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Methotrexate is effective in reactivated colitis and reduces inflammatory alterations in mesenteric adipose tissue during intestinal inflammation
Pharmacological Research 60 (2009) 341–346
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Methotrexate is effective in reactivated colitis and reduces inflammatory alterations in mesenteric adipose tissue during intestinal inflammation Marcia Aparecida Thomaz a , Simone Coghetto Acedo a , Caroline Candida de Oliveira a , José Aires Pereira a , Denise Gonc¸alves Priolli a , Mario José Saad b , José Pedrazzoli Jr a , Alessandra Gambero a,∗ a b
Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Av. São Francisco de Assis 218, 12916-900 Braganc¸a Paulista, SP, Brazil Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas, SP, Brazil
a r t i c l e
i n f o
Article history: Received 30 January 2009 Received in revised form 7 May 2009 Accepted 10 May 2009 Keywords: TNBS Leptin Adiponectin TNF-␣ IL-10 F4/80 TLR-4 iNOS
a b s t r a c t Mesenteric white adipose tissue hypertrophy and modifications in adipocytokine production are described features of Crohn’s disease. Experimentally, mesenteric white adipose tissue alterations, associated with intestinal inflammation, can be induced in a model of reactivated colitis by repeated administration of intrarectal trinitrobenzenosulfonic acid (TNBS) in ethanol solution. Crohn’s disease patients refractory to corticosteroid treatment are frequently treated with methotrexate; however, there is no information regarding the drug’s effect on adipose tissue alterations and in a reactivated colitis experimental model. Thus, we evaluated the effect of methotrexate upon mesenteric WAT alterations and inflammatory features in experimental colitis in rats. Colitis status was evaluated by macroscopic score, histopathological analysis, myeloperoxidase activity, TNF-␣ and IL-10 expression, as well as iNOS and TLR-4 expression in colon samples. The adipose tissue alterations were assessed by TNF-␣, IL-10, leptin and adiponectin production, as well as by macrophage infiltration evaluation. Methotrexate exerts an anti-inflammatory activity in experimental reactivated colitis by regulating the shift from Th1 to Th2 cytokines, reducing TNF-␣ and improving IL-10 production. Additionally, methotrexate reduces other inflammatory parameters in the colon, such as iNOS and TLR-4 expression. In mesenteric white adipose tissue, methotrexate treatment reduces the production of pro- and anti-inflammatory adipocytokines as well as macrophage infiltration, suggesting that immunosuppressant drugs diminish adipose tissue inflammatory alterations associated with intestinal inflammation. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Accumulating evidence suggests that white adipose tissue (WAT) interacts with inflammation and immune processes, based on the fact that WAT cells (adipocytes, matrix cells, macrophage and mast cells) produce and release several bioactive molecules, collectively called adipocytokines [1]. In addition, WAT can be short-term changed during acute infection and inflammation, as well as long-term changed during diseases such as non-insulin dependent diabetes mellitus and Cushing’s syndrome [2]. Low body weight and development of mesenteric WAT hypertrophy are well-described symptoms of Crohn’s disease [3]. Considered as a characteristic feature of Crohn’s disease, fat-wrapping is defined as a mesenteric WAT extension from the mesenteric attachment that partially covers the small and large intestinal circumference, in association with loss of the bowel-mesentery angle
∗ Corresponding author. Tel.: +55 11 4034 8135; fax: +55 11 4034 1825. E-mail address: [email protected] (A. Gambero). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.05.003
[4]. During recent years, the contribution of fat tissue to Crohn’s disease has been investigated. One hypothesis is that the fat hypertrophy represents a defensive reaction against the primary intestinal inflammation [5]. Corroborating this hypothesis, creeping fat in Crohn’s disease produces anti-inflammatory adipocytokines, such as adiponectin [5] and responds to steroid treatment by improving IL-10 production [6]. However, adipocytes from mesenteric WAT in Crohn’s disease specifically express TNF-␣ [7] and adipose tissue secrets high quantities of leptin [5], proinflammatory adipocytokines that indicate adipose tissue as a causative role in intestinal inflammation. In addition, creeping fat in Crohn’s disease is infiltrated by significant amounts of macrophages that also produce inflammatory cytokines, suggesting that mesenteric adipose tissue could have a pathophysiologic relevance [3]. Other proinflammatory substances, i.e. IL-6 and monocyte chemotactic protein-1, have been reported not to be specifically induced in Crohn’s disease, and are also present in other inflammatory intestinal conditions [5]. The general principles for treating active CD take into consideration the activity, site (ileal, ileocolic, colonic and others), and behavior of the disease (inflammatory, structuring, fistulating, course of disease, response to previous medications, side
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effect profile of medication and extraintestinal manifestation) [8]. Approximately 75% of patients with Crohn’s disease or ulcerative colitis will respond to conventional therapy, including corticosteroids [9]. Corticosteroid-refractory patients require treatment with infliximab and immunosuppressants, such as methotrexate [8]. Methotrexate is a folic acid antagonist and blocks the synthesis of purines and pyrimidines by inhibiting several key enzymes, suppressing transmethylation reactions with accumulation of polyamines, reducing antigen-dependent T-cell proliferation and promoting adenosine release with adenosine-mediated suppression of inflammation [10]. The combination of these effects may be responsible for the anti-inflammatory activity of methotrexate. Down-regulation of inflammatory cytokines, mainly TNF-␣, is also suggested as an important action of methotrexate [11]. Experimentally, the mesenteric WAT alterations associated with intestinal inflammation can be observed in a model of colitis, by intrarectal repeated trinitrobenzenosulfonic acid (TNBS) in ethanol solution administration. The mesenteric WAT depot increases and produces high amounts of TNF-␣ during this process [12]. An increase in infiltrating macrophage has also been observed [13]. The scarce reports regarding the action of drugs upon inflammatory alterations in mesenteric WAT from CD have related in steroid-treated patient [5,6]. Thus, this study intends to evaluate the action of methotrexate upon mesenteric WAT alterations induced by experimental colitis, focusing upon the adipocytokine production and inflammatory alterations, such as macrophage infiltration. Additionally, we characterized the effect of methotrexate upon reactivated colitis, a well-described model that resembles the inflammatory characteristics of CD, evaluating inflammatory parameters, such as cytokine production and iNOS and TLR-4 expression in colon. 