Treatment with Met-RANTES decreases bacterial translocation in experimental colitis

The American Journal of Surgery 191 (2006) 77– 83

Scientific papers– clinical (international)

Treatment with Met-RANTES decreases bacterial translocation in experimental colitis Can Kucuk, M.D.a,*, Erdogan Sozuer, M.D.a, Sebnem Gursoy, M.D.b, Ozlem Canoz, M.D.c, Tarık Artıs, M.D.a, Alper Akcan, M.D.a, Hızır Akyıldız, M.D.a, Sebahattin Muhtaroglu, M.D.d a b

Department of General Surgery, Erciyes University School of Medicine, 38039, Kayseri, Turkey Department of Gastroenterology, Erciyes University School of Medicine, 38039, Kayseri, Turkey c Department of Pathology, Erciyes University School of Medicine, 38039, Kayseri, Turkey d Department of Biochemistry, Erciyes University School of Medicine, 38039, Kayseri, Turkey Manuscript received January 26, 2005; revised manuscript April 28, 2005

Abstract Background: During colitis, epithelial function is impaired, leading to increased bacterial translocation. Recent studies have shown the important role of proinflammatory cytokines and chemokines, including RANTES (regulated on activation, normal T-cell expressed and secreted), in inflammatory bowel diseases (IBDs). In this study, we evaluated the role of Met-RANTES, an antagonist of the RANTES receptor, on the impairment of bacterial translocation in a rat model of colitis. Methods: Rats were randomly assigned to 3 groups. group 1 ⫽ control, group 2 ⫽ experimental colitis, and group 3 ⫽ colitis plus Met-RANTES treatment. On day 7 after colitis was induced, plasma tumor necrosis factor-␣ colon tissue myeloperoxidase and portal blood endotoxin levels were measured. Lymph node, liver, and spleen culture quantified bacterial translocation. Results: Met-RANTES treatment resulted in significant decreases in colonic damage as well as bacterial translocation in experimental colitis. Conclusions: These results suggest that chemokine receptor antagonists may potentially be useful in the treatment of IBDs. © 2006 Excerpta Medica Inc. All rights reserved. Keywords: Bacterial translocation; Colitis; Cytokine; Experimental; RANTES

Inflammatory bowel diseases (IBDs), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), are chronic relapsing inflammatory conditions of unknown etiology. IBDs are characterized by chronic inflammation at various sites in the gastrointestinal tract. Both cause diarrhea, which may be profuse and bloody. Disease is characterized by continuous infiltration of affected tissues by inflammatory cells from the circulation. This influx results in further tissue-destructive inflammatory processes [1]. The indigenous intestinal flora and an intact mucosa are vital components of body defenses against luminal pathogenic bacteria. In IBDs, the intestinal mucosal barrier is disrupted by inflammation and ulceration; in these circum* Corresponding author. Tel.: ⫹1-90-532-303-1922; fax: ⫹1-90-352437-4912. E-mail address: [email protected]

stances, translocation of enteric bacteria and their products through the bowel wall to extra intestinal sites may result [2]. With regard to endotoxemia and its relationship with severity of disease, Wellmann et al found endotoxemia in 17 of 18 (94%) CD patients hospitalized for an acute exacerbation, which in their opinion indicated an association between endotoxemia and active disease [3]. In contrast, enteric bacteria have been isolated from the mesenteric lymph node (MLN) complex in patients with CD [4]. Gram-negative bacilli have been cultured in the portal blood and from liver biopsy samples in patients with UC undergoing colectomy [5]. In addition, circulating agglutinating antibodies against various enteric bacteria have been identified in active UC [6]. The exact pathogenesis in IBD is poorly understood, but evidence exists that IBD involves interactions between the immune system, genetic susceptibility, and the environment

0002-9610/06/$ – see front matter © 2006 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2005.10.005

