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Macrophage migration inhibitory factor has a proinflammatory activity via the p38 pathway in glucocorticoid-resistant ulcerative colitis
Clinical Immunology (2006) 120, 335 — 341
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Macrophage migration inhibitory factor has a proinflammatory activity via the p38 pathway in glucocorticoid-resistant ulcerative colitisi Yoh Ishiguro a,*, Tatsuya Ohkawara b, Hirotake Sakuraba a, Kazufumi Yamagata a, Hiroto Hiraga a, Satoko Yamaguchi a, Shinsaku Fukuda a, Akihiro Munakata a, Akio Nakane c, Jun Nishihira d a
First Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan b Department of Gastroenterology and Hematology, Hokkaido University of Graduate School of Medicine, Sapporo, Japan c Department of Bacteriology, Hirosaki University School of Medicine, Hirosaki, Japan d Department of Research and Development, Genetic Lab, Kita-9, Nishi-15, Chuou-ku, Sapporo, Japan Received 17 January 2006; accepted 16 May 2006 Available online 27 June 2006
KEYWORDS Ulcerative colitis; Inflammatory bowel disease; Cytokine; Glucocorticoids
Abstract Macrophage migration inhibitory factor (MIF) is a cytokine that has potent antisteroid effects and might be implicated in the pathogenesis of Ulecrative colitis (UC). We defined the functional role of MIF in the glucocorticoid (GC)-resistant inflammatory response in UC. Twenty-four colonic samples were obtained from GC responsive cases, GC refractory cases, Crohn’s disease and controls. LPMC were isolated from surgical specimens. MIF was strongly expressed at mRNA levels in refractory cases rather than responsive cases with UC and controls. IL-8 production from LPMC was significantly reduced by GC addition in responsive cases but not in refractory cases. In refractory cases, anti-MIF Ab ameliorated GC-resistant IL-8 production and p38-MAPK activity of LPMC. In addition, p38-MAPK antagonist SB230580 also ameliorated GC-resistant IL-8 production. These results suggest that MIF has an additional proinflammatory activity through the p38-MAPK pathway in GC-resistant UC. D 2006 Elsevier Inc. All rights reserved.
Abbreviations: AP-1, activator protein-1; 5-ASA, 5-aminosalicylic acid; CD, Crohn’s disease; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, glucocorticoids; IBD, inflammatory bowel disease; IL, interleukin; JAB-1, Jun activation domain-binding protein-1; LPS, lipopolysaccharride; LPMC, lamina proprial mononuclear cells; MAPK, mitogen activated protein kinase; mAb, monoclonal antibody; NF-nB, nuclear factor kappa B; SASP, sulfasalazine; TNF-a, tumor necrosis factor a; UC, ulcerative colitis B This work was performed at Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan. * Corresponding author. Fax: +81 172 27 0952. E-mail address: [email protected] (Y. Ishiguro). 1521-6616/$ - see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.05.010
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Y. Ishiguro et al.
Introduction Ulcerative colitis (UC) is a chronic, relapsing, inflammatory bowel disease (IBD). About 15% of cases require intravenous steroid administration because of the severity of the attack. As the response rate is about 60% [1], it is of interest to determine the pathogenesis of glucocorticoids (GC) refractoriness, which may also help to predict the prognostic outcome and avoid overuse of GC. Although the etiology of IBD is still unknown, immunological factors, including overproduction of proinflammatory cytokines, are highly likely to have an important role in its pathogenesis. Proinflammatory cytokines are abundant in intractable cases of UC regardless of steroid therapy rather than responsive and other inflammatory controls [2], and the ability of immunosuppressive drugs and recent biological agents that were shown to ameliorate the disease [3,4] supports this hypothesis. Several factors are known to play a role in steroid refractoriness, such as antagonistic glucocorticoid receptor B [5] and P-glycoprotein-1 [6], and other factors may also be involved. Macrophage migration inhibitory protein (MIF) is a cytokine that was first described for its inhibitory effect on the migration of macrophages. Recent in vivo and in vitro studies [7—11] have established the activity of MIF as a proinflammatory cytokine. One of the significant roles of MIF is to overcome the anti-inflammatory effect of GC on proinflammatory cytokine production stimulated by lipopolysaccharide (LPS) [12]. It was also shown that MIF induces matrix metalloproteinase in rheumatoid arthritis synovium via AP-1 [13], and the mortality rate was improved by anti-MIF antibody in a septic shock model [7]. Although MIF has been implicated in pathogenesis in a colitis model [14], and serum levels of MIF in IBD patients are increased [15], more studies are needed to elucidate the role of MIF in UC. In the present study, MIF was significantly higher in refractory cases, and the overcoming effect of MIF on GC treatment was described. Anti-MIF Ab ameliorated GCresistant IL-8 production and p38-MAPK activity in refractory cases. Furthermore, SB203580 ameliorated IL-8 production in the refractory cases. These results suggest that MIF has additional proinflammatory activities through the p38 pathway in refractory cases with UC.
Materials and methods Patient population Because there are several clinical variations of refractory UC including unresponsive fluminant disease, unresponsive chronic disease, and steroid-dependent disease, several criteria have been reported previously [6,16,17]. We defined refractory UC as no response to at least 2 weeks of prior intravenous GC therapy, and as a result, all refractory patients who were assessed in this study were surgically treated. Eight cases of refractory UC (5 male and 3 female; age range 16—56 years, mean age 34 years), and 7 responsive cases (2 males and 5 female, age range 12—62 years, mean age 29 years), 4 cases of active Crohn’s disease admitted for surgical treatment (3 males and 1 males; age
Figure 1 Quantification of MIF RNA expression with real-time PCR. Total RNA was extracted from surgically resected tissues from controls, UC and CD. cDNA was synthesized and real-time PCR was performed. The copy number relative to GAPDH was presented as 1/(2deltaCT). MIF transcripts were significantly highly expressed in refractory cases with UC rather than the responder, controls and CD cases. MIF transcripts in the responder were also higher than in the controls and CD.
