Splanchnic Th2 and Th1 Cytokine Redistribution in Microsurgical Cholestatic Rats

Results The cholestatic rats showed an increase ( P < 0.001) in serum levels of bile acids, total and direct bilirubin, AST, ALT, AST/ALT index, γ-GT, alkaline phosphatase, α 1 - MAP, α 1 -GPA, and LDH ( P < 0.05) in relation to sham-operated rats. TNF-α, IL-1β, IL-4, and IL-10 increased in the ileum ( P < 0.01) and mesenteric lymph complex ( P < 0.001), and decreased in the liver ( P < 0.001). A marked bile proliferation associated with fibrosis ( P < 0.001) and mast cell infiltration was also shown in the liver of cholestatic rats. Conclusion The splanchnic redistribution of cytokines, with an increase of Th 1 and Th 2 production in the small bowel and in the mesenteric lymph complex, supports the key role of inflammatory mechanisms in rats with secondary biliary fibrosis. Key Words microsurgery cholestasis rat Th 1 – Th 2 –cytokines splanchnic Introduction Common bile duct ligation (BDL) in the rat causes secondary biliary cirrhosis and is a model of portal hypertension [1] . Chronic biliary obstruction related to BDL provokes fibrosis and ductular proliferation in the rat liver [2, 3] . This fibroductular reaction is associated with portal hypertension, development of portosystemic collateral circulation, and moderate to severe ascites [2–5] . Cholestatic rats are characterized by an increased release of pro-inflammatory cytokines, increased inflammation, and an excessive pro-inflammatory response to lipopolysaccharide (LPS) [6, 7] . Thus, it has been proposed that low-grade inflammation, related to portal hypertension, switches to high-grade inflammation with the development of severe and life-threatening complications when associated with chronic liver disease [8] . Therefore, after BDL in rats, the association of both pathogenic factors, i.e., portal hypertension and chronic liver disease [2, 3, 4–7] , supports the pivotal role of the experimental model obtained to study the characteristics of the inflammatory response developed. An alternative surgical technique to BDL to perform obstructive cholestasis in the rat consists in the microsurgical resection of the extrahepatic bile tract [9] . When this new technique is used, the postoperative infectious complications are reduced since, contrary to the BDL rats, the rats with microsurgical cholestasis do not develop an infected hilar biliary pseudocyst by dilation of the proximal end of the bile duct, nor the associated sepsis caused by abscesses in the intraperitoneal, hepatic, and pulmonary areas [4, 9] . Therefore, a more selective study of the inflammatory response related to obstructive cholestasis and the associated portal hypertension is made possible with this microsurgical model. Particularly, cholestasis and portal hypertension could produce an inflammatory response of splanchnic origin mediated by mast cells [8] in which T-helper type-2 (Th 2 ) cell associated cytokines as Th 1 cytokines could be involved. Thus, in the current study, we have measured the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-4, considered pro-inflammatory cytokines and IL-10, a potent anti-inflammatory cytokine in the liver, the spleen, the small bowel, and the mesenteric lymph nodes of long-term microsurgical cholestatic rats. In addition, we have assayed the serum levels of an array of liver injury markers and two acute phase proteins; liver fibrosis was also shown by Sirius red stain. Materials and Methods Male Wistar rats, with weights ranging between 230 and 270 g, from the Vivarium of the Complutense University of Madrid were used. The animals were fed a standard laboratory rodent diet (rat/mouse A04 maintenance diet; Panlab, Barcelona, Spain) and water ad libitum. They were housed in a light/dark-controlled room (light cycles: 7:30 a.m.