78

C. Kucuk et al. / The American Journal of Surgery 191 (2006) 77– 83

[1]. Recent studies have shown the important role of proinflammatory cytokines and chemokines, including RANTES (regulated on activation, normal T-cell expressed and secreted), in IBDs [7–9]. Chemokines are proinflammatory proteins that participate in immune and inflammatory responses through the chemoattraction and activation of leukocytes [10]. RANTES is a CC chemokine that promotes the recruitment and activation of inflammatory cells such as monocytes, lymphocytes, mast cells, and eosinophils [11]. RANTES is a ligand for the chemokine receptors CCR1, CCR3, and CCR5, and increased expression of RANTES has been observed in vivo in inflammatory diseases such as glomerulonephritis, adjuvant-induced arthritis, IBDs, and pancreatitis [12,13]. Met-RANTES was initially produced during the synthesis of recombinant RANTES by the extension of the product with a single methionine residue. The resulting protein was found to have no biologic activity but was found to bind to CCR1 and act as a receptor antagonist [14] in conditions such as collagen-induced arthritis, pancreatitis, colitis, and acute renal transplant rejection [8,13]. Met-RANTES has been shown to decrease inflammation. Genetic deletion of CCR1 decreased lung damage in cerulein-induced pancreatitis in mice [15]. Several studies recently conducted demonstrated significant increase in RANTES expression by intestinal mucosa specimens from patients with IBD [8,9,16]. In such patients, there appears to be loss of the normal control mechanisms that regulate chemokine secretion, resulting in increased chemotactic signal, increased recruitment and activation of immunocytes, and ongoing inflammation. Interference with this pathologic chemokine secretion or activity by blocking target receptors represents a potential novel therapeutic strategy in chronic inflammatory conditions such as IBDs [8]. The aim of this study was to determine whether the effect of Met-RANTES had any influence on the impairment of bacterial translocation caused by experimental trinitrobenzene sulphonic acid (TNBS) colitis in rats.

Methods Animal preparation The study was performed at Erciyes University Hakan Cetinsaya Experimental Research Centre. Forty-five male Wistar albino rats bred in the same research center, weighing between 180 and 210 g at 25 to 30 weeks of age, were used for this study. The rats were fed a standard chow pellet diet, had free access to water, and were maintained on a 12-hour light-to-dark cycle. Water only was provided during the 12 hours preceding the experiments. The Animal Care Committee of the University of Erciyes approved all procedures in this study. The animals were divided randomly into 3 groups containing 15 rats each: group 1 (control) ⫽ normal animals, group 2 (colitis) ⫽ induction of

experimental colitis without further treatment, and group 3 (Met-RANTES) ⫽ induction of experimental colitis plus administration of Met-RANTES. Induction of colitis Colitis was induced as previously described [17]. Briefly, rats were lightly anesthetized with halothane, and an infant feeding tube (Unoplast A/S DK 330; Handested, Denmark) fitted onto a blunt 18-gauge needle was inserted rectally. The tip of the tube was placed approximately 8 cm into the colon and 25 mg 2,4,6-trinitrobenzene sulphonic acid (Sigma, St. Louis, Missouri) mixed with 0.25 mL 30% ethanol (TNBS-E) was intracolonically instilled. After instillation, the rats were supported in the supine position until recovery from anesthesia to prevent immediate anal leakage of the instillate. The control group rats received intracolonic saline. Effects of Met-RANTES Met-RANTES (Serono Pharmaceutical Institute, Geneva, Switzerland) is a modified chemokine that binds with high affinity, but does not activate, the chemokine receptor CCR1 [14]. It was administered to rats as an intravenous dose of 200 ␮g/rat/d for 7 days. Colitis and control rats received sterile saline at the same volume and the same times. The 200-␮g dose of Met-RANTES used in this study has been shown to be effective in decreasing inflammation in a rat model of TNBS-E colitis and a rodent model of glomerulonephritis [8,18]. All rats were fed with standard rat chow for 7 days. On day 8 after the beginning of TNBS treatment, the rats were anesthetized using intraperitoneal administration of ketamine hydrochloride (Ketalar; Parke Davis-EWL Istanbul, Turkey) (20 mg/kg body weight), and they breathed spontaneously throughout the procedures. The abdominal skin was disinfected with 1% cetrimide– chlorhexidine solution. All procedures were performed under sterile conditions by 1 surgeon. Midline laparotomy was performed with a 3-cm incision, 2-mL portal and 2-mL systemic blood removal for biochemical analysis and blood cultures. Liver, spleen, and MLNs were removed for the determination of bacterial translocation, and colon was removed for the analysis of myeloperoxidase (MPO) activity and histologic examination. At the end of the surgical procedure, all the rats were killed by overdose ether inhalation. Determination of serum tumor necrosis factor-␣ The level of serum tumor necrosis factor-␣ (TNF-␣) was determined using a specific enzyme-linked immunosorbent assay kit (Rat Micro Titer TNF-␣ Assay Kit, Sigma). The results were expressed as pg/mL.

