2 × 106 cells/dish, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (PBS) or DSS (10 μg/ml) for 1, 2, 3, 4, 5, 10, 30, or 60 min. The negative control cells were treated only with PBS. The activity of caspase-1 in the pM/ was determined by the colorimetric assay kit, as described in the Materials and methods section. Cell viability was determined with an MTT test. Each value is shown as mean ± SD of 3 replicated experiments. Statistical analysis was performed using a non-parametric post-hoc test (Kruskal–Wallis test): ⁎P b 0.05, ⁎⁎P b 0.01 versus Control.

To explore the molecular mechanisms underlying DSSinduced caspase-1 activation and IL-1β protein release, the effects of several protein kinase inhibitors were examined. The concentration of each inhibitor was determined so that cell viability was maintained at 90% or more when treated with maximum concentrations tested (data not shown). PD98059 (50 μM, an inhibitor of MEK1/2), SB203580 (50 μM, an inhibitor of p38 MAPK), and N-acetyl-L-cysteine (NAC, an antioxidant) markedly reduced the DSS-induced caspase-1 activity by 50%, 57%, and 56%, respectively, and IL-1β secretions by 48%, 59%, and 58%, respectively (P b 0.05, each) (Fig. 3A and B), whereas SP600125 (10 μM, JNK1/2 inhibitor), the negative analog of MAPK inhibitors (50 μM) and GF109293X [10 μM, protein kinase C (PKC) inhibitor] did not show any suppression. Further, pre-treatments with actinomycin D and cycloheximide did not affect the activation of caspase-1 or IL-1β protein release (data not shown). DSS triggers phosphorylation of ERK1/2 and p38 MAPK To determine whether DSS triggers the activation of the MAPKs pathways to induce IL-1β release, pM/ were treated with DSS for 30 min, and the phosphorylation of MEK1/2,

10 μg/ml of DSS for 1 to 24 h (Fig. 1A). On the other hand, the levels of IL-1β protein in DSS-treated pM/ were timedependently increased in the media (24-fold at 24 h, P b 0.01), as compared with the vehicle-treated cells (Fig. 1B). Involvement of caspase-1 activation in DSS-induced IL-1β production We then investigated the effects of a caspase-1 inhibitor on DSS-induced IL-1β production by ELISA. As shown in Fig. 2A, IL-1β protein was abundant in the media after a 24-hour treatment with DSS (22-fold, P b 0.01). When pM/ were pre-incubated with a caspase-1 inhibitor (1–100 μM) for 30 min, IL-1β was significantly decreased by 53% to 81% (P b 0.05 and P b 0.01, respectively). DSS treatment led to a transient increase in caspase1 activity, which peaked at 5 min (29-fold, P b 0.01) and then diminished to the basal level at 60 min (Fig. 2B). Cytotoxicity was not seen in any of the experiments (data not shown).

Fig. 3. Effects of kinase inhibitors and an antioxidant on caspase-1 activity (A) and IL-1β production (B). Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 96-well plate at a density of 1 × 106 cells/ well, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (0.5% DMSO, v/v) or 50 μM of PD98059 (PD), 50 μM of SB203580 (SB), 10 μM of SP600125 (SP), 50 μM of negative control (NEG), 10 μM of GF109293X (GF), or 20 mM of NAC. The negative control cells were treated only with 0.5% DMSO. After incubating at 37 °C for 30 min, the pM/ were then treated with DSS (10 μg/ml) for 5 min (caspase-1) or 24 h (IL-1β). IL-1β production (A) and caspase-1 activity (B) were examined by ELISA and a colorimetric assay kit, respectively, as described in the Materials and methods section. Cell viability was determined with an MTT test. Each value is shown as mean ± SD of 3 replicated experiments. Statistical analysis was performed using a non-parametric post-hoc test (Kruskal–Wallis test): ⁎P b 0.01 versus Control, ⁎⁎P b 0.05 versus DSS.

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Fig. 4. DSS activates both p38 MAPK and ERK1/2 pathways. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 35mm dish at a density of 1 × 107 cells/dish, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (PBS) or DSS (10 μg/ml) for 2, 5, 10, or 30 min. The negative control cells were treated only with PBS. The phosphorylation of MEK1/2, ERK1/2, MKK3/6, p38 MAPK, and JNK1/2 was analyzed by Western blotting, as described in the Materials and methods section. The experiments were repeated 3 times independently, with 1 representative result shown. β-actin served as the internal control.