range 18—27 years, mean age 23 years) and 5 cases of colon cancer (4 males and 1 females; age range 55—72 years, mean age 61 years) were enrolled. Some of the specimens from the responsive cases were obtained by biopsy under colonoscopy. The period from disease onset to surgery was 2.6 to 14 years in refractory cases and 1 to 20 years in responsive cases. The total mean dosage of GC was 20.4 g (3.7—50.0 g) in refractory cases, 6.9 g (0—14.0 g) in responsive cases and 0.8 g (0 to 3.4 g) in CD (Fig. 1). The diagnosis of IBD was confirmed using the standard clinical, endoscopic, radiological, and histological criteria. Surgically resected diseased colon from UC and CD patients and tissue specimens from histologically normal mucosal sites from patients with colon cancer were analyzed. All tissue samples were obtained from patients who were provided informed consent, and all the experimental procedures were approved by the Ethics Committee of Hirosaki University. The diagnosis of IBD was confirmed using the standard clinical, endoscopic, radiological, and histological criteria. Surgically resected diseased colon from UC and CD patients and tissue specimens from histologically normal mucosal sites from patients with colon cancer were analyzed. All tissue samples were obtained from patients who were provided informed consent, and all the experimental procedures were approved by the Ethics Committee of Hirosaki University.
Real-time PCR for MIF RNA was isolated from purified epithelium by AGPC methods as previously described [18]. Single-strand cDNA was synthesized using an RT PCR kit (Stratagene, Cedar Creek, TX)
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from 5 Ag of RNA. Reverse transcriptase (RT)-PCR was performed as previously described [18]. For MIF, the primer sequences are detailed in an earlier report [19]. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the primer was purchased from Applied BioSystems (Foster City, CA). The thermal cycler was programmed with a hot start at 948C for 1 min, and the amplification cycles were 948C for 3 min, 608C for 2 min, and 728C for 1 min, and final extension was performed at 728C for 10 min. The copy number relative to GAPDH was presented as 1/(2deltaCT).
Extracts (100 Ag) were fractionated by 15% SDS and electrotransferred to Immobilon p15 membranes (Millipore Corp., Bedford, MA). Protein expression was detected by immunoblot analysis with (1:250; Pharmingen, San Jose, CA) and primary anti-phospho-p38-MAPK antibody (1:250; Cell Signaling Technology Inc., Beverly, MA) and anti-p38-MAPK antibody (1:250; Cell Signaling Technology Inc., Beverly, MA) and horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). The visualization of the blots was performed with chemiluminescent substrate (SuperSignal; Pierce, Rockford, IL). Computer-assisted scanning densitometry was used to analyze the intensity of the immunoreactive bands (ATTO Corp., Tokyo, Japan). Values are expressed in arbitrary units (A.U.) and the ratio of phosphor-p38MAPK/p38-MAPK was calculated.
Immunohistochemistry A single-staining MIF assay was performed as described previously [18]. Briefly, colonic tissues were freshly frozen in Tissue-Tec OCT compound (Miles-Yeda, Rehovot, Israel). Next, 7-Am cryostat sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). Frozen sections were incubated with rabbit anti-MIF IgG (1:2000 dilution) and subsequently incubated with fluorescein isothiocyanateconjugated goat anti-rabbit IgG (H + L) (1:100 dilution; Jackson Immuno Research, West Grove, PA). The images of fluorescein isothiocyanate were acquired with an LSM410 confocal microscope (Carl Zeiss Co., Germany).
Isolation of lamina proprial mononuclear cells (LPMC) LPMC were freshly purified as described previously [2]. In brief, mucosal strips were washed in ice-cold PBS and incubated with 40 ml of RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 25 mM HEPES, 2.5% penicillin—streptomycin—fungizone (PSF; Gibco BRL, Grand Island, NY), 1 mM EDTA at 378C for 60 min. Cell suspensions obtained were passed through a 40-Am nylon mesh (Becton Dickinson, San Diego, CA). Subsequently, isolated LPMC were cultured at 1 106/ml in RPMI-1640 medium with 10% FCS and PSF for 24 hr. The supernatant was subsequently assessed by specific enzyme-linked immunosorbent assay (ELISA).
Cytokine-specific ELISA IL-8 levels was measured by ELISA (R&D Systems, Minneapolis, MN).
Assays for activated transcriptional factors Phospho-p38-MAPK was determined using an ELISA kit (BioSource International Inc., Camarillo, CA), and the assay was performed following the manufacturer’s instructions.
Blocking effect of MIF on IL-8 production p38-MAPK activity For blocking of MIF, anti-MIF monoclonal mouse IgG was used (endotoxin b0.05 Eu/l), and mouse IgG1 (Pharmingen, San Jose, CA) was used as a negative control. To assess whether MIF can overcome GC inhibition of IL-8, LPMC were incubated in the presence of the control IgG or GC (prednisolone: 50 Ag/mL [2]) plus control IgG or GC plus anti-MIF Ab (40 Ag/mL) [20]. The IL-8 in the culture supernatant was subsequently analyzed by specific ELISA. Cells were collected, and the nuclear fraction was extracted. Subsequently, phosho-p38-MAPK activity was assessed as previously described.
Effect of p38-MAPK antagonist on IL-8 production To confirm the role of p38-MAPK in LPMC from refractory cases, we assessed the IL-8 production in the presence or absence of a p38-MAPK antagonist, SB203580 purchased from Sigma-Aldrich Japan (Tokyo, JAPAN). IL-8 production in the LPMC in the presence of the vehicle or the vehicle plus GC (50 Ag/ml of prednisolone) or 10 AM of SB203580 plus GC was evaluated.
Nuclear extraction and cytoplasmic protein extraction
Statistics
To extract cytoplasmic and nuclear LPMC fractions, we used a nuclear extract kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instruction. Each fraction was subjected to ELISA.