C. Kucuk et al. / The American Journal of Surgery 191 (2006) 77– 83

79

Table 1 Colon macroscopic scoring system

Table 2 Histopathologic grading scale of TNBS-E colitis

Score

Criteria

Microscopic findings

0 1 2 3 4 5

No damage Hyperemia Hyperemia and thickening without ulceration Ulceration at a single site Two or more sites of ulceration or inflammation Ulceration or inflammation extending ⬎1 cm along the length of colon Damage covering ⬎2 cm along the length of colon, with the score being increased by 1 for each additional centimeter of involvement

Grade of colitis 0 I

No damage Mild mucosal and/or submucosal inflammatory infiltrate and edema: punctuate mucosal erosions often associated with capillary proliferation, muscularis mucosa intact Grade I changes involving 50% of the specimen Prominent inflammatory infiltrate and edema frequently with deeper areas of ulceration extending through the muscularis mucosa into the submucosa, rare inflammatory cells invading the muscularis propria but without muscle necrosis Grade III changes involving 50% of the specimen Extensive ulceration with coagulative necrosis bordered inferiorly by numerous neutrophils and lesser numbers of mononuclear cells, necrosis extends deeply into the muscularis propria Grade V changes involving 50% of the specimen

6–10

Determination of plasma endotoxin level Plasma endotoxin levels in portal venous blood were assayed using quantitative modification of the Limulusamebocyte-lysate test (Sigma) [19].

II III

IV V

VI

TNBS-E ⫽ 2,4,6-trinitrobenzene plus 0.25 mL 30% ethanol.

Bacterial translocation assay Portions of the liver, spleen, and MLNs were immediately homogenized in saline. Aerobic cultures were performed with blood and eosin-methylene blue agar at 37°C for 24 hours. Anaerobic cultures were performed using anaerobic blood agar in Gas-Pak jars at 37°C for 24 hours. Colonies were identified by standard microbiologic methods. Blood samples, 1 mL, were cultured in tryptic soy medium at 37°C for 7 days. Positive cultures were transferred to blood and EMB agar. Colonies were identified using standard bacteriologic methods. Assessment of colonic damage The distal colon was removed, opened longitudinally, and cleared of fecal material with a gentle spray of 0.9% saline. The freshly opened colonic segments were pinned out on a wax block and examined by a pathologist who was blinded to the treatment. Colon macroscopic score The extent of mucosal damage was assessed using the colon macroscopic scoring system of Wallace et al (Table 1) [20]. Colon microscopic score The tissue samples were fixed with 10% formaldehyde solution for 24 hours and transferred to 70% ethanol solution. The tissues were embedded in paraffin; 4-␮-thick sections were made, and histologic examination was performed under light microscopy after hematoxylin and eosin staining. A pathologist who was unaware of the animals’ groupings evaluated the specimens. Tissues were assessed for the presence and activity of colitis as well as the extent of the tissue damage using a large number of serial sections. Co-

