Interleukin-1β Mediates the Extra-Intestinal Thrombosis Associated with Experimental Colitis
2010; Elsevier BV; Volume: 177; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.100205
ISSN1525-2191
AutoresHideo Yoshida, Janice Russell, Elena Y. Senchenkova, Lidiana De Almeida Paula, D. Neil Granger,
Tópico(s)Inflammasome and immune disorders
ResumoInflammatory bowel diseases (IBDs) are associated with an increased risk for thromboembolism, which is often manifested as deep vein thrombosis or pulmonary embolism, at extra-intestinal sites. Although some of the cytokines that contribute to IBD pathogenesis are also known to alter the coagulation pathway, it remains unclear whether these mediators also contribute to the extra-intestinal thrombosis often associated with IBD. The objective of this study is to evaluate the role of interleukin (IL)-1β in enhanced extra-intestinal thrombosis observed in mice with dextran sodium sulfate (DSS)-induced colitis. IL-1β concentrations were measured in plasma, colon, and skeletal muscle of wild-type (WT) control and colitic mice. Microvascular thrombosis was induced in cremaster muscle microvessels by using a light/dye injury model. The effects of exogenous IL-1β on thrombus formation were determined in control WT mice. DSS-induced thrombogenesis was evaluated in WT mice treated with an IL-1β antibody and in IL-1 receptor-deficient (IL-1r−/−) mice. DSS-induced colonic inflammation in WT mice was associated with enhanced thrombus formation in arterioles. IL-1β concentrations were elevated in inflamed colon and skeletal muscle. Exogenous IL-1β enhanced thrombosis in control mice in a dose-dependent manner. DSS colitic mice treated with the IL-1β antibody as well as IL-1r−/− mice exhibited significantly blunted thrombogenic responses. These findings implicate IL-1β as a mediator of enhanced microvascular thromboses that occur in extra-intestinal tissues during colonic inflammation. Inflammatory bowel diseases (IBDs) are associated with an increased risk for thromboembolism, which is often manifested as deep vein thrombosis or pulmonary embolism, at extra-intestinal sites. Although some of the cytokines that contribute to IBD pathogenesis are also known to alter the coagulation pathway, it remains unclear whether these mediators also contribute to the extra-intestinal thrombosis often associated with IBD. The objective of this study is to evaluate the role of interleukin (IL)-1β in enhanced extra-intestinal thrombosis observed in mice with dextran sodium sulfate (DSS)-induced colitis. IL-1β concentrations were measured in plasma, colon, and skeletal muscle of wild-type (WT) control and colitic mice. Microvascular thrombosis was induced in cremaster muscle microvessels by using a light/dye injury model. The effects of exogenous IL-1β on thrombus formation were determined in control WT mice. DSS-induced thrombogenesis was evaluated in WT mice treated with an IL-1β antibody and in IL-1 receptor-deficient (IL-1r−/−) mice. DSS-induced colonic inflammation in WT mice was associated with enhanced thrombus formation in arterioles. IL-1β concentrations were elevated in inflamed colon and skeletal muscle. Exogenous IL-1β enhanced thrombosis in control mice in a dose-dependent manner. DSS colitic mice treated with the IL-1β antibody as well as IL-1r−/− mice exhibited significantly blunted thrombogenic responses. These findings implicate IL-1β as a mediator of enhanced microvascular thromboses that occur in extra-intestinal tissues during colonic inflammation. Inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism, which is often manifested as deep vein thrombosis or pulmonary embolism. The prothombogenic state that accompanies human IBD is believed to arise from an imbalance between pro- and anti-coagulant factors.1Vrij AA Rijken J van Wersch JW Stockbrügger RW Coagulation and fibrinolysis in inflammatory bowel disease and in giant cell arteritis.Pathophysiol Haemost Thromb. 2003; 33: 75-83Crossref PubMed Scopus (39) Google Scholar, 2van Bodegraven AA Schoorl M Linskens RK Bartels PC Tuynman HA Persistent activation of coagulation and fibrinolysis after treatment of active ulcerative colitis.Eur J Gastroenterol Hepatol. 