ERK1/2, MKK3/6, p38 MAPK, and JNK1/2 was analyzed by Western blot. DSS strikingly induced both MEK1/2 and ERK1/2 activation within 10 min, and that of both MKK3/6 and p38 MAPK within 2 min, as compared with non-treated cells (Fig. 4). Phosphorylation of JNK1/2 was not observed with any of the test conditions. The expression level of each total protein kinase remained constant.

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Fig. 5. DSS induces p38 MAPK activation via ROS generation. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 35mm dish at a density of 1 × 107 cells/dish, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ was pretreated with the vehicle (0.5% DMSO, v/v) or NAC (20 mM) for 30 min, followed by exposure to DSS (10 μg/ml) or H2O2 (1 mM) for 10 min, and the phosphorylation of MEK1/2, ERK1/2, MKK3/6, p38 MAPK, and JNK1/2 was analyzed by Western blotting, as described in the Materials and methods section. The experiments were repeated 3 times independently, with 1 representative result shown. β-actin served as the internal control.

and 15 min using DCFH-DA, a fluorescence probe. The rate of peroxide-positive cells was increased in a time-dependent manner (25% and 68% at 5 and 15 min, P b 0.05 and P b 0.01, respectively) (Fig. 6).

DSS induces p38 MAPK activation, but not that of ERK1/2 and JNK1/2, Via ROS generation Oxidative stress has been demonstrated to induce MAPKs activation in many cell types, therefore, we pre-treated pM/ with the vehicle or NAC for 30 min, followed by exposure to DSS (10 μg/ml) or H2O2 (1 mM) for 10 min, after which the phosphorylation of MAPKKs and MAPKs was studied. Pretreatment with NAC slightly increased DSS-induced MEK1/2 and ERK1/2 activation, as compared with the DSS-treated cells (Fig. 5), whereas NAC notably reduced DSS-induced MKK3/6 and p38 MAPK activities by 48% and 52%, respectively (P b 0.05, each). Further, treatment with exogenous H2O2 (1 mM) for 10 min increased MEK1/2, ERK1/2, MKK3/6, and p38 MAPK activation, while NAC showed no marked suppression. JNK1/2 was not phosphorylated under any of the test conditions. Subsequently, we investigated the time course of ROS generation in pM/ treated with DSS (10 μg/ml) for 5

Fig. 6. DSS induces ROS generation. Cells from peritoneal exudates from nontreated female ICR mice were seeded onto one-chamber slides at a density of 1 × 106 cells/chamber, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (PBS) or DSS (10 μg/ml) for 5 or 15 min. The negative control cells were treated only with PBS. ROS generation was determined using a fluorescence probe, DCFH-DA, as described in the Materials and methods section. The experiments were repeated 3 times independently, with 1 representative result shown. Panel A, Negative control; Panel B, Blank; Panel C, DSS (at 5 min); Panel D, DSS (at 15 min). Approximately 45 cells are presented in a single panel. Original magnification: panels A, B, C, D × 200.

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Involvement of CXCL16 in DSS-induced IL-1β production CXCL16 is a transmembrane protein with an extracellular chemokine domain on a mucin-like stalk that functions as a receptor for oxidized low-density lipoprotein (OxLDL) or DSS in human monocytic THP-1 cells (Minami et al., 2001). The mRNA and protein expressions of CXCL16 in pM/ with or without DSS treatment were examined by RT-PCR, Western blot, and immunocytochemistry methods. Both were found to be expressed in a constitutive manner and their levels were not changed by treatment with DSS for 24 h (Fig. 7A and B). In parallel, CXCL16 protein was located in both the cellular membrane and cytosol in non-treated pM/ (Fig. 7C), and the levels nor theirs localization were not changed by DSS treatment (data not shown). The effects of the anti-mCXCL16 antibody on DSS-induced IL-1β production were evaluated to determine the relevance of this chemokine on that production. When pM/ were preincubated for 24 h with anti-mCXCL16 antibody (2.5 μg/ml),