lonic inflammation was assessed using a modification of the histopathologic grading system of Macpherson and Pfeifer (Table 2) [21]. Myeloperoxidase measurement in colon tissue After being scored, a sample of the distal colon was frozen for subsequent measurement of MPO activity as an index of granulocyte infiltration [20]. The method described by Bradley et al was used to measure MPO activity in colon homogenates [22]. The previously frozen colon tissues were homogenized for 30 seconds in 4 mL 20-mmol/L potassium phosphate buffer, pH 7.4, and centrifuged for 30 minutes at 40,000 g at 4°C. The pellet was resuspended in 4 mL 50-mmol/L potassium phosphate buffer, pH 6.0, containing 0.5g/dL hexadecyltrimethyl ammonium bromide. Samples were sonicated for 90 seconds at full power, incubated in a 60°C water bath for 2 hours and centrifuged. The supernatant (0.1 mL) was added to 2.9 mL 50-mmol/L potassium phosphate buffer, pH 6.0, containing 0.167 mg/mL dianisidine and 0.0005% hydrogen peroxide. Absorbance of 460 nm of visible light (A460) was measured for 3 minutes. The results were expressed as units of enzyme activity per gram of wet tissue weight (U/g tissue). Statistical analysis The data were analyzed using SPSS for Windows version 10.0.1 (SPSS, Chicago, Illinois). Analysis of variance (ANOVA) was used to compare serum TNF-␣, endotoxin, and colon MPO levels among he 3 groups. Posthoc intergroup comparisons were made using Scheffe test. The results were expressed as means ⫾ SD. Mann-Whitney U test was used to compare bacterial translocation and histologic damage score. Statistical significance was defined at P ⬍ .05.

80

C. Kucuk et al. / The American Journal of Surgery 191 (2006) 77– 83

Fig. 1. Serum TNF-␣ and endotoxin concentrations in rats. Values represent mean (⫾ SEM). #P ⬍ .01 versus colitis and Met-RANTES groups. *P ⬍ .01 for Met-Rantes versus colitis groups. RANTES ⫽ regulated on activation, normal T-cell expressed and secreted; TNF-␣ ⫽ tumor necrosis factor-␣.

Results Plasma TNF-␣ and endotoxin levels in the colitis and Met-RANTES groups were significantly increased at day 8 compared with the control groups (P ⬍ .01). The increase in the Met-RANTES group was significantly less than the increase in the colitis group (P ⬍ .01; Fig. 1). In the control group, no bacterial growth was detected at day 8. TNBS-E colitis caused a significant increase in the frequencies of bacterial translocation in liver, spleen, MLNs, and portal blood. Compared with the colitis group, Met-RANTES decreased the bacterial translocation in liver, spleen, MLNs, and portal blood (Table 3) (P ⬍ .01). Species of bacteria isolated from MLNs, liver, spleen, and portal blood were not different between the colitis and MetRANTES–treated groups. Translocating bacteria were identified as Escherichia coli, Pseudomonas aeruginosa, Mycobacterium morgani, and Enterococcus species. The colon MPO levels were higher in the colitis and Met-RANTES groups compared with the control group (P ⬍ .01). However, this increase was less marked in the Met-RANTES group (P ⬍ .01; Fig. 2). Histopathologic evaluation of the control group was in normal limits. In the colitis group, the damage comprised broad mucosal ulcers with a surface layer of necrotic slough, accumulation of mesenteric fat, and fibrinous adhe-

Fig. 2. Colon myeloperoxidase concentrations in rats. Values represent mean (⫾ SEM). #P ⬍ .01 versus colitis and Met-RANTES groups. *P ⬍ .01 for Met-RANTES versus colitis groups. RANTES ⫽ regulated on activation, normal T-cell expressed and secreted.

sions to the bowel. Acute colonic damage with hemorrhage and bowel-wall thickening were also observed. However, the treatment with Met-RANTES significantly decreased the colon macroscopic damage scores (P ⬍ .01; Fig. 3). Microscopic examination showed the very low level of damage or inflammation in rats treated with Met-RANTES, whereas in the colitis rats, there was widespread destruction of the mucosa with extensive, transmural infiltration of neutrophils, monocytes, and lymphocytes. In the rats treated with Met-RANTES in which a macroscopic response was evident, there was minimal damage to the surface epithelium, and there was mild inflammation of the mucosa but no transmural inflammation.