Values are expressed as means F SE of the respective test or control group. Statistical significances between categorical groups were evaluated by Student’s t test. Comparisons between two paired groups were made using the paired t test.
Western blotting
Results
Frozen tissues were homogenated and lysed in lysis buffer purchased from Active Motif (Carlsbad, CA) according to the manufacturer’s instruction. Protein content was determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA).
MIF was strongly induced in refractory cases with UC MIF transcripts were strongly induced in refractory cases with UC (0.368 F 0.053) rather than responder (0.219 F
338 0.023, P b 0.05), controls (0.127 F 0.021, P b 0.01) and CD (0.1465 F 0.053, P b 0.05). MIF transcripts in the responder were also higher than in the controls and CD (P b 0.05, P b 0.05). Immunohistochemical study revealed that MIF-positive cells were compatible with LPMC in all groups (Fig. 2) and increased in refractory cases with UC (Figs. 2B, C) rather than in the controls (Fig. 2A) and responder (Fig. 2D) (magnification 400). The epithelial layer was positively stained in CD but the LPMC were not (Figs. 2E, F).
Blockade of MIF suppressed IL-8 release by LPMC from refractory cases with UC To test the overcoming effect of MIF on GC treatment in UC, we analyzed IL-8 production in LPMC with or without MIF Ab blocking in the presence of GC. IL-8 production was significantly higher in the LPMC taken from refractory UC patients (121.5 F 14.4 ng/ml) than in those from the control subjects (25.0 F 10.0 ng/ml, P b 0.001). GC inhibited IL-8 production in the refractory UC LPMC to a level (66.4 F 11.4 ng/ml, P b 0.05) that was still higher than spontaneous IL-8 production by the control subjects (P b 0.01, Fig. 3), while the level in responsive cases (69.25 F 18.53 ng/ml) was suppressed to the control subjects level (16.26 F 9.07 ng/ml, P = 0.51, vs. Control). The blockade of MIF with anti-MIF Ab suppressed IL-8 production by the LPMC from refractory UC patients (49.2 F 8.75 ng/ml) compared with those treated with control IgG in the presence of GC (P b 0.05, Fig. 3), reaching the levels of cells from the control subjects (P = 0.54, Fig. 3). In contrast, anti-MIF Ab induced about a 30% increase in IL-8 production in the LPMC from the responsive cases (24.0 F 10.1 ng/ml) in the presence of GC (Fig. 3).
Y. Ishiguro et al.
Phosho-p38-MAPK in LPMC and the effect of anti-MIF Ab on transcriptional factors As it has been suggested that IL-8 is regulated via MAPK activation [21], we assessed the level of phosphorylated p38-MAPK in the tissues and the LPMC nuclear fraction (Figs. 4A, B). The ratio of phosphor-p38-MAPK/p38-MAPK was calculated and the levels in samples from refractory UC patients were significantly higher (1.073 F 0.14) than those of responders (0.633 F 0.26, P b 0.05) (Fig. 4A). Accordingly, phosho-p38-MAPK levels in LPMC nuclear fraction were significantly higher in the refractory cases with UC (157.6 F 36.6 U/mg protein) rather than in the responsive cases with UC (68.2 F 12.4 U/mg protein) and the controls (62.3 F 13.2 U/mg protein, P b 0.05, P b 0.05, Fig. 4B). Next, we assessed the effect of anti-MIF Ab on these transcriptional factors in responsive and refractory cases in the presence of GC. Phosho-p38-MAPK was upregulated in responsive cases in the presence of GC plus control IgG (133.1 F 9.726%, P b 0.05 vs. control IgG), while reduced by anti-MIF Ab combined with GC (43.75 F 0.75%, P b 0.01 vs. control IgG) (Fig. 4C). Phosho-p38-MAPK was reduced in responsive cases in the presence of GC (33.96 F 11.71%, P b 0.05 vs. control IgG), while upregulated in the presence of GC and anti-MIF Ab in responsive cases in accordance with IL-8 production (124.5 F 49.8%) (Fig. 4C).
p38-MAPK antagonist reduced on IL-8 production in refractory cases with UC To confirm the role of p38-MAPK in the LPMC from refractory cases, we assessed IL-8 production in the presence of GC with or without an antagonist, SB 203580
Figure 2 Immunohistochemical study. Frozen sections were incubated with rabbit anti-MIF IgG and subsequently incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (H + L). The images of fluorescein isothiocyanate were then acquired. Positive staining cells were compatible with LPMC, and increased in refractory cases with UC (B, C) rather than in the controls (A) and responder (D) (magnification 400). The epithelial layer was positively stained in CD but the LPMC were not (E, F).
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Figure 3 Blocking MIF on LPMC IL-8 production in refractory and responsive UC. LPMC from UC cases were cultured with 40 Ag/ml of anti-MIF antibody or control IgG in the presence of 50 Ag/ml of prednisolone or medium alone. After 24 h, the supernatant was assayed with specific ELISA to assess IL-8 production. LPMC IL-8 production was higher in refractory cases rather than in control subjects and responders. In the presence of GC, IL-8 production was still higher in refractory UC, but anti-MIF Ab treatment significantly suppressed this rather than IgG treatment, reaching the levels of spontaneous production found in the control subjects. Eight refractory cases, 3 responder cases, and 5 controls were analyzed.
(Fig. 5). IL-8 production was significantly suppressed by SB203580 combined with GC (46.9 F 11.5%, P b 0.05, vs. vehicle) but not GC alone (79.8 F 6.5%, n.s., vs. vehicle). In responsive cases, GC significantly suppressed IL-8 production (30.3 F 0.4%, P b 0.05, vs. vehicle), and SB230580 also suppressed IL-8 production (23.1 F 0.5%, P b 0.05, vs. vehicle) (Fig. 5).