2002; 14: 413-418Crossref PubMed Scopus (40) Google Scholar A similar procoagulant/prothrombogenic phenotype is evident in animal models of experimental colitis, which exhibit elevated blood levels of thrombin-antithrombin complexes, reduced protein C, and decreased antithrombin III.3Anthoni C Russell J Wood KC Stokes KY Vowinkel T Kirchhofer D Granger DN Tissue factor: a mediator of inflammatory cell recruitment, tissue injury, and thrombus formation in experimental colitis.J Exp Med. 2007; 204: 1595-1601Crossref PubMed Scopus (72) Google Scholar, 4Scaldaferri F Sans M Vetrano S Graziani C De Cristofaro R Gerlitz B Repici A Arena V Malesci A Panes J Grinnell BW Danese S Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease.J Clin Invest. 2007; 117: 1951-1960Crossref PubMed Scopus (107) Google Scholar, 5Onomura M Tsukada H Fukuda K Kodama M Nakamura H Hosokawa M Ohya M Seino Y Effect of argatroban on trinitrobenzene sulfonic acid-induced colitis.J Gastroenterol Hepatol. 2000; 15: 931-938Crossref PubMed Scopus (8) Google Scholar These changes in coagulant-anticoagulant factors are accompanied by enhanced thrombus formation in microvessels of organs distant to the colon, such as skeletal muscle.3Anthoni C Russell J Wood KC Stokes KY Vowinkel T Kirchhofer D Granger DN Tissue factor: a mediator of inflammatory cell recruitment, tissue injury, and thrombus formation in experimental colitis.J Exp Med. 2007; 204: 1595-1601Crossref PubMed Scopus (72) Google Scholar, 6Yoshida H Russell J Stokes KY Yilmaz CE Esmon CT Granger DN Role of the protein C pathway in the extraintestinal thrombosis associated with murine colitis.Gastroenterology. 2008; 135: 882-888Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 7Yoshida H Russell J Granger DN Thrombin mediates the extraintestinal thrombosis associated with experimental colitis.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G904-G908Crossref PubMed Scopus (13) Google Scholar Although the contributions of different pro- (tissue factor, thrombin) and anti- (activated protein C) coagulants to colitis-enhanced extra-intestinal thrombosis have been evaluated,3Anthoni C Russell J Wood KC Stokes KY Vowinkel T Kirchhofer D Granger DN Tissue factor: a mediator of inflammatory cell recruitment, tissue injury, and thrombus formation in experimental colitis.J Exp Med. 2007; 204: 1595-1601Crossref PubMed Scopus (72) Google Scholar, 6Yoshida H Russell J Stokes KY Yilmaz CE Esmon CT Granger DN Role of the protein C pathway in the extraintestinal thrombosis associated with murine colitis.Gastroenterology. 2008; 135: 882-888Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 7Yoshida H Russell J Granger DN Thrombin mediates the extraintestinal thrombosis associated with experimental colitis.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G904-G908Crossref PubMed Scopus (13) Google Scholar the chemical and/or cellular signal produced by the inflamed colon that promotes thrombosis at distant sites remain undefined. A variety of cytokines have been implicated in the pathogenesis of IBD. Interleukin-1beta (IL-1β) has received considerable attention as a potential mediator of inflammatory cell infiltration and mucosal barrier disruption that accompanies gut inflammation.8Raddatz D Toth S Schwörer H Ramadori G Glucocorticoid receptor signaling in the intestinal epithelial cell lines IEC-6 and Caco-2: evidence of inhibition by interleukin-1beta.Int J Colorectal Dis. 2001; 16: 377-383Crossref PubMed Scopus (21) Google Scholar IL-1β also appears to promote inflammation by stimulating the production of other cytokines (eg, IL-6) and chemokines (eg, CXCL1, CXCL8, IL-8).9Witowski J Tayama H Ksiazek K Wanic-Kossowska M Bender TO Jörres A Human peritoneal fibroblasts are a potent source of neutrophil-targeting cytokines: a key role of IL-1beta stimulation.Lab Invest. 2009; 89: 414-424Crossref PubMed Scopus (21) Google Scholar, 10Kwon KH Murakami A Hayashi R Ohigashi H Interleukin-1beta targets interleukin-6 in progressing dextran sulfate sodium-induced experimental colitis.Biochem Biophys Res Commun. 