Comments CD and UC are the 2 major clinical forms of IBD in developed countries, affecting mostly young people in a chronic and often debilitating way. Symptoms include diarrhea, weight loss, and abdominal pain. An increase in incidence has been noticed in the last decade, and the actual combined prevalence of IBDs is now estimated to be 150 to 200/100,000 people in Europe and North America [23]. Although experimental animal models of colitis are of value in eliciting some of the mechanisms of IBDs, the different models differ in their relative use [7]. Most animal models reported lack of certain characteristics of IBDs and

Table 3 Incidence of bacterial translocation in the different groups Distant organ MLNs Liver Spleen Portal blood Systemic blood

Control* 0† 0† 0† 0‡ 0‡

Colitis*

Met-RANTES*

11 9 7 7 5

4# 2# 2# 1# 0#

MLNs ⫽ mesenteric lymph nodes; RANTES ⫽ regulated on activation, normal T-cell expressed and secreted. * Number of positive tests of all animals tested (15 rats group). † P ⬍ .01 versus colitis and Met-RANTES groups. ‡ P ⬎ .05 versus Met-RANTES groups. # P ⬍ .01 versus colitis group.

Fig. 3. Colon macroscopic and microscopic scores of rats. Values represent mean (⫾ SEM). #P ⬍ .01 versus colitis and Met-RANTES groups. *P ⬍ .01 for Met-RANTES versus colitis groups. RANTES ⫽ regulated on activation, normal T-cell expressed and secreted.

C. Kucuk et al. / The American Journal of Surgery 191 (2006) 77– 83

model acute events rather than the chronic features typical of IBDs. TNBS colitis has been well characterized and has clinical, biochemical, and pathologic similarities to colonic CD. Animals with TNBS colitis have responded to drugs useful in IBDs [20]. This observation, together with the availability of a quantitative scoring system, makes it a useful system for the evaluation of new therapeutic agents. We therefore used TNBS-induced colitis, which has the dual advantage of chronicity and acuteness so often seen in patients with IBD. Some other advantages of this model of colitis are its reasonable reproducibility and its value in assessing the efficacy of therapeutic agents commonly used in the treatment of colitis [7,20]. The gastrointestinal tract is continuously exposed to toxins, microbes, microbial products, and other antigens. The intestinal epithelium plays several important functions, such as acting as a physical barrier that limits the entry of luminal substances and microorganisms into the lamina propria [24], in protecting an organism from the potential deleterious effects of these agents. Berg et al described bacterial translocation as the passage of viable bacteria through the epithelial mucosa into the lamina propria and then to the MLNs and possibly other tissues [25]. Originally, bacterial translocation meant not only the migration of intestinal bacteria through the gut (the mechanisms and routes of which have never been identified) but also the consequences of this phenomenon, ie, the infection of extraintestinal tissue with a subsequent systemic inflammatory response, possibly even leading to sepsis with multiple-organ dysfunction [26]. The latter indicates that the immune system is no longer capable of eliminating migrating bacteria and/or inflowing toxins and considers the fact that gut-barrier dysfunction may be attributed to both morphologic and functional disorders. There appear to be 3 main mechanisms that promote bacterial translocation: impaired host defenses, physical damage of the intestinal mucosa, and disruption of the ecology of the indigenous gut flora, resulting in intestinal bacterial overgrowth [25]. Along with an obvious physical disruption of the epithelial barrier by inflammation and ulceration and, as outlined previously, disturbed bacterial defenses with loss of communal stability and decreased colonization resistance [27], at least 2 of these factors are features of IBDs. Several human studies have indicated that bacterial translocation also occurs in IBDs. Eade and Brooke demonstrated portal bacteremia in 24% and systemic bacteremia in 1% of patients who underwent elective colectomy for ulcerative colitis [5]. They also found a positive correlation between endotoxin plasma levels and clinical disease severity. In addition, bacteria were cultured from liver biopsy samples in 11% of these patients. Ambrose et al cultured intestinal bacteria from the ileal serosa in 27% and from the MLNs in 33% of patients undergoing surgery for CD [4]. In our study, both systemic and portal bacteremia were demonstrated, and it was shown that bacteria could be cultured from MLNs, liver, and spleen in the