–7:30 p.m.) with an average temperature (22 ± 2 °C) and humidity (65%–70%), in groups of three to four animals. The experimental procedures employed in this study were based on the principles and practices of the 1986 European Guide for the Care and Use of Laboratory Animals, in accordance to Ethical Guidelines from European Community Council Directive (86/609/EEC) and published in Spanish Royal Decree 1201/2005. Experimental Design The animals were randomly divided into two groups: sham-operated (SO) (group I: n = 32) and microsurgical cholestasis (MC) (group II: n = 44), in which the resection of the extrahepatic biliary tract was performed [9] . All the animals were sacrificed by anesthesia overdose at 6 wk of evolution, and body (BW), liver (LW), spleen (SW), and testes (TW) weights were determined. Surgical Procedure Rats were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine (12 mg/kg) i.m. Surgery was performed under aseptic but not sterile conditions. In group I (SO), the bile duct and its lobular branches were dissected without resection. The study group (MC), underwent extrahepatic biliary tract resection using a binocular operatory microscope (OPMI 1-FR; Zeiss, Madrid, Spain), as previously described [9] . Briefly, the common bile duct was ligated (silk 4/0) and sectioned close to its intrapancreatic portion. Once the bile duct was sectioned, it was shifted upwards. The dissection and section between ligatures of the biliary branches that drain each hepatic lobe were made in the caudo-craneal direction. First, the biliary branch of the caudate lobe, and then the two biliary branches of the right lateral lobe, were dissected, ligated, and sectioned close to the hepatic parenchyma. The upward dissection of the extrahepatic biliary tract made it possible to individualize, ligate, and section the biliary branches draining the middle lobe and, finally, the biliary branch of the left lateral lobe. The abdomen was closed in two layers by continuous running sutures with an absorbable suture (3/0 polyglycolic acid) and silk (3/0). Buprenorphine (0.05 mg/Kg/8 h s.c.) was administered for analgesia the first day of the postoperative period. Portosystemic Collateral Circulation Method Portosystemic collateral circulation was studied as follows. First, a midline abdominal incision was performed, and then the areas in which the collateral venous circulation is normally developed, i.e., splenorenal, gastroesophageal, and colorectal were carefully studied for the presence of increased collateral veins [10] . Portal Vein Pressure Measurements Splenic pulp pressure, an indirect measurement of portal pressure (PP), was measured by inserting a fluid-filled 20-gauge needle into the splenic parenchyma [11] . The needle was joined to a PE-50 tube and then connected to a pressure recorder (PowerLab 200 ML201) and to a transducer (Sensonor SN-844) with a chart V 4,0 computer program (ADI Instruments, Castle Hill, NSW, Australia), and was calibrated before each experiment. Previous studies have demonstrated the excellent correlation between splenic pulp pressure and PP [12] . Gross Mesenteric Vein Study Three grades of mesenteric venous vasculopathy were considered; grade 0: normal aspects of the branches of the superior mesenteric vein, without dilation and tortuosity secondary to the superior mesenteric vein compression; grade 1: dilation and tortuosity of these branches secondary to this maneuver, and grade 2: in which the dilation and tortuosity of the branches of the upper mesenteric vein were spontaneous [13] . Serum Biochemical Tests Blood samples were drawn by puncture with a 22-gauge needle of the infrahepatic inferior vena cava. After 15 min of centrifugation at 1500 g , the serum was separated and transferred to sterile polypropylene tubes. The serum was frozen at –40 °C until bile acids (BA), total and direct bilirubin (DB), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-glutamyltranspeptidase (GGTP), creatinin, urea, and albumin were assayed in an autoanalyzer. Serum levels of alfa-1 acid glicoprotein (α-1AGP) and alfa-1 major acid protein (α-1MAP), or thiostatin, were measured using an enzyme-linked immunosorbent assay (ELISA) kit specific for rat proteins according to the manufacturer's instructions. TNF-α, IL-1β, IL-4, and IL-10 Splanchnic Levels Segments from the distal ileum (1 cm from the ileo-caecal junction), the liver (middle lobe), the spleen, and the mesenteric lymph complex were quickly taken and frozen on dry ice. Frozen organ samples were weighed on a Mettler balance (model AE 100; Mettler Instruments