Discussion The mechanism of GC refractoriness of UC is still unclear. Large amounts of proinflammatory cytokines such as IL-1B, IL-8, IL-6 and TNF-A in the chronic phase in spite of GC therapy might be involved [2,22], and many of the transcriptional factors regulate these inflammatory networks. GC has been shown to inhibit the signaling in these pathways [23—25], whereas the RelA p65 subunit of NF-KB [26], p38-MAPK [26] and AP-1 [27,28] antagonize the effect of GC. MIF is also thought to be involved in this pathway through activation of proinflammatory cytokines. MIF is first described as a migratory inhibiting factor for macrophages that is produced by T cells [12] and fibroblast-like synovial cells from rheumatoid arthritis (RA) patients [19]. From the studies in a septic shock model [7], RA [19] and CD [14,15], MIF has been shown to contribute to the production of a large amount of proinflammatory cytokines and to be involved in GC resistance. However, the functional role of MIF and its interrelationship with transcriptional factors in UC, especially in refractory cases is still unclear. Herein, we showed a significant role of MIF in GC refractory UC. MIF is highly induced in GC refractory cases
Figure 4 Phosho-p38-MAPK was elevated in the tissue levels and nuclear fraction of LPMC taken from refractory UC and Anti-MIF Ab reduced this activity. (A) The ratio of phospho-p38MAPK/p38-MAPK was calculated from the Western blot analysis about the tissue specimens. The levels in samples from refractory UC patients were significantly higher than those of responders. *P b 0.05 vs. Responders, (B) the nuclear fraction was extracted from freshly isolated LPMC and was assessed for phosho-p38-MAPK. Phospho-p38-MAPK was significantly enhanced in the refractory cases compared to the control subjects and responsive cases. Four control cases, three refractory cases and three responder cases were analyzed. *P b 0.05 vs. Responders, Controls (C) LPMC were incubated for 24 hr in the presence of control IgG or GC (50 Ag/ml of prednisolone) with control IgG or GC plus 40 Ag/ml of anti-MIF Ab. The level of phosho-p38MAPK in the nuclear fraction was measured by ELISA. In refractory cases with UC, control IgG plus GC enhanced the % activity of phospho-p38-MAPK compared with control IgG alone (P b 0.01), whereas Anti-MIF Ab plus GC significantly ameliorated the activity (P b 0.05). An inverse pattern was obtained in responsive cases. Three refractory cases and three responder cases were analyzed. The error bars represent 1 SE, *P b 0.05, **P b 0.01 vs. Control.
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Figure 5 Blocking effect of p38-MAPK on IL-8 production. The effect of SB203580 on IL-8 production by LPMC from refractory and responsive UC cases was assessed. LPMC was cultured for 24 h in the presence of a vehicle or GC (50 Ag/ml of prednisolone) plus vehicle or GC plus 10 AM of SB230580. In refractory cases, the GC plus vehicle did not suppress IL-8 production compared with the vehicle alone, but GC plus SB230580 significantly reduced IL-8 production compared to the vehicle. In responsive cases, GC significantly suppressed IL-8 production. SB230580 also suppressed IL-8 production. Data were representative of four refractory cases and three responsive cases. The error bars represent 1 SE, *P b 0.05 vs. Controls.
at transcriptional levels and MIF regulates the GC-resistant IL-8 production associated with the p38-MAPK pathway. IL-8 production by LPMC from refractory cases with UC was reduced by GC but remained higher than that in responsive cases and the spontaneous production level found in controls. When anti-MIF Ab, but not control IgG, was added to the culture medium, IL-8 levels were significantly suppressed closely to the control levels. From these findings, it was concluded that MIF overcomes the inhibition of IL-8 production by GC in refractory UC. As shown in a former in vitro study where MIF was induced in the presence of GC, the results that MIF was highly induced in refractory cases might be resultant from treatment with a larger amount of GC at least partially. Although MIF is shown to be induced and have an important role in CD [14], lower expression of MIF in CD may be relevant to the low dose GC therapy in this study. These results suggest that GC itself may induce MIF expression in IBD and may have a greater role in GC therapy. Because p38 is an agonist of LPS [29] and MIF is reported to activate TLR4 [30], we focused on phosho-p38-MAPK. The present study revealed that p38-MAPK was reduced in the presence of GC in responsive cases but not in refractory cases, and anti-MIF Ab ameliorated p38-MAPK activity in refractory cases in accordance with the IL-8 production. Since p38-MAPK contributes to TNF-A-stimulated IL-8 secretion by intestinal epithelial cells via a post-transcriptional mechanism that involves stabilization of the IL-8 transcript [20], and the same mechanism was also involved in fibroblast [31,32], it is possible that MIF may regulate IL-8 production via the p38-MAPK pathway. The present study showed that nuclear phosho-p38-MAPK was significantly upregulated in refractory cases and its antagonist, SB203580, downregulated IL-8 production in the LPMC taken from refractory cases, even in the presence of GC, suggesting that GC-
Y. Ishiguro et al. resistant IL-8 production is mediated at least partially via the p38-MAPK pathway. Inhibition of MIF activity downregulates the GC-resistant p38MAPK activity in GC-resistant patients (open bars), whereas this inhibition abolishes the GC-mediated suppression of p38MAPK activity in GC response patients (solid bars) (Fig. 4C). Therefore, it is likely that MIF plays distinct roles in the GC-resistant vs. response cases of UC patients. Bucala [11] has suggested that MIF signaling is mediated through two distinct pathways; distinct signaling cascades are induced by MIF that interacts with intracytoplasmic receptor after its endocytosis vs. that binds to the surface receptor. Therefore, it is possible that MIF may differentially induce the signaling cascades depending on GCresistant vs. -response patients. This possibility would be examined in our future studies. In conclusion, the present study revealed that the p38MAPK pathway is one of the other pathways of MIF in the GC unresponsive inflammatory response. Thus, manipulating this inflammatory pathway in the chronic phase would be beneficial for treatment of refractory cases with UC.