2. Materials and methods 2.1. Animals Male, Wistar rats (200–250 g) free of specific pathogens were obtained from CEMIB (State University of Campinas, Campinas, SP, Brazil). The experiments were performed in accordance with the principles outlined by the Brazilian College for Animal Experimentation (COBEA). The rats were maintained in a room with controlled humidity and temperature and were exposed to 12-h light-dark cycles. Twelve hours prior to an experiment, the animals were deprived of food (standard chow), but not water. Studies were carried out using 4–7 rats per group. 2.1.1. Reactivated colitis induced by TNBS and treatment protocol The animals were anesthetized with ketamine/xylazine (1:1, v/v), and colitis was induced by intracolonic instillation of 3 mg of TNBS dissolved in 0.3 ml of 50% ethanol (Sigma, St. Louis, MO). The solution was injected into the colon, 8 cm proximal to the anus using a catheter. The instillation procedure required only a few seconds and the rats were maintained in a vertical position until they recovered from the anesthesia. The same procedure was repeated 14 and 28 days after the first TNBS instillation. Control rats received saline by the same protocol. One half of the colitis animals received methotrexate, intraperitoneally (3 mg kg−1 ; Sigma, St. Louis, MO), divided into two administrations on the 28th and 31th days of the protocol and were then sacrificed at the end of the treatment (35th day). The animals were maintained in collective cages. 2.1.2. Colitis characterization by macroscopic damage, microscopic assessment and myeloperoxidase activity Colons were removed immediately from animals sacrificed by cervical dislocation. The colons were opened longitudinally and
evaluated by two observers unaware of the experimental groups for macroscopically visible damage, as described by Bell et al. [14]. The criteria for assessment of macroscopic colonic damage of each animal were: no damage (no points); hyperemia, no ulcers (1 point); linear ulcer with no significant inflammation (2 points); linear ulcer with inflammation at one site (3 points); two or more sites of ulceration/inflammation (4 points); two or more major sites of ulceration and inflammation or one site of ulceration/inflammation extending 1 cm along the length of the colon (5 points); if damage covers 2 cm along the length of the colon, the score is increased by 1 for each additional centimeter of involvement (6–10 points). The specimens were fixed in 10% buffered formalin and embedded in paraffin. Two sections of 4 m in thickness were cut and stained with hematoxylin–eosin (H&E) for histological evaluation. Colon samples obtained longitudinally from a site of macroscopically detectable inflammation (or a corresponding site in the tissue with no macroscopically detectable inflammation) were homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. For the myeloperoxidase (MPO) assay, 50 L of each sample were added to 200 L of o-dianisidine solution (0.167 mg/mL o-dianisidine dihydrochloride, 0.0005% hydrogen peroxide in 50 mM phosphate buffer, pH 6.0) immediately prior to reading the change in absorbance at 460 nm over 5 min using a microplate reader (Multiscan MS, Labsystems). 2.1.3. Short-term culture of total adipose tissue specimens Secretion of adipocytokines was evaluated by measuring the amount of adipocytokines released from WAT cells to the incubation buffer without any stimulus, using commercial EIA kits (TNF-␣ and IL-10 from GE Healthcare, UK; leptin and adiponectin from B-Bridge International, Mountain View, CA). Mesenteric WAT was dissected from proximal colon immediately after laparotomy. Biopsies from mesenteric adipose tissue were minced and incubated with constant shaking (60 cycles/min) for 4 h at 37 ◦ C in a 5% CO2 atmosphere with Medium 199 (Gibco, Gaithersburg, MD) supplemented with 1% bovine serum albumin. After 4 h, the culture medium was collect and stored at −80 ◦ C. 2.1.4. Protein extraction and Western immunoblot analysis For evaluation of colonic and WAT additional inflammatory markers, the abdominal cavities of anesthetized rats were opened and adipose tissue fragments from mesenteric adipose tissue and colon biopsies were excised and immediately homogenized in solubilizing buffer at 4 ◦ C [1% Triton X-100, 100 mM Tris–HCl (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium orthovanadate, 2.0 mM PMSF, and 0.1 mg aprotinin/ml]. Insoluble material was removed by centrifugation for 20 min, at 9000 × g, at 4 ◦ C. The protein concentration of the supernatants was determined by the Biuret method. The extracts were treated with Laemmli sample buffer containing 100 mM dithiothreitol and heated in a boiling water bath for 5 min, after which samples were subjected to SDS-PAGE in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For immunoblot experiments, 0.15 mg of protein extracts from each tissue were separated by SDS-PAGE, transferred to nitrocellulose membranes and blocked for 2 h with blocking buffer (Blocking agent 5% (GE Healthcare) in Tris buffer solution). Membranes were then incubated overnight with antiTNF-␣, anti-iNOS, anti-IL-10, anti--actin and anti-F4/80 (1:1000; Santa Cruz, USA). The nitrocellulose membranes were developed using commercial chemoluminescent Kits (GE Healthcare, UK). Band intensities were quantified by optical densitometry (Scion Image software, ScionCorp, Frederick, MD) of the developed autoradiography.
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Table 1 Parameters evaluation in controls, colitis and methotrexate treated animals. Group
Body weight (g)
Damage score (0–10)
Control Colitis Methotrexate
403.8 ± 5.7 346.2 ± 12.9** 360.0 ± 11.9
1.8 ± 0.5 6.2 ± 0.6** 3.2 ± 0.8#
Myeloperoxidase activity (U/g tissue) 1.32 ± 0.18 17.95 ± 2.33** 8.11 ± 2.70#
Data are expressed as mean ± SEM. ** p < 0.01 when compared with control group. # p < 0.05 when compared with colitis (n = 5).
2.2. Statistical analysis All data are expressed as the mean ± SEM Comparisons among groups of data were performed using one-way ANOVA followed by Dunnett multiple comparisons test. An associated probability (p value) of less than 5% was considered to be significant. 3. Results 3.1. Colitis evaluation The colitis was characterized by a higher macroscopic score and by an increased myeloperoxidase activity in colons from
TNBS/ethanol rats. Myeloperoxidase activity is a biochemical marker of infiltrating neutrophils. The colitis animals also present a reduced body weight, when compared with healthy animals (Table 1). The histological analyses of colon samples from colitis animals revealed the presence of ulcerations, infiltrating inflammatory cells in mucosa and submucosa and edema in the submucosal area (Fig. 1B and C). In some cases, as shown in Fig. 1C, it was possible to observe a transmural inflammation. These alterations were not found in the colon samples from controls, where a typical morphology could be observed (Fig. 1A). Methotrexate-treated animals presented a reduced level of macroscopically observed damage and myeloperoxidase activity when compared with colitis non-treated animals. The treatment protocol used did not modify
Fig. 1. Histopathological appearance of colonic tissue in rats. (A) Normal colonic mucosa of control rats [hematoxylin and eosin (H&E) 40×]. (B and C) Colonic mucosa of colitis rats. Large ulcerations (U) and submucosa edema with infiltrating inflammatory cells (arrows) (H&E 100×). (D and E) MTX-treated colitis rats. Moderated infiltrating inflammatory cells (arrows) (H&E 40× and 100×, respectively).