81

TNBS-E colitis group. All organ cultures were positive for enteric species. The extent of this translocation of bacteria from colonic lumen correlated positively with the colon macroscopic and microscopic score, suggesting that in this model, disruption of the physical barrier is an important factor in promoting bacterial translocation. Chemokines are small (8 to 14 kd) proinflammatory proteins that have a broad range of activities involved in the recruitment and function of specific populations of leukocytes at sites of inflammation; they also have an important role in the initiation and maintenance of the host inflammatory response [28]. These specialized cytokines play a critical part in the generation of cellular inflammation, both in the protective responses to invading pathogens and in the pathologic processes associated with infection and immunemediated diseases. Chemokines are more than simple chemotactic factors because they are also implicated in leukocyte activation, angiogenesis, and antimicrobial functions. The important role of chemokines in a wide range of inflammatory diseases suggests that they may be useful targets for therapeutic intervention. Many studies have claimed that chemokines may play an important role in the immunopathogenesis of IBDs based on the findings of increased expression of several chemokines in the colonic mucosa of IBD patients [9,29]. For example, Mazzucchelli et al described an increase in the numbers of MCP-1– and RANTES-expressing cells in the intestinal mucosa of patients with IBD [9]. Ajuebor et al examined 4 chemokines during the chronic phase of colitis, and only RANTES tissue levels were significantly increased [8]. mRNA expression for the RANTES receptors CCR1 and CCR5 was also significantly increased during the chronic phase of colitis. RANTES is the natural ligand of CCR5. During intestinal inflammation, CCR5 expression is upregulated on highly activated Th1-type intestinal lamina propria lymphocytes and intraepithelial lymphocytes, supporting the view that CCR5 plays an important role in lymphocyte localization within the intestinal mucosa [30]. Met-RANTES is one of a number of chemokine analogues that act as RANTES antagonists [31]. The ability of Met-RANTES to decrease tissue injury in this study is consistent with its effects in models of arthritis and renal inflammation. Moreover, a role for RANTES in the progression from acute to chronic colitis, which our results strongly suggest, is consistent with the conclusions of Lloyd et al with respect to the role of RANTES in murine crescentic nephritis [32]. The effectiveness of Met-RANTES in abrogating colitis suggests that the CCR1 and/or CCR5 are key receptors in mediating the effects of RANTES. CCR1 has been reported to be expressed on activated T cells, monocytes, eosinophils, dendritic cells, and neutrophils. In contrast, CCR5 has been shown to be expressed on activated T cells, monocytes, macrophages, and dendritic cells but not on neutrophils [28]. The expression of CCR1 on neutrophils may account for the earlier increase in the expression of this receptor than of CCR5 after induction of colitis because