2005; 337: 647-654Crossref PubMed Scopus (104) Google Scholar, 11Medzhitov R Preston-Hurlburt P Kopp E Stadlen A Chen C Ghosh S Janeway CA MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways.Mol Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar A less studied characteristic of IL-1β is its procoagulant actions. The cytokine is known to induce the expression of tissue factor while down-regulating the protein C pathway, which are features of the prothrombogenic response elicited in the microvasculature of dextran sodium sulfate (DSS) colitic mice.3Anthoni C Russell J Wood KC Stokes KY Vowinkel T Kirchhofer D Granger DN Tissue factor: a mediator of inflammatory cell recruitment, tissue injury, and thrombus formation in experimental colitis.J Exp Med. 2007; 204: 1595-1601Crossref PubMed Scopus (72) Google Scholar, 4Scaldaferri F Sans M Vetrano S Graziani C De Cristofaro R Gerlitz B Repici A Arena V Malesci A Panes J Grinnell BW Danese S Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease.J Clin Invest. 2007; 117: 1951-1960Crossref PubMed Scopus (107) Google Scholar, 6Yoshida H Russell J Stokes KY Yilmaz CE Esmon CT Granger DN Role of the protein C pathway in the extraintestinal thrombosis associated with murine colitis.Gastroenterology. 2008; 135: 882-888Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar These properties of IL-1β, coupled to its ability to stimulate the production of other powerful procoagulant cytokines like IL-6,12van der Poll T van Deventer SJ Cytokines and anticytokines in the pathogenesis of sepsis.Infect Dis Clin North Am. 1999; 13: 413-426Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar suggest that IL-1β is a viable candidate mediator of the distant organ thrombogenesis that accompanies colonic inflammation. The overall objective of this study was to determine whether IL-1β contributes to the enhanced extra-intestinal thrombosis in experimental colitis. To achieve this objective, we evaluated (1) IL-1β levels in colon, skeletal muscle, and plasma of control and DSS colitic mice by using a cytometric bead array; (2) the thrombogenic response of cremaster muscle arterioles to light/dye-induced endothelial injury after administration of different doses of recombinant murine IL-1β; (3) thrombus formation in wild-type (WT) colitic mice receiving a polyclonal anti-IL-1β antibody (Ab), (4) thrombus formation in IL-1 receptor deficient mice with DSS colitis, and (5) in mice deficient in myeloid differentiation protein-88 (MyD88), a cytoplasmic adaptor molecule that enables the IL-1 receptor to activate nuclear factor kappa B and elicit the production of different cytokines and chemokines.11Medzhitov R Preston-Hurlburt P Kopp E Stadlen A Chen C Ghosh S Janeway CA MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways.Mol Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar Our findings suggest that IL-1β mediates the enhanced extra-intestinal thrombosis associated with colonic inflammation via a MyD88-independent signaling mechanism. A total of 98 male C57BL/6J mice, including 10 IL-1 receptor-1 deficient (IL-1r−/−) mice, seven MyD88 deficient (MyD88−/−) mice, and five recombinase activating gene-1 (RAG-1−/−) mice were purchased from Jackson Laboratory Bar Harbor, ME for this study. All mice were housed under specific pathogen-free conditions in standard cages and fed standard laboratory chow and water until the desired age (6 to 8 weeks). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center and were performed according to the criteria outlined by the National Institutes of Health. Colitis was induced, as previously described,13Vowinkel T Kalogeris TJ Mori M Krieglstein CF Granger DN Impact of dextran sulfate sodium load on the severity of inflammation in experimental colitis.Dig Dis Sci. 2004; 49: 556-564Crossref PubMed Scopus (107) Google Scholar by feeding mice 3% (w/v) DSS (40,000 molecular weight; MP Biomedicals, Solon, OH) dissolved in filter-purified drinking water. The first day of DSS feeding was defined as day 0, and the mice were maintained