Corp., Hightstown, NJ) and transferred to 5 mL polypropylene tubes (Falcon; Becton Dickinson, Lincoln Park, NJ) containing lysis buffer (4 °C) at a ratio of 10 mL buffer/L g of wet tissue. Lysis buffer was 1 mmol/L phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Company, Madrid, Spain), and 1 μg/mL pepstatin A (Sigma Chemical Company), aprotinin (Sigma Chemical Company), anti-pain (Sigma Chemical Company), and leupeptin in 1× phosphate-buffered saline solution with pH 7.2 (Biofluids, Rockville, MD) containing 0.05% sodium azide (Sigma Chemical Company). The samples were homogenized for 30 s with an electrical homogenizer (Polytron; Brinkmann Instruments, Westminster, NY) at maximum speed, and the tubes were immediately frozen in liquid nitrogen. The samples were homogenized three times for optimal processing. The supernatants were frozen at –80 °C to allow the formation of macromolecular aggregates. After thawing at 4 °C, the aggregates were pelleted at 3000 g (4 °C) and, the final organ homogenate volume was measured with a graduate pipette [14] . The homogenates were stored at –80 °C until assayed for the quantitative levels of rat TNF-α, IL-1β, IL-4, and IL-10, in two groups of six animals each (SO- and cholestatic rats) using commercially available enzyme linked immunosorbent assay specific kits (BioNOVA Cientifica Ltd. Madrid, Spain). Histopathologic Examinations Representative samples of the middle liver lobe were collected and fixed in phosphate-buffered neutral formalin (10%) and embedded in paraffin. Five μ-thick sections were stained with hematoxylin and eosin (H and E), Giemsa, and Sirius red, which specifically stains collagen components [15] . An image analysis system was used to objectively assess fibrosis and the collagen content of the hepatic tissue from the sections stained with Sirius red. A minimum area of hepatic tissue was digitalized to 15 mm 2 through microscopic images with a 10× lens. Next, the area stained with Sirius red was assessed with regard to the total area of hepatic tissue by using Leica Q Win software [16] . Statistical Analysis A statistical analysis was performed using SPSS software (Statistical Package for the Social Sciences, version 14.00, Chicago, IL). The results are expressed as the mean ± the standard deviation. Student's t -test for unpaired data was used for the statistical comparison of the variables between the different groups. The results are considered significant if P < 0.05. Results Body and Organ Weights The body weight increase after 6 wk of evolution was lower ( P < 0.001) in cholestatic rats (46.39 ± 38.42 g) in relation to sham-operated rats (109.76 ± 20.56 g).Liver weight (14.01 ± 3.35 g versus 11.72 ± 1.31 g) and the ratio of liver weight to body weight (LW/BW × 100; 4.72 ± 0.83 versus 3.15 ± 0.29) was higher ( P < 0.001) in rats with cholestasis in regards to SO rats. All these animals presented splenomegaly (2.10 ± 0.61 versus 0.95 ± 0.13 g; P < 0.001). Cholestatic rats showed testicular atrophy (2.21 ± 0.77 g versus 3.62 ± 0.28 g; P < 0.001). Portal Pressure, Portosystemic Collateral Circulation, and Grade of Mesenteric Venous Vasculopathy Portal pressure was higher ( P < 0.001 in cholestatic rats (13.43 ± 2.33 mm Hg) in relation to SO rats (6.78 ± 2.35 mm Hg). All the cholestatic rats developed portosystemic collateral circulation, whether pararectal (100%; n = 44), paraesophageal (97.20%; n = 43), splenorenal superior (100%; n = 44), or splenorenal inferior (95.4%; n = 42). In addition, other signs of portal hypertension, such as mesenteric venous vasculopathy, was present in 93.17% of the rats with cholestasis, whether grade I (81.81%; n = 36) or grade II (11.36%; n = 5). Liver Function The cholestatic rats showed an increase ( P < 0.001) of specific serum levels markers for hepatobiliary injury, like total and direct bilirubin, bile acids, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), AST/ALT index, and gamma-glutamil transpeptidase (γ-GT) and lactatedehydrogenase (LDH; P < 0.05) with regard to SO rats. Urea increased ( P < 0.05), whereas albumin levels decreased ( P < 0.001) in rats with cholestasis ( Table 1 ). Serum Acute Phase Proteins Serum levels of α 1 -MAP, or