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Fig. 3. Level of iNOS protein in colon from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on colon protein extracts with antibodies against iNOS (A) and -actin (B). The results are representative of one experiment. Lower panels show the densitometry quantification of iNOS (C) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. **p < 0.01 when compared with control group; # p < 0.05 when compared with colitis group.
3.1.1. Adipocytokine production Mesenteric WAT from colitis animals released higher amounts of TNF-␣ and IL-10, when compared with the same depot obtained from healthy animals (Table 2). Our experimental procedure does not modify the mesenteric WAT production of leptin and adiponectin. After methotrexate treatment, significant reductions were observed in ex vivo TNF-␣, IL-10 and adiponectin release (Table 2).
Fig. 2. Levels of TNF-␣ and IL-10 in colon from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on colon protein extracts with antibodies against TNF-␣ (A), IL-10 (D) and -actin (B and E). The results are representative of one experiment. Lower panels show the densitometry quantification of TNF-␣ (C) and IL-10 (F) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. *p < 0.05 and **p < 0.01 when compared with control group; # p < 0.05 and ## p < 0.01 when compared with colitis group.
the animals final body weight (Table 1). Microscopic analysis also revealed a reduction in inflammatory alterations. A reduction in edema was also observed, as well as some infiltrating inflammatory cells (Fig. 1D and E). The cytokine expression in colonic tissue was evaluated in all groups. TNBS/ethanol administrations were able to induce a greater TNF-␣ and a lower IL-10 expression in the colon sample. Methotrexate treatment was able to reduce TNF-␣ and, in addition, restore IL-10 expression to the same levels as those observed for control animals (Fig. 2). Additional inflammatory markers were also evaluated, such as, iNOS expression. Colitis animals presented a higher level of iNOS protein in the colon and methotrexate efficiently reduced this level (Fig. 3). The Toll-like receptor (TLR)-4 was also increased in colitis animals and its level was reduced after treatment (Fig. 4).
Fig. 4. Level of TLR-4 protein in colon from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on colon protein extracts with antibodies against TLR-4 (A) and -actin (B). The results are representative of one experiment. Lower panels show the densitometry quantification of TLR-4 (C) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. *p < 0.05 and **p < 0.01 when compared with control group; # p < 0.05 and ## p < 0.01 when compared with colitis group.
M.A. Thomaz et al. / Pharmacological Research 60 (2009) 341–346 Table 2 Release of adipocytokines from mesenteric adipose tissue in control, colitis and methotrexate treated groups. Control TNF-␣ (pg ml) IL-10 (ng ml) Leptin (g ml) Adiponectin (g ml)
58.25 0.98 4.13 2.30
± ± ± ±
Colitis 6.51 0.25 0.96 0.13
157.49 3.27 3.89 2.29
Methotrexate ± ± ± ±
6.63** 0.96* 0.74 0.15
93.73 1.28 4.47 1.55
± ± ± ±
14.04# 0.14# 0.67 0.20#
Data are expressed as mean ± SEM (n = 5). * p < 0.05, when compared with control group. ** p < 0.01, when compared with control group. # p < 0.05, when compared with colitis group.
Fig. 5. Level of F4/80 protein in mesenteric adipose tissue from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on adipose tissue protein extracts with antibodies against F4/80 (A) and -actin (B). The results are representative of one experiment. Lower panels show the densitometry quantification of F4/80 (C) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. **p < 0.01 when compared with control group; # p < 0.05 when compared with colitis group.
3.1.2. Macrophage infiltration into mesenteric adipose tissue In colitis animals, the mesenteric adipose tissue presented a higher level of F4/80 protein expression, indicating the presence of infiltrated macrophages (Fig. 5). Methotrexate treatment was able to reduce this infiltration. 4. Discussion Administration of a hapten, combined with a barrier breaker, such as ethanol, into the colon results in mucosal injury and inflammation that resemble the colonic inflammation present in inflammatory bowel disease in humans. This injury gradually subsides over the weeks, but it can be reactivated with an additional TNBS/ethanol administration [15]. In the active phase, it is possible to observe a transmural inflammation associated with diarrhea, weight loss, and an imbalance activation of Th1- and Th2-lymphocytes, resulting in an induction of an IL-12-driven inflammation with a massive Th1-mediated response, including TNF-␣ production [16–18]. Anti-inflammatory/immunosuppressant drugs, such as, dexamethasone, cyclosporine A and 5-aminosalicylic acid reduced colonic damage score and MPO activity in a reactivated colitis model [15]. However, there are no previous reports on the actions of methotrexate in reactivated or acute colitis in rats. In acute dinitrobenzenesulfonic acid (DNBS)-induced colitis in mice, methotrexate at dosage of 0.5 mg/kg/day produces controversial
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results [19]. In dextran sulfate sodium (DSS)-induced colitis in mice, methotrexate, at dosage of 1 mg/kg/day, reduces the inflammatory score [20]. In our study, the dosage chosen was based on several literature reports regarding the immunosuppressant and anti-inflammatory actions of methotrexate in arthritis models, including the ability of methotrexate to inhibit TNF-␣ in these models [21,22]. Methotrexate, at a dosage of 3 mg/kg/week, was able to significantly reduce the inflammatory score, histopathological alterations, MPO activity and colonic TNF-␣ expression, confirming that the dosage chosen was effective for TNF-␣ inhibition. Additionally, we found a reduced IL-10 level in the colon of colitis animals and methotrexate was able to reverse this reduction. IL-10 is a Th2 cytokine, produced by a variety of cells including B and T cells, macrophages, mast cells and intestinal epithelial cells, with a keyrole in the maintenance of intestinal immune homeostasis by its immunomodulatory activity [23]. The immunomodulatory activity of IL-10 is based upon its ability to inhibit both the synthesis of proinflammatory cytokines (IL-1, TNF-␣ and IL-6) and of Th2 cell-derived cytokines (IL-4 and IL-5) [24]. Dexamethasone and curcumin treatment in TNBS colitis also results