on the DSS until day 6. The 3% DSS regimen, unlike higher DSS doses, is not associated with mortality. Control mice received filtered water alone (without DSS). In a separate group of experiments, the light/dye-induced microvascular thrombosis responses were compared between WT mice with DSS-induced colitis and mice with colitis induced by the adoptive transfer (i.p.) of CD4+ T-lymphocytes (1 × 106 cells) obtained from IL-10−/− mice into RAG−/− recipients. IL-10−/− splenocytes were enriched for CD4+ T cells (>85%) by using a commercially available negative selection kit specifically for CD4+ T cells (Dynal, Carlsbad, CA), as previously described.14Ostanin DV Bao J Koboziev I Gray L Robinson-Jackson SA Kosloski-Davidson M Price VH Grisham MB T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G135-G146Crossref PubMed Scopus (307) Google Scholar The T-cell transfer model produces murine colitis at 8 weeks following adoptive transfer of the IL-10−/− CD4+ T cells. Body weights, fecal status, presence of occult blood in the stools, and peri-anal bleeding were observed and recorded every day while the mice received DSS. Occult blood was detected by using guaiac paper (ColoScreen; Helena Laboratories, Beaumont, TX).13Vowinkel T Kalogeris TJ Mori M Krieglstein CF Granger DN Impact of dextran sulfate sodium load on the severity of inflammation in experimental colitis.Dig Dis Sci. 2004; 49: 556-564Crossref PubMed Scopus (107) Google Scholar Disease activity index (DAI), a measure of disease severity ranging between 0 and 4, was calculated from data collected on stool consistency, presence or absence of fecal blood, and weight loss, as previously described.13Vowinkel T Kalogeris TJ Mori M Krieglstein CF Granger DN Impact of dextran sulfate sodium load on the severity of inflammation in experimental colitis.Dig Dis Sci. 2004; 49: 556-564Crossref PubMed Scopus (107) Google Scholar The DAI was measured daily to confirm that DSS treatment resulted in clinical responses that are consistent with colitic disease activity. IL-1β levels in plasma, colon, and skeletal muscle (quadriceps) were measured by using a cytometric bead array. To obtain the plasma samples, the right carotid artery was cannulated with a phosphatidylethanolamine 10 tube (BD, Flanklin Lakes, NJ). A blood sample was withdrawn and collected in an Eppendorf tube, which was then centrifuged at 5000 rpm × 10 minutes to separate the plasma. Tissue samples were promptly mixed with PBS containing a protease inhibitor (Sigma Chemicals, St. Louis, MO) and thoroughly homogenized. The homogenate was centrifuged at 10,000 rpm × 5 minutes to separate the supernatant. IL-1β concentrations in the supernatants and plasma samples were measured with the cytometric bead array as per the manufacturer's instruction (BD Biosciences, San Jose, CA). The detection limit of the cytometric bead array for mouse IL-1β is 10 pg/ml. On day 6 of DSS (colitis) or water (control) treatment, mice were anesthetized by using 50 mg/kg body wt. (i.p.) pentobarbital, with supplemental doses of 12.5 mg/kg, given as needed. The right internal jugular vein was cannulated for intravenous administration of fluorescein isothiocyanate (FITC) dextran, and the right carotid artery was cannulated for measurement of systemic blood pressure. Body temperature was maintained at 36.5°C to 37.5°C during the entire experiment with a homeothermic blanket and monitored with a rectal temperature probe. An incision was made in the scrotal skin to expose the left cremaster muscle. A lengthwise incision was made on the surface of the cremaster muscle. The testicle and epididymis were separated from the muscle. The muscle was spread out on the pedestal and the edges of the muscle were moderately extended with sutured threads.15Hickey MJ Kanwar S McCafferty DM Granger DN Eppihimer MJ Kubes P Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.J Immunol. 