Acknowledgment This work was supported in part by the Research Committee of Inflammatory Bowel Disease provided by the Japanese Ministry of Health, Labor, and Welfare.
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Macrophage migration inhibitory factor has a proinflammatory activity via the p38 pathway in glucocorticoid-resistant ulcerative colitisi Yoh Ishiguro a,*, Tatsuya Ohkawara b, Hirotake Sakuraba a, Kazufumi Yamagata a, Hiroto Hiraga a, Satoko Yamaguchi a, Shinsaku Fukuda a, Akihiro Munakata a, Akio Nakane c, Jun Nishihira d a
First Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan b Department of Gastroenterology and Hematology, Hokkaido University of Graduate School of Medicine, Sapporo, Japan c Department of Bacteriology, Hirosaki University School of Medicine, Hirosaki, Japan d Department of Research and Development, Genetic Lab, Kita-9, Nishi-15, Chuou-ku, Sapporo, Japan Received 17 January 2006; accepted 16 May 2006 Available online 27 June 2006
KEYWORDS Ulcerative colitis; Inflammatory bowel disease; Cytokine; Glucocorticoids
Abstract Macrophage migration inhibitory factor (MIF) is a cytokine that has potent antisteroid effects and might be implicated in the pathogenesis of Ulecrative colitis (UC). We defined the functional role of MIF in the glucocorticoid (GC)-resistant inflammatory response in UC. Twenty-four colonic samples were obtained from GC responsive cases, GC refractory cases, Crohn’s disease and controls. LPMC were isolated from surgical specimens. MIF was strongly expressed at mRNA levels in refractory cases rather than responsive cases with UC and controls. IL-8 production from LPMC was significantly reduced by GC addition in responsive cases but not in refractory cases. In refractory cases, anti-MIF Ab ameliorated GC-resistant IL-8 production and p38-MAPK activity of LPMC. In addition, p38-MAPK antagonist SB230580 also ameliorated GC-resistant IL-8 production. These results suggest that MIF has an additional proinflammatory activity through the p38-MAPK pathway in GC-resistant UC. D 2006 Elsevier Inc. All rights reserved.
Abbreviations: AP-1, activator protein-1; 5-ASA, 5-aminosalicylic acid; CD, Crohn’s disease; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, glucocorticoids; IBD, inflammatory bowel disease; IL, interleukin; JAB-1, Jun activation domain-binding protein-1; LPS, lipopolysaccharride; LPMC, lamina proprial mononuclear cells; MAPK, mitogen activated protein kinase; mAb, monoclonal antibody; NF-nB, nuclear factor kappa B; SASP, sulfasalazine; TNF-a, tumor necrosis factor a; UC, ulcerative colitis B This work was performed at Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan. * Corresponding author. Fax: +81 172 27 0952. E-mail address: [email protected] (Y. Ishiguro). 1521-6616/$ - see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.05.010
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Y. Ishiguro et al.
Introduction Ulcerative colitis (UC) is a chronic, relapsing, inflammatory bowel disease (IBD). About 15% of cases require intravenous steroid administration because of the severity of the attack. As the response rate is about 60% [1], it is of interest to determine the pathogenesis of glucocorticoids (GC) refractoriness, which may also help to predict the prognostic outcome and avoid overuse of GC. Although the etiology of IBD is still unknown, immunological factors, including overproduction of proinflammatory cytokines, are highly likely to have an important role in its pathogenesis. Proinflammatory cytokines are abundant in intractable cases of UC regardless of steroid therapy rather than responsive and other inflammatory controls [2], and the ability of immunosuppressive drugs and recent biological agents that were shown to ameliorate the disease [3,4] supports this hypothesis. Several factors are known to play a role in steroid refractoriness, such as antagonistic glucocorticoid receptor B [5] and P-glycoprotein-1 [6], and other factors may also be involved. Macrophage migration inhibitory protein (MIF) is a cytokine that was first described for its inhibitory effect on the migration of macrophages. Recent in vivo and in vitro studies [7—11] have established the activity of MIF as a proinflammatory cytokine. One of the significant roles of MIF is to overcome the anti-inflammatory effect of GC on proinflammatory cytokine production stimulated by lipopolysaccharide (LPS) [12]. It was also shown that MIF induces matrix metalloproteinase in rheumatoid arthritis synovium via AP-1 [13], and the mortality rate was improved by anti-MIF antibody in a septic shock model [7]. Although MIF has been implicated in pathogenesis in a colitis model [14], and serum levels of MIF in IBD patients are increased [15], more studies are needed to elucidate the role of MIF in UC. In the present study, MIF was significantly higher in refractory cases, and the overcoming effect of MIF on GC treatment was described. Anti-MIF Ab ameliorated GCresistant IL-8 production and p38-MAPK activity in refractory cases. Furthermore, SB203580 ameliorated IL-8 production in the refractory cases. These results suggest that MIF has additional proinflammatory activities through the p38 pathway in refractory cases with UC.
Materials and methods Patient population Because there are several clinical variations of refractory UC including unresponsive fluminant disease, unresponsive chronic disease, and steroid-dependent disease, several criteria have been reported previously [6,16,17]. We defined refractory UC as no response to at least 2 weeks of prior intravenous GC therapy, and as a result, all refractory patients who were assessed in this study were surgically treated. Eight cases of refractory UC (5 male and 3 female; age range 16—56 years, mean age 34 years), and 7 responsive cases (2 males and 5 female, age range 12—62 years, mean age 29 years), 4 cases of active Crohn’s disease admitted for surgical treatment (3 males and 1 males; age
Figure 1 Quantification of MIF RNA expression with real-time PCR. Total RNA was extracted from surgically resected tissues from controls, UC and CD. cDNA was synthesized and real-time PCR was performed. The copy number relative to GAPDH was presented as 1/(2deltaCT). MIF transcripts were significantly highly expressed in refractory cases with UC rather than the responder, controls and CD cases. MIF transcripts in the responder were also higher than in the controls and CD.