in increased colonic IL-10 production, parallel to TNF-␣ inhibition [25]. The mechanism by which low dose methotrexate exerts its anti-inflammatory effect in rheumatoid arthritis patients seems also to be related to an induction of IL-10 secretion (a Th2 cytokine) and a reduction in Th1 profile in mononuclear cells [26]. We also verified the ability of methotrexate to modify additional intestinal inflammatory parameters, such as iNOS and Toll-like receptor (TLR)-4. It is thought that Crohn’s disease arises from a dysregulated mucosal immune response to luminal bacteria. TLRs, which are pattern-recognition receptors expressed by both immune and non-immune cells, play a pivotal role in host/microbial interactions and have two distinct functions that protect against infection and control tissue homeostasis, depending on the recognition of pathogens or commensals. An increased TLR4 expression was found in the terminal ileum of CD patients with active disease [27]. In TNBS-colitis, the level of colonic TLR-4 protein was also up-regulated [28,29]. We observed that methotrexate treatment effectively normalized the up-regulation of TLR-4 in colitis animals, suggesting that this pathway could contribute to attenuate the gut inflammation. An elevated level of peroxynitrite, produced from increased colonic iNOS expression and activity, is a major contributor to colon epithelial apoptosis during inflammation [30]. Methotrexate treatment also contributed to decrease the iNOS expression in the colon, as described for other anti-inflammatory substances [31]. Finally, we evaluated the ability of methotrexate to modify the production of adipocytokine in mesenteric WAT during colitis. Methotrexate was able to significantly reduce the production of TNF-␣ by WAT, suggesting that the antiinflammatory/immunosuppressant activity of methotrexate acts not only upon colonic tissue but also on adjacent adipose tissue. The mesenteric WAT also participated in this experimental model by increasing production of IL-10, an immunomodulatory cytokine. Methotrexate was also responsible for reducing IL-10 production as well as decreasing the basal adiponectin production; both of these adipocytokine could have protective roles during intestinal inflammation. These alterations in cytokine production, observed in mesenteric WAT after methotrexate treatment, correlated with a decreased macrophage infiltration. Thus, methotrexate could act directly on adipose tissue cells, such as adipocytes, macrophages and stromal cells, inhibiting the cytokine production in an unspecific manner and resulting in a decreased production of pro- and anti-inflammatory mediators. Another possibility is that the reduction in the production of TNF-␣ and IL-10 by WAT could be related to reducing amounts of macrophages in this depot. Migration of leukocytes from blood to tissue is a regulated multi-step process,
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involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. The monocyte migration to adipose tissue is believed to be dependent upon monocyte chemoattractant protein (MCP)1 production, and mesenteric WAT is a depot with a high MCP-1 production ability [32]. Intercellular adhesion molecule (ICAM-1), interacting with MAC-1 (a 2 -integrin), seems to be involved in leukocyte migration to adipose tissue [33–34]. Methotrexate may act by inhibiting these early events, preventing the migration of monocytes to adipose tissue [35]. Adiponectin is mainly expressed by mature adipocytes and exerts anti-inflammatory effects on macrophages and endothelial cells. Adiponectin and TNF-␣ suppress each other’s production and antagonize each other’s actions in their target tissue [36]. Thus, a reduction of macrophage infiltration with concomitant reduction of TNF-␣ production in mesenteric WAT, associated with an improvement in colonic inflammatory status, could result in an absence of a local inflammatory response, leading to a decreased production of adiponectin. In conclusion, we suggest that methotrexate exerts therapeutic effects on intestinal inflammation by regulating the shift from Th1 to Th2 cytokines and by reducing other inflammatory parameters in the colon and diminishing the inflammatory environment of mesenteric WAT. Acknowledgements We thank FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, Brazil) for financial support. References [1] Karmiris K, Koutroubakis IE, Kouroumalis EA. Leptin, adiponectin, resistin, and ghrelin—implications for inflammatory bowel disease. Mol Nutr Food Res 2008;52:855–66. [2] Pond CM. Long-term changes in adipose tissue in human disease. Proc Nutr Soc 2001;60:365–74. [3] Sheehan AL, Warren BF, Gear MW, Shepherd NA. Fat-wrapping in Crohn’s disease: pathological basis and relevance to surgical practice. Br J Surg 1992;79:955–8. [4] Peyrin-Biroulet L, Chamaillard M, Gonzalez F, Beclin E, Decourcelle C, Antunes L, et al. Mesenteric fat in Crohn’s disease: a pathogenetic hallmark or an innocent bystander? Gut 2007;56:577–83. [5] Paul G, Schäffler A, Neumeier M, Fürst A, Bataillle F, Buechler C, et al. Profiling adipocytokine secretion from creeping fat in Crohn’s disease. Inflamm Bowel Dis 2006;12:471–7. [6] Schäffler A, Fürst A, Büchler C, Paul G, Rogler G, Schölmerich J, et al. Secretion of RANTES (CCL5) and interleukin-10 from mesenteric adipose tissue and from creeping fat in Crohn’s disease: regulation by steroid treatment. J Gastroenterol Hepatol 2006;21:1412–8. [7] Desreumaux P, Ernst O, Geboes K, Gambiez L, Berrebi D, Müller-Alouf H, et al. Inflammatory alterations in mesenteric adipose tissue in Crohn’s disease. Gastroenterology 1999;117:73–81. [8] Travis SP, Stange EF, Lémann M, Oresland T, Chowers Y, Forbes A, et al. European evidence based consensus on the diagnosis and management of Crohn’s disease: current management. Gut 2006;55:i16–35. [9] Faubion Jr WA, Loftus Jr EV, Harmsen WS, Zinsmeister AR, Sandborn WJ. The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study. Gastroenterology 2001;121:255–60. [10] Tian H, Cronstein BN. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis. Bull Hosp Jt Dis (NYU) 2007;65:168–73. [11] Majumdar S, Aggarwal BB. Methotrexate suppresses NF-kappaB activation through inhibition of IkappaBalpha phosphorylation and degradation. J Immunol 2001;167:2911–20. [12] Gambero A, Maróstica M, Abdalla Saad MJ, Pedrazzoli Jr J. Mesenteric adipose tissue alterations resulting from experimental reactivated colitis. Inflamm Bowel Dis 2007;13:1357–64.