1999; 162: 1137-1143PubMed Google Scholar The surface of the exposed cremaster muscle was suffused continuously with bicarbonate-buffered saline, with a pH 7.35 to 7.45. The microvasculature was observed by using an upright microscope (BX51WI; Olympus, Tokyo, Japan) with a 40× water immersion objective lens (LUMPlanFI/IR 40×/0.80 w, Japan). The light and fluorescent microscopic images were projected onto a monitor (TRINITRON PVM-2030; Sony, Tokyo, Japan) through a color video camera (Hitachi VK-C150; Hitachi, Tokyo, Japan) or a charge-coupled device video camera (Hamamatsu XC-77; Hamamatsu, Tokyo, Japan), respectively. The images were recorded by using a DVD recorder (JVC SR-MV50, NJ). A video timer (Panasonic Time-Date Generator WJ-810; Panasonic, Tokyo, Japan) was connected to the monitor to record time and date. The diameters of the cremaster vessels were measured by video analysis software (ImageJ 1.37v; NIH, Public Domain software) on a personal computer (G4 Macintosh; Apple, Cupertino, CA). Red blood cell velocity (VRBC) in the microvessels was measured by using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University, College Station, TX). Blood flow was calculated from the product of mean red blood cell velocity (Vmean = VRBC/1.6) and cross-sectional area, assuming cylindrical geometry. Wall shear rate was calculated based on the Newtonian definition: wall shear rate = 8 (Vmean/DV). In each mouse, four to five venular segments with a diameter of 35 to 50 μm, at least 100 μm in length, and a wall shear rate ≥500 per second were randomly selected for study in the cremaster muscle. Leukocytes were classified according to quality or duration of their interaction with the venular wall as either free-flowing, rolling, or adherent.16Vowinkel T Wood KC Stokes KY Russell J Tailor A Anthoni C Senninger N Krieglstein CF Granger DN Mechanisms of platelet and leukocyte recruitment in experimental colitis.Am J Physiol Gastrointest Liver Physiol. 2007; 293: G1054-G1060Crossref PubMed Scopus (57) Google Scholar Rolling leukocytes were defined as cells crossing the 100 μm venular segment at a velocity that is significantly lower than the center line blood flow. Adherent leukocytes were defined as remaining stationary for ≥30 seconds.17Ishikawa M Sekizuka E Yamaguchi N Nakadate H Terao S Granger DN Minamitani H Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature.Am J Physiol Heart Circ Physiol. 2007; 292: H2306-H2315Crossref PubMed Scopus (46) Google Scholar The number of rolling or adherent leukocytes were expressed as number of cells per minute per millimeter of vessel area, calculated from diameter and length, assuming cylindrical vessel shape. Second or third-order venules and arterioles (one to three per mouse), meeting the characteristics described above, were randomly selected in each cremaster muscle to study thrombus formation. Then, 10 ml/kg of 5% FITC-dextran (150,000 MW; Sigma Chemicals) was slowly injected into the intravenous cannula and allowed to circulate for 10 minutes. Photoactivation of FITC-dextran (excitation: 495 nm; emission: 519 nm) within the microvessels was achieved by epi-illumination using a 175-W xenon lamp (Lambda LS, Sutter, CA) and a fluorescein filter cube (HQ-FITC, Chroma Technology Company, Rockingham, VT). The excitation power density was measured daily (ILT 1700 Radiometer, SED033 detector; International Light, Peabody, MA) and maintained within 1% of 0.74 W/cm2, as previously described.18Rumbaut RE Randhawa JK Smith CW Burns AR Mouse cremaster venules are predisposed to light/dye-induced thrombosis independent of wall shear rate, CD18, ICAM-1, or P-selectin.Microcirculation. 2004; 11: 239-247Crossref PubMed Scopus (41) Google Scholar, 19Rumbaut RE Slaff DW Burns AR Microvascular thrombosis models in venules and arterioles in vivo.Microcirculation. 