range 18—27 years, mean age 23 years) and 5 cases of colon cancer (4 males and 1 females; age range 55—72 years, mean age 61 years) were enrolled. Some of the specimens from the responsive cases were obtained by biopsy under colonoscopy. The period from disease onset to surgery was 2.6 to 14 years in refractory cases and 1 to 20 years in responsive cases. The total mean dosage of GC was 20.4 g (3.7—50.0 g) in refractory cases, 6.9 g (0—14.0 g) in responsive cases and 0.8 g (0 to 3.4 g) in CD (Fig. 1). The diagnosis of IBD was confirmed using the standard clinical, endoscopic, radiological, and histological criteria. Surgically resected diseased colon from UC and CD patients and tissue specimens from histologically normal mucosal sites from patients with colon cancer were analyzed. All tissue samples were obtained from patients who were provided informed consent, and all the experimental procedures were approved by the Ethics Committee of Hirosaki University. The diagnosis of IBD was confirmed using the standard clinical, endoscopic, radiological, and histological criteria. Surgically resected diseased colon from UC and CD patients and tissue specimens from histologically normal mucosal sites from patients with colon cancer were analyzed. All tissue samples were obtained from patients who were provided informed consent, and all the experimental procedures were approved by the Ethics Committee of Hirosaki University.
Real-time PCR for MIF RNA was isolated from purified epithelium by AGPC methods as previously described [18]. Single-strand cDNA was synthesized using an RT PCR kit (Stratagene, Cedar Creek, TX)
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from 5 Ag of RNA. Reverse transcriptase (RT)-PCR was performed as previously described [18]. For MIF, the primer sequences are detailed in an earlier report [19]. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the primer was purchased from Applied BioSystems (Foster City, CA). The thermal cycler was programmed with a hot start at 948C for 1 min, and the amplification cycles were 948C for 3 min, 608C for 2 min, and 728C for 1 min, and final extension was performed at 728C for 10 min. The copy number relative to GAPDH was presented as 1/(2deltaCT).
Extracts (100 Ag) were fractionated by 15% SDS and electrotransferred to Immobilon p15 membranes (Millipore Corp., Bedford, MA). Protein expression was detected by immunoblot analysis with (1:250; Pharmingen, San Jose, CA) and primary anti-phospho-p38-MAPK antibody (1:250; Cell Signaling Technology Inc., Beverly, MA) and anti-p38-MAPK antibody (1:250; Cell Signaling Technology Inc., Beverly, MA) and horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). The visualization of the blots was performed with chemiluminescent substrate (SuperSignal; Pierce, Rockford, IL). Computer-assisted scanning densitometry was used to analyze the intensity of the immunoreactive bands (ATTO Corp., Tokyo, Japan). Values are expressed in arbitrary units (A.U.) and the ratio of phosphor-p38MAPK/p38-MAPK was calculated.
Immunohistochemistry A single-staining MIF assay was performed as described previously [18]. Briefly, colonic tissues were freshly frozen in Tissue-Tec OCT compound (Miles-Yeda, Rehovot, Israel). Next, 7-Am cryostat sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). Frozen sections were incubated with rabbit anti-MIF IgG (1:2000 dilution) and subsequently incubated with fluorescein isothiocyanateconjugated goat anti-rabbit IgG (H + L) (1:100 dilution; Jackson Immuno Research, West Grove, PA). The images of fluorescein isothiocyanate were acquired with an LSM410 confocal microscope (Carl Zeiss Co., Germany).
Isolation of lamina proprial mononuclear cells (LPMC) LPMC were freshly purified as described previously [2]. In brief, mucosal strips were washed in ice-cold PBS and incubated with 40 ml of RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 25 mM HEPES, 2.5% penicillin—streptomycin—fungizone (PSF; Gibco BRL, Grand Island, NY), 1 mM EDTA at 378C for 60 min. Cell suspensions obtained were passed through a 40-Am nylon mesh (Becton Dickinson, San Diego, CA). Subsequently, isolated LPMC were cultured at 1 106/ml in RPMI-1640 medium with 10% FCS and PSF for 24 hr. The supernatant was subsequently assessed by specific enzyme-linked immunosorbent assay (ELISA).
Cytokine-specific ELISA IL-8 levels was measured by ELISA (R&D Systems, Minneapolis, MN).
Assays for activated transcriptional factors Phospho-p38-MAPK was determined using an ELISA kit (BioSource International Inc., Camarillo, CA), and the assay was performed following the manufacturer’s instructions.
Blocking effect of MIF on IL-8 production p38-MAPK activity For blocking of MIF, anti-MIF monoclonal mouse IgG was used (endotoxin b0.05 Eu/l), and mouse IgG1 (Pharmingen, San Jose, CA) was used as a negative control. To assess whether MIF can overcome GC inhibition of IL-8, LPMC were incubated in the presence of the control IgG or GC (prednisolone: 50 Ag/mL [2]) plus control IgG or GC plus anti-MIF Ab (40 Ag/mL) [20]. The IL-8 in the culture supernatant was subsequently analyzed by specific ELISA. Cells were collected, and the nuclear fraction was extracted. Subsequently, phosho-p38-MAPK activity was assessed as previously described.
Effect of p38-MAPK antagonist on IL-8 production To confirm the role of p38-MAPK in LPMC from refractory cases, we assessed the IL-8 production in the presence or absence of a p38-MAPK antagonist, SB203580 purchased from Sigma-Aldrich Japan (Tokyo, JAPAN). IL-8 production in the LPMC in the presence of the vehicle or the vehicle plus GC (50 Ag/ml of prednisolone) or 10 AM of SB203580 plus GC was evaluated.
Nuclear extraction and cytoplasmic protein extraction
Statistics
To extract cytoplasmic and nuclear LPMC fractions, we used a nuclear extract kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instruction. Each fraction was subjected to ELISA.
Values are expressed as means F SE of the respective test or control group. Statistical significances between categorical groups were evaluated by Student’s t test. Comparisons between two paired groups were made using the paired t test.