thiostatin, and α 1 -GPA were higher ( P < 0.001) in relation to SO rats ( Fig. 1 ). Liver Histopathogic Study The hepatic parenchyma in SO rats did not show any impairment ( Fig. 2 ), while the rat liver after 6 wk of obstructive cholestasis showed a severe destruction of the hepatic parenchyma with intense biliary duct proliferation in areas I and II of the hepatic acinus, necrotic and apoptotic hepatocytes ( Fig. 2 ), and intense fibrosis, which was quantified by digital image analysis ( P < 0.001) ( Fig. 3 ). In cholestatic livers, there was an increase of mast cell infiltration, particularly in portal spaces and in the fibrous septa, close to both proliferated biliary ducts and fibrosis ( Fig. 2 ). Cytokine Levels in Splanchnic Organs and in Mesenteric Lymph Nodes In the cholestatic rats, TNF-α, IL-1β, IL-4, and IL-10 hepatic levels decreased ( P < 0.001) in relation to SO rats. Spleen levels of these cytokines are comparable in rats with cholestasis and sham-operated. Interestingly, the ileum and mesenteric lymph nodes of cholestatic rats showed a decrease of TNF-α ( P < 0.01 and P < 0.001, respectively), IL-1β ( P < 0.01 and P < 0.001, respectively), IL-4 ( P < 0.01 and P < 0.05, respectively), and IL-10 ( P < 0.05 and P < 0.001, respectively) levels with regards to SO rats ( Figs. 4, 5, 6, 7 and 8 ). Discussion The results obtained in the current study of the splanchnic levels of Th 1 - (TNF-α and IL-1β) and Th 2 - (IL-4 e IL-10) cytokines in long-term microsurgical extrahepatic cholestatic rats showed a significant increase in the small bowel (ileum) and in the mesenteric lymph nodes compared with the decrease in their liver ( Figs. 4, 5, 6, 7 and 8 ). The increased levels of TNF-α, IL-1β, IL-4 and IL-10 in mesenteric lymph nodes and ileum in this experimental model of obstructive cholestasis could be attributed to the inflammatory response, mediated by mast cells, which would be triggered by portal hypertension [17] . We have previously demonstrated that in rats with long-term prehepatic portal hypertension, a mast cell hyperplasia occurred throughout the gastrointestinal tract, although it was higher in the ileum and the mesenteric lymph nodes [13] . Mast cells produce a vast array of cytokines, including TNF-α, IL-4, and IL-10 [18] . Mast cells have the unique capacity to store presynthesized TNF-α and, therefore, can spontaneously release this cytokine after they are activated [19] . It has been hypothesized that TNF-α causes vasodilatation through both the PGI 2 and NO pathways [20] . If so, the release of the stored TNF-α by activated mast cells may be involved in the development of the hyperdynamic splanchnic state in this model of obstructive cholestasis [20] . Cholestasis has a deteriorating effect on the intestinal mucosa [21, 22] . Indeed short-term cholestatic rats by BDL suffer a marked loss of tight junction associated proteins in the ileal mucosa, with subsequent increased transepithelial permeability [23] . These significant morphologic impairments are associated with increased vulnerability of the mucosa to toxic and oxidative injuries [23] . On the contrary, antioxidant treatments significantly improved barrier function [23, 24] . The suppression of bile secretion into the intestine enhances bacterial translocation from the intestinal lumen to mesenteric lymph nodes and is considered one of the main events in the pathogenesis of endotoxemia and sepsis-related complications in biliary obstruction [21, 22, 25] . Intestinal hyperproduction of TNF-α may play a role in the process of bacterial translocation because in cirrhotic rats with ascites, the administration of anti-TNF-α monoclonal antibody decreases the incidence of bacterial translocation [26] . The existence of endotoxemia by an increased intestinal absorption of luminal endotoxin in the obstructive cholestatic surgical models [22, 27] would convert the gastrointestinal tract into the “engine” of a number of end organ effects like those proposed by Carrico et al. in the pathophysiology of the multiple-organ-failure syndrome in critically ill septic patients [28] . The increased small bowel production of TNF-α and IL-1β in rats with long-term (2–3 mo) prehepatic portal hypertension