the IL-1β level was significantly reduced by 74% (P b 0.05), while the anti-CD36 antibody tended to decrease IL-1β production. Non-specific IgG and other scavenger receptor antibodies did not have an effect on IL-1β production (Fig. 7D). Discussion Various pro-inflammatory cytokines have been implicated in the pathogenesis of IBD, including UC and CD (Balding et al., 2004; Stokkers and Hommes, 2004). In particular, IL-1β may play a pivotal role in the onset of experimental colitis and human IBD (Balding et al., 2004; Ludwiczek et al., 2004), however, the molecular mechanisms underlying its production remain unclear. The present results showed for the first time that DSS caused a post-translational enhancement of IL-1β secretion from murine pM/, presumably through binding with CXCL16, ROS generation, the resultant activation of both the p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation (Fig. 8). DSS induced the activation of p38 MAPK within 2 min (Fig. 4) and intracellular peroxide formation was detectable after 5 min (Fig. 6). However, that discrepancy may have been due to the sensitivity of the fluorescent probe DCFH-DA. A scavenger receptor (SR-PSOX) that binds phosphatidylserine and oxidized lipoprotein has been reported to recognize negative-charged molecules, such as OxLDL and DSS, in phorbol ester-stimulated THP-1 human monocytes based on their uptake (Shimaoka et al., 2003). It is a transmembrane-type chemokine, selectively expressed on antigen presenting cells such as dendritic cells and macrophages, and was identified as the ligand for an orphan G-protein coupled chemokine receptor/ CXC chemokine receptor 6 (Shimaoka et al., 2003, 2004a,b). Surprisingly, SR-PSOX was indicated to be biochemically and functionally identical to CXCL16 (Shimaoka et al., 2003, Fig. 7. Involvement of CXCL16 in DSS-induced IL-1β production. (A-C) Levels of CXCL16 mRNA expression and protein in DSS-stimulated pM/. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 35-mm dish (RT-PCR and Western blot analysis) or one-chamber slides (immunocytochemistry) at a density of 1 × 107 cells/dish or 1 × 106 cells/ chamber, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (PBS) or DSS (10 μg/ml) for 1, 3, 6, 12, or 24 h (24 h for immunocytochemistry). The negative control cells were treated only with PBS. CXCL16 mRNA and its protein were examined by RT-PCR (A), Western blot analysis (B), and immunocytochemistry (C), as described in the Materials and methods section. The experiments were repeated 3 times independently, with 1 representative result shown. HPRT and β-actin served as the internal controls. Panel a, anti-CXCL16 antibody; Panel b, PI; Panel c, merge. Original magnification: panels a–c × 1000. (D) Effects of anti-mCXCL16 antibody on IL-1β production in pM/. Cells from peritoneal exudates from non-treated female ICR mice were seeded onto a 96-well plate at a density of 1 × 106 cells/well, and then cultured at 37 °C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM/ were treated with the vehicle (normal IgG), anti-CXCL16, anti-SR-A, anti-CD36, or anti-CD68 antibodies (2.5 μg/ml, each). The negative control cells were treated only with normal IgG. After incubating at 37 °C for 30 min, the pMφ were then treated with DSS (10 μg/ml) for 24 h. The concentration of IL-1β in the supernatant of the medium (50 μl) without dilution was determined using a mouse IL-1β ELISA kit, as described in the Materials and methods section. Cell viability was determined with an MTT test. Each value is shown as mean ± SD of 3 replicated experiments. Statistical analysis was performed using a non-parametric post-hoc test (Kruskal–Wallis test): ⁎P b 0.01 versus Control, ⁎⁎P b 0.05 versus DSS.

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Fig. 8. Proposed scheme of molecular events involved in the DSS-induced IL-1β production pathway in murine pM/. DSS may be recognized by CXCL16, after which ROS are intracellularly generated by an unknown mechanism. This leads to the activation of the MKK3/6 and p38 MAPK pathways for caspase-1 activation, which is responsible for the conversion of proIL-1β to biologically active IL-1β. Alternatively, DSS treatment may induce the activation of the MEK1/2 and ERK1/2 pathways, which might also be associated with caspase-1 activation and IL-1β release.