Western blotting
Results
Frozen tissues were homogenated and lysed in lysis buffer purchased from Active Motif (Carlsbad, CA) according to the manufacturer’s instruction. Protein content was determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA).
MIF was strongly induced in refractory cases with UC MIF transcripts were strongly induced in refractory cases with UC (0.368 F 0.053) rather than responder (0.219 F
338 0.023, P b 0.05), controls (0.127 F 0.021, P b 0.01) and CD (0.1465 F 0.053, P b 0.05). MIF transcripts in the responder were also higher than in the controls and CD (P b 0.05, P b 0.05). Immunohistochemical study revealed that MIF-positive cells were compatible with LPMC in all groups (Fig. 2) and increased in refractory cases with UC (Figs. 2B, C) rather than in the controls (Fig. 2A) and responder (Fig. 2D) (magnification 400). The epithelial layer was positively stained in CD but the LPMC were not (Figs. 2E, F).
Blockade of MIF suppressed IL-8 release by LPMC from refractory cases with UC To test the overcoming effect of MIF on GC treatment in UC, we analyzed IL-8 production in LPMC with or without MIF Ab blocking in the presence of GC. IL-8 production was significantly higher in the LPMC taken from refractory UC patients (121.5 F 14.4 ng/ml) than in those from the control subjects (25.0 F 10.0 ng/ml, P b 0.001). GC inhibited IL-8 production in the refractory UC LPMC to a level (66.4 F 11.4 ng/ml, P b 0.05) that was still higher than spontaneous IL-8 production by the control subjects (P b 0.01, Fig. 3), while the level in responsive cases (69.25 F 18.53 ng/ml) was suppressed to the control subjects level (16.26 F 9.07 ng/ml, P = 0.51, vs. Control). The blockade of MIF with anti-MIF Ab suppressed IL-8 production by the LPMC from refractory UC patients (49.2 F 8.75 ng/ml) compared with those treated with control IgG in the presence of GC (P b 0.05, Fig. 3), reaching the levels of cells from the control subjects (P = 0.54, Fig. 3). In contrast, anti-MIF Ab induced about a 30% increase in IL-8 production in the LPMC from the responsive cases (24.0 F 10.1 ng/ml) in the presence of GC (Fig. 3).
Y. Ishiguro et al.
Phosho-p38-MAPK in LPMC and the effect of anti-MIF Ab on transcriptional factors As it has been suggested that IL-8 is regulated via MAPK activation [21], we assessed the level of phosphorylated p38-MAPK in the tissues and the LPMC nuclear fraction (Figs. 4A, B). The ratio of phosphor-p38-MAPK/p38-MAPK was calculated and the levels in samples from refractory UC patients were significantly higher (1.073 F 0.14) than those of responders (0.633 F 0.26, P b 0.05) (Fig. 4A). Accordingly, phosho-p38-MAPK levels in LPMC nuclear fraction were significantly higher in the refractory cases with UC (157.6 F 36.6 U/mg protein) rather than in the responsive cases with UC (68.2 F 12.4 U/mg protein) and the controls (62.3 F 13.2 U/mg protein, P b 0.05, P b 0.05, Fig. 4B). Next, we assessed the effect of anti-MIF Ab on these transcriptional factors in responsive and refractory cases in the presence of GC. Phosho-p38-MAPK was upregulated in responsive cases in the presence of GC plus control IgG (133.1 F 9.726%, P b 0.05 vs. control IgG), while reduced by anti-MIF Ab combined with GC (43.75 F 0.75%, P b 0.01 vs. control IgG) (Fig. 4C). Phosho-p38-MAPK was reduced in responsive cases in the presence of GC (33.96 F 11.71%, P b 0.05 vs. control IgG), while upregulated in the presence of GC and anti-MIF Ab in responsive cases in accordance with IL-8 production (124.5 F 49.8%) (Fig. 4C).
p38-MAPK antagonist reduced on IL-8 production in refractory cases with UC To confirm the role of p38-MAPK in the LPMC from refractory cases, we assessed IL-8 production in the presence of GC with or without an antagonist, SB 203580
Figure 2 Immunohistochemical study. Frozen sections were incubated with rabbit anti-MIF IgG and subsequently incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (H + L). The images of fluorescein isothiocyanate were then acquired. Positive staining cells were compatible with LPMC, and increased in refractory cases with UC (B, C) rather than in the controls (A) and responder (D) (magnification 400). The epithelial layer was positively stained in CD but the LPMC were not (E, F).
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Figure 3 Blocking MIF on LPMC IL-8 production in refractory and responsive UC. LPMC from UC cases were cultured with 40 Ag/ml of anti-MIF antibody or control IgG in the presence of 50 Ag/ml of prednisolone or medium alone. After 24 h, the supernatant was assayed with specific ELISA to assess IL-8 production. LPMC IL-8 production was higher in refractory cases rather than in control subjects and responders. In the presence of GC, IL-8 production was still higher in refractory UC, but anti-MIF Ab treatment significantly suppressed this rather than IgG treatment, reaching the levels of spontaneous production found in the control subjects. Eight refractory cases, 3 responder cases, and 5 controls were analyzed.
(Fig. 5). IL-8 production was significantly suppressed by SB203580 combined with GC (46.9 F 11.5%, P b 0.05, vs. vehicle) but not GC alone (79.8 F 6.5%, n.s., vs. vehicle). In responsive cases, GC significantly suppressed IL-8 production (30.3 F 0.4%, P b 0.05, vs. vehicle), and SB230580 also suppressed IL-8 production (23.1 F 0.5%, P b 0.05, vs. vehicle) (Fig. 5).