82

C. Kucuk et al. / The American Journal of Surgery 191 (2006) 77– 83

neutrophil recruitment is an early event in the TNBS model. In our study, treatment during this period with MetRANTES resulted in a significant decrease of the severity of colitis. In addition, Met-RANTES produced a statistically significant decrease of bacterial translocation and serum endotoxin and colonic MPO levels compared with the colitis group. Although a number of proinflammatory cytokines (interleukin (IL)-1, IL-6, and TNF-␣) have been implicated in IBDs, TNF-␣) has received the most attention and investigation, having been implicated in IBDs since studies revealed increased levels in Crohn’s disease patients [33,34]. TNF-␣ is a proinflammatory cytokine that has been shown to be one of the most significant factors participating in the inflammatory process of the bowel of patients with IBD. TNF-␣ induces the production in cascade of a number of other cytokines including adhesion molecules, arachidonic acid metabolites, and activation of immune and nonimmune cells [35]. A recently published study showed that the administration of avian TNF antibodies effectively treated experimental colitis in rats [36]. Increased TNF-␣ production is also a feature of all of the murine models of IBDs, and anti–TNF-␣ has been shown to decrease pathology and mortality in some of these systems [34]. TNF-␣ and IL-1 have also been detected in stool from IBD patients, representing an index of intestinal inflammation [37]. It is possible that TNF-␣ plays an important role for the development of colitis in rats treated with TNBS. In our study, TNF-␣ levels parallel to the increased inflammation and bacterial translocation were increased in the colitis group. However, Met-RANTES therapy significantly decreased the levels of serum TNF-␣ and bacterial translocation. In summary, the results of the present study further demonstrate the important role of the RANTES in regulating epithelial function. The marked increase in bacterial translocation in postcolitis rats was reversed by selective inhibition of RANTES. The beneficial effects of RANTES antagonism in experimental colitis suggest that chemokine receptor antagonists are potentially useful therapeutic approaches to the treatment of IBDs.

References [1] McDonald TT, Monteleone G, Pender SL. Recent developments in the immunology of inflammatory bowel disease. Scand J Immunol 2000;51:2–9. [2] Gardiner KR, Halliday MI, Barclay GR, et al. Significance of systemic endotoxaemia in inflammatory bowel disease. Gut 1995;36: 897–901. [3] Wellmann W, Fink PC, Benner F, et al. Endotoxaemia in active Crohn’s disease. Treatment with whole gut irrigation and 5-aminosalicylic acid. Gut 1986;27:814 –20. [4] Ambrose NS, Johnson M, Burdon DW, et al. Incidence of pathogenic bacteria from mesenteric lymph nodes and ileal serosa during Crohn’s disease surgery. Br J Surg 1984;71:623–5. [5] Eade MN, Brooke BN. Portal bacteraemia in cases of ulcerative colitis submitted to colectomy. Lancet 1969;I:1008 –9.