2004a). To the best of our knowledge, the present results are the first to demonstrate that mRNA and protein of CXCL16 are expressed in a constitutive manner in murine pM/, and our results with the anti-mCXCL16 antibody (Fig. 7D) imply that this molecule is one of the receptors for DSS involved with transducing pro-inflammatory signaling. Intriguingly, orally administered DSS was previously reported to be taken up by macrophages in inflamed mucosa, spleens, and mesenteric lymph nodes in mice with chronic colitis (Okayasu et al., 1990). Further, Mietus-Snyder et al. reported that oxidative stress may up-regulate scavenger receptor expression, thereby promoting the uptake of LDL in macrophages (Mietus-Snyder et al., 2000), whereas we did not see any increase in CXCL16 expression levels following DSS treatment (Fig. 7A, B). ROS are well-known injury mediators of inflammatory cascades that play a principle role in the development of IBD (Dijkstra et al., 1998). In the present study, we showed that DSSinduced ROS generation may be necessary for p38 MAPK activation and resultant IL-1β production (Figs. 5 and 6). Although we have no data to explain how DSS induces ROS generation, it is well documented that NADPH oxidase is the major enzyme for ROS generation in phagocytic cells (Park, 2003). In addition, while we demonstrated that H2O2-induced MAPKs activations were not attenuated by NAC (Fig. 5), the precise reason remains unclear. Nevertheless, the central roles of ROS in the development of IBD are evident. For example, the involvement of ROS in the pathogenesis of DSS-induced colitis has been reported (Korkina et al., 2003) and DSS increased the level of an oxidative DNA damage biomarker, 8-oxo-7,8-dihydro-2′-deoxyguanosine, in rat colonic mucosa (Tardieu et al., 1998). Sustained production of ROS during colonic inflammation may overwhelm the endogenous antioxidant defense system and, in accordance with that notion, there are several independent reports of decreased antioxidant levels in patients with IBD (Buffinton and Doe, 1995; Koutroubakis et al., 2004). Large numbers of phagocytic leuko-

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cytes, such as neutrophils, eosinophils, monocytes, and macrophages, in the mucosal interstitium are reported to be the source of ROS (Moslen, 1994), and there is ample evidence that ROS induce IL-1β production. For example, silica-induced generation of ROS and reactive nitrogen species resulted in the activation of cell signaling pathways, including ERK phosphorylation, and increased the expression of IL-1β (Fubini and Hubbard, 2003). The activated IL-1β signaling cascade, in turn, stimulates the production of ROS (Hwang et al., 2004). MAPKs activation has been implicated in the pathogenesis of IBD (Waetzig et al., 2002). Our data from pharmacological blockade (Fig. 3) and Western blotting (Fig. 4) assays strongly suggest that DSS induces IL-1β via the activation of p38 MAPK and ERK1/2, however, not that of JNK or PKC. It is of importance to note that both p38 MAPK and ERK1/2 have been found to be significantly activated in the inflamed colonic mucosa of IBD patients (Salh et al., 2003). Similarly, pharmacological inhibition of p38 MAPK was demonstrated to be beneficial for the treatment of IBD (ten Hove et al., 2002) and IL-1β was reduced by inhibition of the phosphorylation of p38 MAPK in pM/ from C57BL/6 mice (Kelly et al., 2003). Further, the p38 MAPK signaling cascade regulates the induction of proinflammatory genes in a transcriptional manner, including IL1β, TNF-α, IL-6, and interferon-γ (van den Blink et al., 2001). p38 MAPK activation is involved in androgen-independent proliferation of human prostate cancer cells by regulating IL-6 secretion (Shida et al., 2007). In addition, IBD is characterized by leukocytic infiltration, particularly by activated T cells and macrophages, both of which are responsible for the production of pro-inflammatory cytokines (Kucharzik et al., 2001). Our findings support the above-mentioned observations and suggest that infiltrated pM/ are the predominant biological source of activated p38 MAPK. Several lines of evidence have indicated that ROS induce MAPKs activation in many cell types (de Bernardo et al., 2004; Kim et al., 2003; Kong et al., 1998; Schweyer et al., 2004; Shimizu et al., 1999). For example, Schweyer et al. provided experimental evidence that the phosphorylation of both MEK1/2 and ERK1/2 was mediated by ROS in human malignant testicular germ cell lines (Schweyer et al., 2004), while neuronal death induced by glutathione depletion is due to a ROS-dependent activation of the ERK1/2 signaling pathway in glial cells (de Bernardo et al., 2004). Kong et al. also demonstrated that the activation of MAPKs and caspase-1 may involve oxidative modification of glutathione (Kong et al., 1998). In addition, inflammatory stimuli-induced activation of caspase-1 was shown to be mediated through p38 MAPK in murine brain microglia (Kim et al., 2003) and Shimizu et al. demonstrated that the activation of p38 MAPK leads to caspase-1 activation in the process of apoptosis of keratinocytes (Shimizu et al., 1999). These findings are consistent with our current hypothesis that both activated ERK1/2 and p38 MAPK trigger the activation of caspase-1, thereby promoting the release of matured IL-1β protein from pM/. However, we can not rule out the possibility that ROS may directly activate caspase-1 (Fig. 8), since it has a redoxsensitive cysteine residue (Miller et al., 1997). In addition, disturbingly, the time courses of the increase of IL-1β production