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Methotrexate is effective in reactivated colitis and reduces inflammatory alterations in mesenteric adipose tissue during intestinal inflammation Marcia Aparecida Thomaz a , Simone Coghetto Acedo a , Caroline Candida de Oliveira a , José Aires Pereira a , Denise Gonc¸alves Priolli a , Mario José Saad b , José Pedrazzoli Jr a , Alessandra Gambero a,∗ a b
Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Av. São Francisco de Assis 218, 12916-900 Braganc¸a Paulista, SP, Brazil Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas, SP, Brazil
a r t i c l e
i n f o
Article history: Received 30 January 2009 Received in revised form 7 May 2009 Accepted 10 May 2009 Keywords: TNBS Leptin Adiponectin TNF-␣ IL-10 F4/80 TLR-4 iNOS
a b s t r a c t Mesenteric white adipose tissue hypertrophy and modifications in adipocytokine production are described features of Crohn’s disease. Experimentally, mesenteric white adipose tissue alterations, associated with intestinal inflammation, can be induced in a model of reactivated colitis by repeated administration of intrarectal trinitrobenzenosulfonic acid (TNBS) in ethanol solution. Crohn’s disease patients refractory to corticosteroid treatment are frequently treated with methotrexate; however, there is no information regarding the drug’s effect on adipose tissue alterations and in a reactivated colitis experimental model. Thus, we evaluated the effect of methotrexate upon mesenteric WAT alterations and inflammatory features in experimental colitis in rats. Colitis status was evaluated by macroscopic score, histopathological analysis, myeloperoxidase activity, TNF-␣ and IL-10 expression, as well as iNOS and TLR-4 expression in colon samples. The adipose tissue alterations were assessed by TNF-␣, IL-10, leptin and adiponectin production, as well as by macrophage infiltration evaluation. Methotrexate exerts an anti-inflammatory activity in experimental reactivated colitis by regulating the shift from Th1 to Th2 cytokines, reducing TNF-␣ and improving IL-10 production. Additionally, methotrexate reduces other inflammatory parameters in the colon, such as iNOS and TLR-4 expression. In mesenteric white adipose tissue, methotrexate treatment reduces the production of pro- and anti-inflammatory adipocytokines as well as macrophage infiltration, suggesting that immunosuppressant drugs diminish adipose tissue inflammatory alterations associated with intestinal inflammation. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Accumulating evidence suggests that white adipose tissue (WAT) interacts with inflammation and immune processes, based on the fact that WAT cells (adipocytes, matrix cells, macrophage and mast cells) produce and release several bioactive molecules, collectively called adipocytokines [1]. In addition, WAT can be short-term changed during acute infection and inflammation, as well as long-term changed during diseases such as non-insulin dependent diabetes mellitus and Cushing’s syndrome [2]. Low body weight and development of mesenteric WAT hypertrophy are well-described symptoms of Crohn’s disease [3]. Considered as a characteristic feature of Crohn’s disease, fat-wrapping is defined as a mesenteric WAT extension from the mesenteric attachment that partially covers the small and large intestinal circumference, in association with loss of the bowel-mesentery angle
∗ Corresponding author. Tel.: +55 11 4034 8135; fax: +55 11 4034 1825. E-mail address: [email protected] (A. Gambero). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.05.003
[4]. During recent years, the contribution of fat tissue to Crohn’s disease has been investigated. One hypothesis is that the fat hypertrophy represents a defensive reaction against the primary intestinal inflammation [5]. Corroborating this hypothesis, creeping fat in Crohn’s disease produces anti-inflammatory adipocytokines, such as adiponectin [5] and responds to steroid treatment by improving IL-10 production [6]. However, adipocytes from mesenteric WAT in Crohn’s disease specifically express TNF-␣ [7] and adipose tissue secrets high quantities of leptin [5], proinflammatory adipocytokines that indicate adipose tissue as a causative role in intestinal inflammation. In addition, creeping fat in Crohn’s disease is infiltrated by significant amounts of macrophages that also produce inflammatory cytokines, suggesting that mesenteric adipose tissue could have a pathophysiologic relevance [3]. Other proinflammatory substances, i.e. IL-6 and monocyte chemotactic protein-1, have been reported not to be specifically induced in Crohn’s disease, and are also present in other inflammatory intestinal conditions [5]. The general principles for treating active CD take into consideration the activity, site (ileal, ileocolic, colonic and others), and behavior of the disease (inflammatory, structuring, fistulating, course of disease, response to previous medications, side
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effect profile of medication and extraintestinal manifestation) [8]. Approximately 75% of patients with Crohn’s disease or ulcerative colitis will respond to conventional therapy, including corticosteroids [9]. Corticosteroid-refractory patients require treatment with infliximab and immunosuppressants, such as methotrexate [8]. Methotrexate is a folic acid antagonist and blocks the synthesis of purines and pyrimidines by inhibiting several key enzymes, suppressing transmethylation reactions with accumulation of polyamines, reducing antigen-dependent T-cell proliferation and promoting adenosine release with adenosine-mediated suppression of inflammation [10]. The combination of these effects may be responsible for the anti-inflammatory activity of methotrexate. Down-regulation of inflammatory cytokines, mainly TNF-␣, is also suggested as an important action of methotrexate [11]. Experimentally, the mesenteric WAT alterations associated with intestinal inflammation can be observed in a model of colitis, by intrarectal repeated trinitrobenzenosulfonic acid (TNBS) in ethanol solution administration. The mesenteric WAT depot increases and produces high amounts of TNF-␣ during this process [12]. An increase in infiltrating macrophage has also been observed [13]. The scarce reports regarding the action of drugs upon inflammatory alterations in mesenteric WAT from CD have related in steroid-treated patient [5,6]. Thus, this study intends to evaluate the action of methotrexate upon mesenteric WAT alterations induced by experimental colitis, focusing upon the adipocytokine production and inflammatory alterations, such as macrophage infiltration. Additionally, we characterized the effect of methotrexate upon reactivated colitis, a well-described model that resembles the inflammatory characteristics of CD, evaluating inflammatory parameters, such as cytokine production and iNOS and TLR-4 expression in colon. 2. Materials and methods 2.1. Animals Male, Wistar rats (200–250 g) free of specific pathogens were obtained from CEMIB (State University of Campinas, Campinas, SP, Brazil). The experiments were performed in accordance with the principles outlined by the Brazilian College for Animal Experimentation (COBEA). The rats were maintained in a room with controlled humidity and temperature and were exposed to 12-h light-dark cycles. Twelve hours prior to an experiment, the animals were deprived of food (standard chow), but not water. Studies were carried out using 4–7 rats per group. 