suggests that these pro-inflammatory cytokines would enhance intestinal inflammation and, therefore, they could be considered as etiopathogenic mediators in portal hypertensive enteropathy [13] . If so, these pro-inflammatory mediators would also be involved in the development of a hypertensive enteropathy in the rats with obstructive cholestasis. The intestinal increase of IL-4 and IL-10 in microsurgical cholestatic rats could be related with the participation of mast cells [18, 29, 30] both in the decreased intestinal barrier integrity and in their sensitization or increased susceptibility to toxins [23] . Particularly, the exposure of mast cells to bacterial components, such as lipopolysaccharide (LPS), would induce their activation with subsequent increase in Th2 cytokine production [31] . Thus, when they are activated by LPS in the intestine, it would explain why mast cells migrate and massively infiltrate the mesenteric lymph nodes. Hepatic MMC hyperplasia has been demonstrated in rats with long-term (21 to 30 d) obstructive cholestasis [32–34] . Mast cell hyperplasia is associated with the proliferation of bile ductules during extrahepatic cholestasis. At the same time, the recanalization of the ligated common bile duct leads to an abrupt and transient increase in the number of mast cells linked with a rapid increase in the number of apoptotic biliary epithelial cells. These findings suggest that mast cells accumulating in the portal triads may contribute to liver remodeling through the induction of apoptosis [35] . In long-term extrahepatic microsurgical cholestasis, the predominating hepatic alteration is the marked ductular proliferation with a mild portal inflammatory infiltration in addition to apoptosis and fibrosis in the long-term [16, 36] . This evolution of the damaged cholestatic liver towards fibrosis is why the process can be compared with other repair processes, such as wound healing [37–41] . Thus, in short-term extrahepatic cholestasis in rodents, the liver inflammatory component with intense oxidative stress [23, 24, 42] , activation of the transcription factor NF-κB with white blood cell chemoattractant [43–46] , and increased pro-inflammatory cytokine release, i.e., TNF-α, IL-1β, and IL-6 [47, 48] , as well as increased sensibility to LPS [48–51] or to the gram-negative pathogen Escherichia coli [52] would predominate. Paradoxically, in this early evolutive phase, bile duct ligation also protects the liver from ischemic injury and inflammation because of the impaired NF-κB activation by ischemia [53] . Between 3 and 4 wk of evolution of post-bile duct ligation in the rat, even though the liver pro-inflammatory response persists, with an increase of TNF-α and IL-1β levels, IL-10 also increases, whereas IFN-γ decreases [54] . The increased hepatic sensibility to LPS is also maintained [7, 55] , but with low responsiveness of the inflammatory cells, related to a decreased free radical production and phagocytic activity of these cells [56] . Liver mucosal mast cell hyperplasia and degranulation, with the release of profibrogenic mediators, correlate in this evolutive phase of extrahepatic cholestasis in the rat with the time of most active collagen synthesis [34, 57] . In later evolutive phases (6 wk), the sustained cholestatic injury induces micronodular cirrhosis. Fibrosis correlates with ductular reaction and is produced by periductular/ductular matrix-producing myofibroblasts [58] . In this phase of the hepatic healing process, matrix remodeling and scar formation could predominate with increased serum TGF-β levels, a potent profibrotic cytokine [59] . The decrease in liver cytokines, particularly pro-inflammatory cytokine, shown in the rats with microsurgical cholestasis, would therefore be explained. The inexistence of an infected hilar biliary pseudocyst in this microsurgical model [4, 9] compared with what occurs in the bile duct-ligated models would also favor a reduced hepatic inflammatory response. However, the liver maintains its ability to synthesize acute phase proteins. In rats with long-term bile duct ligation, LPS also increases the serum concentration of α 1 -acid glycoprotein (α 1 AGP) and hepatoglobin, indicating