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(evident only after hours) and the increase in caspase-1 activity (peaking already after 5 min before returning to base level at 60 min) are very different. Moreover, ERK1/2 phosphorylation is only evident after 10 min of DSS treatment, i.e. after the peak of caspase-1 activity. Further studies are needed to better determine the mechanisms involved in the time difference of IL-1β, caspase-1, and MAPKs activation caused by DSS treatment. Conclusion We demonstrated that DSS enhanced IL-1β secretion in a post-translational manner via binding with CXCL16, ROS generation, the resultant activation of both the p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation in murine pM/. Since IL-1β is activated by caspase-1 at the early stage of the cascade that leads to intestinal inflammation, caspase-1 inhibitors and pharmacological agents that attenuate oxidative stress and ERK/p38 MAPK activation may be useful in therapeutic strategies for IBD. Acknowledgement We thank Dr. Masaya Nagao of Laboratory of Biosignals and Response, Kyoto University for technical support of confocal laser microscopy. This study was supported by grants-in-aid for cancer research from the Ministry of Health, Labor and Welfare of Japan, and from the Ministry of Agriculture, Forestry, and Fisheries (MAFF) Food Research Project “Integrated Research on Safety and Physiological Function of Food”. References Balding, J., Livingstone, W.J., Conroy, J., Mynett-Johnson, L., Weir, D.G., Mahmud, N., 2004. Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms. Mediators of Inflammation 13 (3), 181–187. Brynskov, J., Nielsen, O.H., Ahnfelt-Ronne, I., Bendtzen, K., 1992. Cytokines in inflammatory bowel disease. Scandinavian Journal of Gastroenterology 27 (11), 897–906. Buffinton, G.D., Doe, W.F., 1995. Depleted mucosal antioxidant defenses in inflammatory bowel disease. Free Radical Biology & Medicine 19 (6), 911–918. Cappello, M., Keshav, S., Prince, C., Jewell, D.P., Gordon, S., 1992. Detection of mRNAs for macrophage products in inflammatory bowel disease by in situ hybridisation. Gut 33 (9), 1214–1219. Chang, L., Karin, M., 2001. Mammalian MAP kinase signalling cascades. Nature 410 (6824), 37–40. Cominelli, F., Dinarello, C.A., 1989. Interleukin-1 in the pathogenesis of and protection from inflammatory bowel disease. Biotherapy 1 (4), 369–375. de Bernardo, S., Canals, S., Casarejos, M.J., Solano, R.M., Menendez, J., Mena, M.A., 2004. Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures. Journal of Neurochemistry 91 (3), 667–682. Dieleman, L.A., Ridwan, B.U., Tennyson, G.S., Beagley, K.W., Bucy, R.P., Elson, C.O., 1994. Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice. Gastroenterology 107 (6), 1643–1652. Dieleman, L.A., Palmen, M.J., Akol, H., Bloemena, E., Pena, A.S., Meuwissen, S.G., Van Rees, E.P., 1998. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clinical and Experimental Immunology 114 (3), 385–391. Dijkstra, G., Moshage, H., van Dullemen, H.M., de Jager-Krikken, A., Tiebosch, A.T., Kleibeuker, J.H., 1998. Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease. Journal of Pathology 186 (4), 416–421.

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