2.1.1. Reactivated colitis induced by TNBS and treatment protocol The animals were anesthetized with ketamine/xylazine (1:1, v/v), and colitis was induced by intracolonic instillation of 3 mg of TNBS dissolved in 0.3 ml of 50% ethanol (Sigma, St. Louis, MO). The solution was injected into the colon, 8 cm proximal to the anus using a catheter. The instillation procedure required only a few seconds and the rats were maintained in a vertical position until they recovered from the anesthesia. The same procedure was repeated 14 and 28 days after the first TNBS instillation. Control rats received saline by the same protocol. One half of the colitis animals received methotrexate, intraperitoneally (3 mg kg−1 ; Sigma, St. Louis, MO), divided into two administrations on the 28th and 31th days of the protocol and were then sacrificed at the end of the treatment (35th day). The animals were maintained in collective cages. 2.1.2. Colitis characterization by macroscopic damage, microscopic assessment and myeloperoxidase activity Colons were removed immediately from animals sacrificed by cervical dislocation. The colons were opened longitudinally and
evaluated by two observers unaware of the experimental groups for macroscopically visible damage, as described by Bell et al. [14]. The criteria for assessment of macroscopic colonic damage of each animal were: no damage (no points); hyperemia, no ulcers (1 point); linear ulcer with no significant inflammation (2 points); linear ulcer with inflammation at one site (3 points); two or more sites of ulceration/inflammation (4 points); two or more major sites of ulceration and inflammation or one site of ulceration/inflammation extending 1 cm along the length of the colon (5 points); if damage covers 2 cm along the length of the colon, the score is increased by 1 for each additional centimeter of involvement (6–10 points). The specimens were fixed in 10% buffered formalin and embedded in paraffin. Two sections of 4 m in thickness were cut and stained with hematoxylin–eosin (H&E) for histological evaluation. Colon samples obtained longitudinally from a site of macroscopically detectable inflammation (or a corresponding site in the tissue with no macroscopically detectable inflammation) were homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. For the myeloperoxidase (MPO) assay, 50 L of each sample were added to 200 L of o-dianisidine solution (0.167 mg/mL o-dianisidine dihydrochloride, 0.0005% hydrogen peroxide in 50 mM phosphate buffer, pH 6.0) immediately prior to reading the change in absorbance at 460 nm over 5 min using a microplate reader (Multiscan MS, Labsystems). 2.1.3. Short-term culture of total adipose tissue specimens Secretion of adipocytokines was evaluated by measuring the amount of adipocytokines released from WAT cells to the incubation buffer without any stimulus, using commercial EIA kits (TNF-␣ and IL-10 from GE Healthcare, UK; leptin and adiponectin from B-Bridge International, Mountain View, CA). Mesenteric WAT was dissected from proximal colon immediately after laparotomy. Biopsies from mesenteric adipose tissue were minced and incubated with constant shaking (60 cycles/min) for 4 h at 37 ◦ C in a 5% CO2 atmosphere with Medium 199 (Gibco, Gaithersburg, MD) supplemented with 1% bovine serum albumin. After 4 h, the culture medium was collect and stored at −80 ◦ C. 2.1.4. Protein extraction and Western immunoblot analysis For evaluation of colonic and WAT additional inflammatory markers, the abdominal cavities of anesthetized rats were opened and adipose tissue fragments from mesenteric adipose tissue and colon biopsies were excised and immediately homogenized in solubilizing buffer at 4 ◦ C [1% Triton X-100, 100 mM Tris–HCl (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium orthovanadate, 2.0 mM PMSF, and 0.1 mg aprotinin/ml]. Insoluble material was removed by centrifugation for 20 min, at 9000 × g, at 4 ◦ C. The protein concentration of the supernatants was determined by the Biuret method. The extracts were treated with Laemmli sample buffer containing 100 mM dithiothreitol and heated in a boiling water bath for 5 min, after which samples were subjected to SDS-PAGE in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For immunoblot experiments, 0.15 mg of protein extracts from each tissue were separated by SDS-PAGE, transferred to nitrocellulose membranes and blocked for 2 h with blocking buffer (Blocking agent 5% (GE Healthcare) in Tris buffer solution). Membranes were then incubated overnight with antiTNF-␣, anti-iNOS, anti-IL-10, anti--actin and anti-F4/80 (1:1000; Santa Cruz, USA). The nitrocellulose membranes were developed using commercial chemoluminescent Kits (GE Healthcare, UK). Band intensities were quantified by optical densitometry (Scion Image software, ScionCorp, Frederick, MD) of the developed autoradiography.
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Table 1 Parameters evaluation in controls, colitis and methotrexate treated animals. Group
Body weight (g)
Damage score (0–10)
Control Colitis Methotrexate
403.8 ± 5.7 346.2 ± 12.9** 360.0 ± 11.9
1.8 ± 0.5 6.2 ± 0.6** 3.2 ± 0.8#
Myeloperoxidase activity (U/g tissue) 1.32 ± 0.18 17.95 ± 2.33** 8.11 ± 2.70#
Data are expressed as mean ± SEM. ** p < 0.01 when compared with control group. # p < 0.05 when compared with colitis (n = 5).
2.2. Statistical analysis All data are expressed as the mean ± SEM Comparisons among groups of data were performed using one-way ANOVA followed by Dunnett multiple comparisons test. An associated probability (p value) of less than 5% was considered to be significant. 3. Results 3.1. Colitis evaluation The colitis was characterized by a higher macroscopic score and by an increased myeloperoxidase activity in colons from
TNBS/ethanol rats. Myeloperoxidase activity is a biochemical marker of infiltrating neutrophils. The colitis animals also present a reduced body weight, when compared with healthy animals (Table 1). The histological analyses of colon samples from colitis animals revealed the presence of ulcerations, infiltrating inflammatory cells in mucosa and submucosa and edema in the submucosal area (Fig. 1B and C). In some cases, as shown in Fig. 1C, it was possible to observe a transmural inflammation. These alterations were not found in the colon samples from controls, where a typical morphology could be observed (Fig. 1A). Methotrexate-treated animals presented a reduced level of macroscopically observed damage and myeloperoxidase activity when compared with colitis non-treated animals. The treatment protocol used did not modify
Fig. 1. Histopathological appearance of colonic tissue in rats. (A) Normal colonic mucosa of control rats [hematoxylin and eosin (H&E) 40×]. (B and C) Colonic mucosa of colitis rats. Large ulcerations (U) and submucosa edema with infiltrating inflammatory cells (arrows) (H&E 100×). (D and E) MTX-treated colitis rats. Moderated infiltrating inflammatory cells (arrows) (H&E 40× and 100×, respectively).