[6] Heddle RJ, Shearman DJC. Serum antibodies to Escherichia coli in subjects with ulcerative colitis. Clin Exp Immunol 1979;38:22–30. [7] Olson CO, Sartor RB, Tennyson GS, et al. Experimental models of inflammatory bowel disease. Gastroenterology 1995;109:1344 – 67. [8] Ajuebor MN, Hogaboam CM, Kunkel SL, et al. The chemokine RANTES is a crucial mediator of the progression from acute to chronic colitis in the rat. J Immunol 2001;166:552– 8. [9] Mazzucchelli L, Hauser C, Zgraggen K, et al. Differential in situ expression of the genes encoding the chemokines MCP-1 and RANTES in human inflammatory bowel disease. J Pathol 1996;178: 201– 6. [10] Baggiolini M. Chemokines and leukocyte traffic. Nature 1998;392: 565– 8. [11] Das AM, Ajuebor MN, Flower RJ, et al. Contrasting roles for RANTES and macrophage inflammatory protein-1 a (MIP-1a) in a murine model of allergic peritonitis. Clin Exp Immunol 1999;117: 223–9. [12] Mazzucchelli L, Hauser C, Zgraggen K, et al. Differential in situ expression of the genes encoding the chemokines MCP-1 and RANTES in human inflammatory bowel disease. J Pathol 1996;178: 201– 6. [13] Bhatia M, Proudfoot AE, Wells TN, et al. Treatment with MetRANTES reduces lung injury in caerulein-induced pancreatitis. Br J Surg 2003;90:698 –704. [14] Proudfoot AE, Power CA, Hoogewerf AJ, et al. Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist. J Biol Chem 1996;271:2599 – 603. [15] Gerard C, Frossard JL, Bhatia M, et al. Targeted disruption of the beta-chemokine receptor CCR1 protects against pancreatitis-associated lung injury. J Clin Invest 1997;100:2022–7. [16] McCormack G, Moriarty D, O’Donoghue DP, et al. Tissue cytokine and chemokine expression in inflammatory bowel disease. Inflamm Res 2001;50:491–5. [17] Morris GP, Beck PL, Herridge MS, et al. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 1989;96:795– 803. [18] Panzer U, Schneider A, Wilken J, et al. The chemokine receptor antagonist AOP-RANTES reduces monocyte infiltration in experimental glomerulonephritis. Kidney Int 1999;56:2107–15. [19] Garcia-Lafuente A, Antolin M, Guarner F, et al. Incrimination of anaerobic bacteria in the induction of experimental colitis. Am J Physiol 1997;272:10 –15. [20] Wallace JL, Mac Naughton WK, Morris GP, et al. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 1989;96:29 –36. [21] Macpherson BR, Pfeifer CJ. Experimental production of diffuse colitis in rats. Digestion 1978;17:35–50. [22] Bradley PP, Priebat DA, Christensen RD, et al. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206 –9. [23] Calkins BM, Mendelhoff AI. The epidemiology of idiopathic inflammatory bowel disease. In: Kirsner JB, Shorter RG, editors. Inflammatory Bowel Disease. Baltimore, MD: Williams and Wilkins; 1995: 31– 68. [24] Zamuner R, Warrier N, Buret AG, et al. Cyclooxygenase 2 mediates post-inflammatory colonic secretory and barrier dysfunction. Gut 2003;52:1714 –20. [25] Berg RD, Garlington AW. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infect Immun 1979;23: 403–11. [26] Stechmiller JK, Treloar D, Allen N. Gut dysfunction in critically ill patients: a review of the literature. Am J Crit Care 1997;6:204 –9. [27] Gardiner KR, Erwin PJ, Anderson NH, et al. Colonic bacteria and bacterial translocation in experimental colitis. Br J Surg 1993;80: 512– 6.

C. Kucuk et al. / The American Journal of Surgery 191 (2006) 77– 83 [28] Luster AD. Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med 1998;338:436 – 45. [29] MacDermott RP, Sanderson IR, Reinecker HC. The central role of chemokines (chemotactic cytokines) in the immunopathogenesis of ulcerative colitis and Crohn’s disease. Inflamm Bowel Dis 1998;4: 54 – 67. [30] Agace WW, Roberts AI, Wu L, et al. Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation. Eur J Immunol 2000;30:819 –26. [31] Adams DH, Lloyd AR. Chemokines: leucocyte recruitment and activation cytokines. Lancet 1997;349:490 –5. [32] Lloyd CM, Dorf ME, Proudfoot A, et al. Role of MCP-1 and RANTES in inflammation and progression to fibrosis during murine crescentic nephritis. J Leukocyte Biol 1997;62:676 – 80.

83

[33] Blam ME, Stein RB, Lichtenstein GR. Integrating anti-tumor necrosis factor therapy in inflammatory bowel disease: current and future perspectives. Am J Gastroenterol 2001;96:1977–97. [34] Kam LY, Targan SR. TNF-alpha antagonists for the treatment of Crohn’s disease. Expert Opin Pharmacother 2000;4:615–22. [35] Louis E. The immuno-inflammatory reaction in Crohn’s disease and ulcerative colitis: characterisation, genetics and clinical application. Focus on TNF alpha. Acta Gastroenterol Belg 2001;64:1–5. [36] Worledge KL, Godiska R, Barrett TA, et al. Oral administration of avian tumor necrosis factor antibodies effectively treats experimental colitis in rats. Dig Dis Sci 2000;45:2298 –305. [37] Braegger CP, Nicholls S, Murch SM, et al. Tumor necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet 1992; 339:89 –91.