Discussion The mechanism of GC refractoriness of UC is still unclear. Large amounts of proinflammatory cytokines such as IL-1B, IL-8, IL-6 and TNF-A in the chronic phase in spite of GC therapy might be involved [2,22], and many of the transcriptional factors regulate these inflammatory networks. GC has been shown to inhibit the signaling in these pathways [23—25], whereas the RelA p65 subunit of NF-KB [26], p38-MAPK [26] and AP-1 [27,28] antagonize the effect of GC. MIF is also thought to be involved in this pathway through activation of proinflammatory cytokines. MIF is first described as a migratory inhibiting factor for macrophages that is produced by T cells [12] and fibroblast-like synovial cells from rheumatoid arthritis (RA) patients [19]. From the studies in a septic shock model [7], RA [19] and CD [14,15], MIF has been shown to contribute to the production of a large amount of proinflammatory cytokines and to be involved in GC resistance. However, the functional role of MIF and its interrelationship with transcriptional factors in UC, especially in refractory cases is still unclear. Herein, we showed a significant role of MIF in GC refractory UC. MIF is highly induced in GC refractory cases
Figure 4 Phosho-p38-MAPK was elevated in the tissue levels and nuclear fraction of LPMC taken from refractory UC and Anti-MIF Ab reduced this activity. (A) The ratio of phospho-p38MAPK/p38-MAPK was calculated from the Western blot analysis about the tissue specimens. The levels in samples from refractory UC patients were significantly higher than those of responders. *P b 0.05 vs. Responders, (B) the nuclear fraction was extracted from freshly isolated LPMC and was assessed for phosho-p38-MAPK. Phospho-p38-MAPK was significantly enhanced in the refractory cases compared to the control subjects and responsive cases. Four control cases, three refractory cases and three responder cases were analyzed. *P b 0.05 vs. Responders, Controls (C) LPMC were incubated for 24 hr in the presence of control IgG or GC (50 Ag/ml of prednisolone) with control IgG or GC plus 40 Ag/ml of anti-MIF Ab. The level of phosho-p38MAPK in the nuclear fraction was measured by ELISA. In refractory cases with UC, control IgG plus GC enhanced the % activity of phospho-p38-MAPK compared with control IgG alone (P b 0.01), whereas Anti-MIF Ab plus GC significantly ameliorated the activity (P b 0.05). An inverse pattern was obtained in responsive cases. Three refractory cases and three responder cases were analyzed. The error bars represent 1 SE, *P b 0.05, **P b 0.01 vs. Control.
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Figure 5 Blocking effect of p38-MAPK on IL-8 production. The effect of SB203580 on IL-8 production by LPMC from refractory and responsive UC cases was assessed. LPMC was cultured for 24 h in the presence of a vehicle or GC (50 Ag/ml of prednisolone) plus vehicle or GC plus 10 AM of SB230580. In refractory cases, the GC plus vehicle did not suppress IL-8 production compared with the vehicle alone, but GC plus SB230580 significantly reduced IL-8 production compared to the vehicle. In responsive cases, GC significantly suppressed IL-8 production. SB230580 also suppressed IL-8 production. Data were representative of four refractory cases and three responsive cases. The error bars represent 1 SE, *P b 0.05 vs. Controls.
at transcriptional levels and MIF regulates the GC-resistant IL-8 production associated with the p38-MAPK pathway. IL-8 production by LPMC from refractory cases with UC was reduced by GC but remained higher than that in responsive cases and the spontaneous production level found in controls. When anti-MIF Ab, but not control IgG, was added to the culture medium, IL-8 levels were significantly suppressed closely to the control levels. From these findings, it was concluded that MIF overcomes the inhibition of IL-8 production by GC in refractory UC. As shown in a former in vitro study where MIF was induced in the presence of GC, the results that MIF was highly induced in refractory cases might be resultant from treatment with a larger amount of GC at least partially. Although MIF is shown to be induced and have an important role in CD [14], lower expression of MIF in CD may be relevant to the low dose GC therapy in this study. These results suggest that GC itself may induce MIF expression in IBD and may have a greater role in GC therapy. Because p38 is an agonist of LPS [29] and MIF is reported to activate TLR4 [30], we focused on phosho-p38-MAPK. The present study revealed that p38-MAPK was reduced in the presence of GC in responsive cases but not in refractory cases, and anti-MIF Ab ameliorated p38-MAPK activity in refractory cases in accordance with the IL-8 production. Since p38-MAPK contributes to TNF-A-stimulated IL-8 secretion by intestinal epithelial cells via a post-transcriptional mechanism that involves stabilization of the IL-8 transcript [20], and the same mechanism was also involved in fibroblast [31,32], it is possible that MIF may regulate IL-8 production via the p38-MAPK pathway. The present study showed that nuclear phosho-p38-MAPK was significantly upregulated in refractory cases and its antagonist, SB203580, downregulated IL-8 production in the LPMC taken from refractory cases, even in the presence of GC, suggesting that GC-
Y. Ishiguro et al. resistant IL-8 production is mediated at least partially via the p38-MAPK pathway. Inhibition of MIF activity downregulates the GC-resistant p38MAPK activity in GC-resistant patients (open bars), whereas this inhibition abolishes the GC-mediated suppression of p38MAPK activity in GC response patients (solid bars) (Fig. 4C). Therefore, it is likely that MIF plays distinct roles in the GC-resistant vs. response cases of UC patients. Bucala [11] has suggested that MIF signaling is mediated through two distinct pathways; distinct signaling cascades are induced by MIF that interacts with intracytoplasmic receptor after its endocytosis vs. that binds to the surface receptor. Therefore, it is possible that MIF may differentially induce the signaling cascades depending on GCresistant vs. -response patients. This possibility would be examined in our future studies. In conclusion, the present study revealed that the p38MAPK pathway is one of the other pathways of MIF in the GC unresponsive inflammatory response. Thus, manipulating this inflammatory pathway in the chronic phase would be beneficial for treatment of refractory cases with UC.
Acknowledgment This work was supported in part by the Research Committee of Inflammatory Bowel Disease provided by the Japanese Ministry of Health, Labor, and Welfare.
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