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Fig. 3. Level of iNOS protein in colon from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on colon protein extracts with antibodies against iNOS (A) and -actin (B). The results are representative of one experiment. Lower panels show the densitometry quantification of iNOS (C) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. **p < 0.01 when compared with control group; # p < 0.05 when compared with colitis group.
3.1.1. Adipocytokine production Mesenteric WAT from colitis animals released higher amounts of TNF-␣ and IL-10, when compared with the same depot obtained from healthy animals (Table 2). Our experimental procedure does not modify the mesenteric WAT production of leptin and adiponectin. After methotrexate treatment, significant reductions were observed in ex vivo TNF-␣, IL-10 and adiponectin release (Table 2).
Fig. 2. Levels of TNF-␣ and IL-10 in colon from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on colon protein extracts with antibodies against TNF-␣ (A), IL-10 (D) and -actin (B and E). The results are representative of one experiment. Lower panels show the densitometry quantification of TNF-␣ (C) and IL-10 (F) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. *p < 0.05 and **p < 0.01 when compared with control group; # p < 0.05 and ## p < 0.01 when compared with colitis group.
the animals final body weight (Table 1). Microscopic analysis also revealed a reduction in inflammatory alterations. A reduction in edema was also observed, as well as some infiltrating inflammatory cells (Fig. 1D and E). The cytokine expression in colonic tissue was evaluated in all groups. TNBS/ethanol administrations were able to induce a greater TNF-␣ and a lower IL-10 expression in the colon sample. Methotrexate treatment was able to reduce TNF-␣ and, in addition, restore IL-10 expression to the same levels as those observed for control animals (Fig. 2). Additional inflammatory markers were also evaluated, such as, iNOS expression. Colitis animals presented a higher level of iNOS protein in the colon and methotrexate efficiently reduced this level (Fig. 3). The Toll-like receptor (TLR)-4 was also increased in colitis animals and its level was reduced after treatment (Fig. 4).
Fig. 4. Level of TLR-4 protein in colon from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on colon protein extracts with antibodies against TLR-4 (A) and -actin (B). The results are representative of one experiment. Lower panels show the densitometry quantification of TLR-4 (C) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. *p < 0.05 and **p < 0.01 when compared with control group; # p < 0.05 and ## p < 0.01 when compared with colitis group.
M.A. Thomaz et al. / Pharmacological Research 60 (2009) 341–346 Table 2 Release of adipocytokines from mesenteric adipose tissue in control, colitis and methotrexate treated groups. Control TNF-␣ (pg ml) IL-10 (ng ml) Leptin (g ml) Adiponectin (g ml)
58.25 0.98 4.13 2.30
± ± ± ±
Colitis 6.51 0.25 0.96 0.13
157.49 3.27 3.89 2.29
Methotrexate ± ± ± ±
6.63** 0.96* 0.74 0.15
93.73 1.28 4.47 1.55
± ± ± ±
14.04# 0.14# 0.67 0.20#
Data are expressed as mean ± SEM (n = 5). * p < 0.05, when compared with control group. ** p < 0.01, when compared with control group. # p < 0.05, when compared with colitis group.
Fig. 5. Level of F4/80 protein in mesenteric adipose tissue from control, colitis and methotrexate treated animals (methotrexate). Western blot analysis was performed on adipose tissue protein extracts with antibodies against F4/80 (A) and -actin (B). The results are representative of one experiment. Lower panels show the densitometry quantification of F4/80 (C) levels, normalized by densitometry quantification of -actin level for the same sample. Data are expressed as the mean ± SEM of four experiments. **p < 0.01 when compared with control group; # p < 0.05 when compared with colitis group.
3.1.2. Macrophage infiltration into mesenteric adipose tissue In colitis animals, the mesenteric adipose tissue presented a higher level of F4/80 protein expression, indicating the presence of infiltrated macrophages (Fig. 5). Methotrexate treatment was able to reduce this infiltration. 4. Discussion Administration of a hapten, combined with a barrier breaker, such as ethanol, into the colon results in mucosal injury and inflammation that resemble the colonic inflammation present in inflammatory bowel disease in humans. This injury gradually subsides over the weeks, but it can be reactivated with an additional TNBS/ethanol administration [15]. In the active phase, it is possible to observe a transmural inflammation associated with diarrhea, weight loss, and an imbalance activation of Th1- and Th2-lymphocytes, resulting in an induction of an IL-12-driven inflammation with a massive Th1-mediated response, including TNF-␣ production [16–18]. Anti-inflammatory/immunosuppressant drugs, such as, dexamethasone, cyclosporine A and 5-aminosalicylic acid reduced colonic damage score and MPO activity in a reactivated colitis model [15]. However, there are no previous reports on the actions of methotrexate in reactivated or acute colitis in rats. In acute dinitrobenzenesulfonic acid (DNBS)-induced colitis in mice, methotrexate at dosage of 0.5 mg/kg/day produces controversial
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results [19]. In dextran sulfate sodium (DSS)-induced colitis in mice, methotrexate, at dosage of 1 mg/kg/day, reduces the inflammatory score [20]. In our study, the dosage chosen was based on several literature reports regarding the immunosuppressant and anti-inflammatory actions of methotrexate in arthritis models, including the ability of methotrexate to inhibit TNF-␣ in these models [21,22]. Methotrexate, at a dosage of 3 mg/kg/week, was able to significantly reduce the inflammatory score, histopathological alterations, MPO activity and colonic TNF-␣ expression, confirming that the dosage chosen was effective for TNF-␣ inhibition. Additionally, we found a reduced IL-10 level in the colon of colitis animals and methotrexate was able to reverse this reduction. IL-10 is a Th2 cytokine, produced by a variety of cells including B and T cells, macrophages, mast cells and intestinal epithelial cells, with a keyrole in the maintenance of intestinal immune homeostasis by its immunomodulatory activity [23]. The immunomodulatory activity of IL-10 is based upon its ability to inhibit both the synthesis of proinflammatory cytokines (IL-1, TNF-␣ and IL-6) and of Th2 cell-derived cytokines (IL-4 and IL-5) [24]. Dexamethasone and