a normal hepatic contribution to this part of the acute phase response [60] . In this chronic inflammatory phase of liver cholestasis, mast cells can be considered key elements in the process of transforming sinusoidal endothelial cells into capillary-type endothelial cells, with the development of continuous collagen sinusoidal basement membranes. This leads to a process called “sinusoidal capillarization” [61] . Excluding this alteration, mast cells seem to exert a less profound influence on liver fibrosis after bile duct ligation in rodents [39, 62] . However, many of the other multiple functions carried out by mast cells could be assayed in experimental secondary biliary cirrhosis, for example, their ability to limit the extent or duration of chronic inflammation [18] , the ability to induce tolerance [63] , and favor the growth of tumors [18] . The response of the murine liver to biliary obstructive injury implies its transcriptional reprogramming favoring the activation of gene regulating metabolism, cell proliferation, and matrix remodeling in a time-restricted and sequential fashion [64] . This evolution results in deranged hepatic energy metabolism, as well as in a loss of antioxidant power by the liver [23, 24, 44, 64] . Both factors could progressively aggravate the inflammatory response evolution in the gastrointestinal tract, and induce high long-term mortality through the development of sepsis related complications. In tissues with chronic inflammation, there is evidence indicating that the normal homeostatic regulation of cytokines does not occur, since new and complex interactions are developed [65] . It has also been postulated that an imbalance between Th1 and Th2 cytokine production is involved in chronic liver disease. Thus, high serum levels of anti-inflammatory cytokines in patients with type C chronic liver disease has been attributed to a predominant Th2 response associated with a weak Th1 response [66] . Moreover, it has been demonstrated that the hepatocyte cytokine profile became increasingly Th1 in nature in rats that have received an oral administration of the biliary toxin α-naphthy-lisothiocyanate (ANIT). In this experimental model of cholangitis, a progressive shift to a Th1 dominant hepatic cytokine profile is associated with hepatic predominant Th2 cytokine mRNA levels. This discrepancy may be caused by the complexities of cytokine synthesis and release, which are regulated and influenced by various factors. The milieu of the cytokine-producing cells and the type and amount of stimuli for cytokine production stand out among these factors [67] . Thus, studying the cytokine mRNA levels in the different splanchnic areas in which we have assayed the Th1 and Th2 cytokines is worthwhile in our experimental model of microsurgical cholestasis. Similarly, it could be suspected that in experimental secondary biliary cirrhosis, a chronic liver inflammatory condition with new types of interrelationships in the gut-spleen liver axis could be established. If so, cytokines would be involved in this new context, but expressing different functions, which could explain their splanchnic redistribution. Acknowledgments The authors thank Maria Elena Vicente for preparing the manuscript, Elizabeth Mascola for translating it into English, and librarians of Complutense University Medical School, particularly the Director, Juan Carlos Domínguez Martínez, and María José Valdemoro. This study was carried out in part with grants from the Department of Health , Institute of Health Sciences , and the Autonomous Government of Castilla-La Mancha (ref. no. PI-2007/64 ), and Mutua Madrileña Research Foundation (ref. no. PA 3077/2008 ). References 1 J.G. Abraldes M. Pasarin J.C. Garcia-Pagan Animal models of portal hypertension World J Gastroenterol 12 2006 6577 2 J. Kountouras B.H. Billing P.J. Scheuer Prolonged bile duct obstruction: A new experimental model for cirrhosis in the rat Br J Exp Pathol 65 1984 305 3 S. Milani H. Herbst D. Schuppan Vimentin expression of newly formed rat bile duct epithelial cells in secondary biliary fibrosis Virchows Arch 415 1989 237 4 M.A. Aller M. Duran L. 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