curcumin treatment in TNBS colitis also results in increased colonic IL-10 production, parallel to TNF-␣ inhibition [25]. The mechanism by which low dose methotrexate exerts its anti-inflammatory effect in rheumatoid arthritis patients seems also to be related to an induction of IL-10 secretion (a Th2 cytokine) and a reduction in Th1 profile in mononuclear cells [26]. We also verified the ability of methotrexate to modify additional intestinal inflammatory parameters, such as iNOS and Toll-like receptor (TLR)-4. It is thought that Crohn’s disease arises from a dysregulated mucosal immune response to luminal bacteria. TLRs, which are pattern-recognition receptors expressed by both immune and non-immune cells, play a pivotal role in host/microbial interactions and have two distinct functions that protect against infection and control tissue homeostasis, depending on the recognition of pathogens or commensals. An increased TLR4 expression was found in the terminal ileum of CD patients with active disease [27]. In TNBS-colitis, the level of colonic TLR-4 protein was also up-regulated [28,29]. We observed that methotrexate treatment effectively normalized the up-regulation of TLR-4 in colitis animals, suggesting that this pathway could contribute to attenuate the gut inflammation. An elevated level of peroxynitrite, produced from increased colonic iNOS expression and activity, is a major contributor to colon epithelial apoptosis during inflammation [30]. Methotrexate treatment also contributed to decrease the iNOS expression in the colon, as described for other anti-inflammatory substances [31]. Finally, we evaluated the ability of methotrexate to modify the production of adipocytokine in mesenteric WAT during colitis. Methotrexate was able to significantly reduce the production of TNF-␣ by WAT, suggesting that the antiinflammatory/immunosuppressant activity of methotrexate acts not only upon colonic tissue but also on adjacent adipose tissue. The mesenteric WAT also participated in this experimental model by increasing production of IL-10, an immunomodulatory cytokine. Methotrexate was also responsible for reducing IL-10 production as well as decreasing the basal adiponectin production; both of these adipocytokine could have protective roles during intestinal inflammation. These alterations in cytokine production, observed in mesenteric WAT after methotrexate treatment, correlated with a decreased macrophage infiltration. Thus, methotrexate could act directly on adipose tissue cells, such as adipocytes, macrophages and stromal cells, inhibiting the cytokine production in an unspecific manner and resulting in a decreased production of pro- and anti-inflammatory mediators. Another possibility is that the reduction in the production of TNF-␣ and IL-10 by WAT could be related to reducing amounts of macrophages in this depot. Migration of leukocytes from blood to tissue is a regulated multi-step process,
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involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. The monocyte migration to adipose tissue is believed to be dependent upon monocyte chemoattractant protein (MCP)1 production, and mesenteric WAT is a depot with a high MCP-1 production ability [32]. Intercellular adhesion molecule (ICAM-1), interacting with MAC-1 (a 2 -integrin), seems to be involved in leukocyte migration to adipose tissue [33–34]. Methotrexate may act by inhibiting these early events, preventing the migration of monocytes to adipose tissue [35]. Adiponectin is mainly expressed by mature adipocytes and exerts anti-inflammatory effects on macrophages and endothelial cells. Adiponectin and TNF-␣ suppress each other’s production and antagonize each other’s actions in their target tissue [36]. Thus, a reduction of macrophage infiltration with concomitant reduction of TNF-␣ production in mesenteric WAT, associated with an improvement in colonic inflammatory status, could result in an absence of a local inflammatory response, leading to a decreased production of adiponectin. In conclusion, we suggest that methotrexate exerts therapeutic effects on intestinal inflammation by regulating the shift from Th1 to Th2 cytokines and by reducing other inflammatory parameters in the colon and diminishing the inflammatory environment of mesenteric WAT. Acknowledgements We thank FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo, Brazil) for financial support. References [1] Karmiris K, Koutroubakis IE, Kouroumalis EA. Leptin, adiponectin, resistin, and ghrelin—implications for inflammatory bowel disease. Mol Nutr Food Res 2008;52:855–66. [2] Pond CM. Long-term changes in adipose tissue in human disease. Proc Nutr Soc 2001;60:365–74. [3] Sheehan AL, Warren BF, Gear MW, Shepherd NA. Fat-wrapping in Crohn’s disease: pathological basis and relevance to surgical practice. Br J Surg 1992;79:955–8. [4] Peyrin-Biroulet L, Chamaillard M, Gonzalez F, Beclin E, Decourcelle C, Antunes L, et al. Mesenteric fat in Crohn’s disease: a pathogenetic hallmark or an innocent bystander? Gut 2007;56:577–83. [5] Paul G, Schäffler A, Neumeier M, Fürst A, Bataillle F, Buechler C, et al. Profiling adipocytokine secretion from creeping fat in Crohn’s disease. Inflamm Bowel Dis 2006;12:471–7. [6] Schäffler A, Fürst A, Büchler C, Paul G, Rogler G, Schölmerich J, et al. Secretion of RANTES (CCL5) and interleukin-10 from mesenteric adipose tissue and from creeping fat in Crohn’s disease: regulation by steroid treatment. J Gastroenterol Hepatol 2006;21:1412–8. [7] Desreumaux P, Ernst O, Geboes K, Gambiez L, Berrebi D, Müller-Alouf H, et al. Inflammatory alterations in mesenteric adipose tissue in Crohn’s disease. Gastroenterology 1999;117:73–81. [8] Travis SP, Stange EF, Lémann M, Oresland T, Chowers Y, Forbes A, et al. European evidence based consensus on the diagnosis and management of Crohn’s disease: current management. Gut 2006;55:i16–35. [9] Faubion Jr WA, Loftus Jr EV, Harmsen WS, Zinsmeister AR, Sandborn WJ. The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study. Gastroenterology 2001;121:255–60. [10] Tian H, Cronstein BN. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis. Bull Hosp Jt Dis (NYU) 2007;65:168–73. [11] Majumdar S, Aggarwal BB. Methotrexate suppresses NF-kappaB activation through inhibition of IkappaBalpha phosphorylation and degradation. J Immunol 2001;167:2911–20. [12] Gambero A, Maróstica M, Abdalla Saad MJ, Pedrazzoli Jr J. Mesenteric adipose tissue alterations resulting from experimental reactivated colitis. Inflamm Bowel Dis 2007;13:1357–64.
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