2005; 12: 259-274Crossref PubMed Scopus (79) Google Scholar Epi-illumination was continuously applied to the vessels, and thrombus formation was quantified by determining (1) the time of onset of platelet deposition/aggregation within the microvessel (onset time), and (2) the time required for complete flow cessation for ≥60 seconds (cessation time). Epi-illumination was discontinued once blood flow ceased in the vessel under study. The results of each vessel type (venules, arterioles) were averaged between 1 and 3 thrombi produced in each mouse. The light/dye method was used to monitor thrombus formation in the following experimental groups: (1) control WT mice receiving an intrascrotal injection of 0.2 ml of saline at 4 hours before vessel photoactivation; (2) WT mice receiving an intrascrotal injection of recombinant mouse IL-1β (Calbiochem, La Jolla, CA) at a concentration of either 0.2, 1.0, or 5.0 μg/kg (dissolved in 0.2 ml of normal saline) at 4 hours before photoactivation; (3) DSS-treated WT mice receiving (i.p., 24 hours before photoactivation) 100 μg/mouse of a polyclonal anti-mouse IL-1β antibody (goat IgG; R&D Systems, Minneapolis, MN) dissolved 0.2 ml of normal saline; (4) DSS-treated WT mice receiving (i.p., 24 hours before photoactivation) 100 μg/mouse of a control antibody (goat IgG; R&D Systems) dissolved 0.2 ml of normal saline; (5) water-treated interleukin-1 receptor deficient (IL-1r−/−) mice; (6) DSS-treated IL-1r−/− mice; and (7) DSS-treated MyD88−/− mice. A separate group (n = 3) of mice were rendered neutropenic by intraperitoneal injection (150 μg) of anti-neutrophil serum (RB6–8C5, rat anti-mouse Ly-6G; eBioscience, San Diego, CA) 24 hours before the induction of light/dye injury. Anti-neutrophil treatment yielded a 93.7 ± 3.6% reduction in blood neutrophil count. In another group of control (n = 5) and colitic (n = 5) mice, we quantified the rolling and firm adherence of leukocytes in cremaster venules. Additional water (n = 5) or DSS (n = 5) fed mice were used to collect colon, skeletal muscle, and plasma samples for determination of IL-1β concentration. Freshly collected platelet rich plasma from donor mice was used to monitor platelet aggregation velocity following agonist exposure by using a Laser Particle Analyzer (Lumex Ltd., St. Petersburg, Russia). Initially, the EC50 value (concentration required to achieve half-maximum velocity) for aggregation velocity of wild type platelets in response to thrombin (0.25; 0.5; 1; and 4 U/ml) was determined as previously described.20Mindukshev IV Jahatspanian IE Goncharov NV Jenkins RO Krivchenko AI A new method for studying platelets, based upon the low-angle light scattering technique. 1. Theoretical and experimental foundations of the method.Spectroscopy. 2005; 19: 235-246Crossref Scopus (19) Google Scholar Thrombin was chosen as the platelet agonist because previous work has implicated this pro-coagulant as a critical mediator of DSS colitis-enhanced microvascular thrombosis.7Yoshida H Russell J Granger DN Thrombin mediates the extraintestinal thrombosis associated with experimental colitis.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G904-G908Crossref PubMed Scopus (13) Google Scholar This method yielded an EC50 of 0.63 U/ml, which was subsequently used to compare the aggregation velocity of platelets derived from the following experimental groups: (1) WT mice (n = 6) placed on tap water (WT-Control); (2) WT mice (n = 6) placed on 3% DSS (WT-DSS); (3) WT-DSS mice (n = 5) treated with the IL-1β antibody (as described above); and (4) IL-1 receptor-deficient mice (n = 5) treated with DSS (IL-1r−/− -DSS). Data were analyzed by using standard statistical analysis, ie, one-way analysis of variance and Fisher's post hoc test. All values are reported as means ± SE from four to seven mice. All mice placed on 3% DSS exhibited a significantly increased DAI without mortality. There was no statistical difference in DAI on day 6 of DSS treatment between any of experimental groups. Figure 1 shows the changes in time of onset of thrombosis and the time to flow cessation induced by light/dye injury in cremaster muscle arterioles and venules of WT control and DSS-treated (colitic) mice. As previously reported,3Anthoni C Russell J Wood KC Stokes KY Vowinkel T Kirchhofer D Granger DN Tissue factor: a mediator of inflammatory cell recruitment, tissue injury, and thrombus formation in experimental colitis.J Exp Med. 2007; 204: 1595-1601Crossref PubMed Scopus (72) Google Scholar, 6Yoshida H Russell J Stokes KY Yilmaz CE Esmon CT Granger DN Role of the protein C pathway in the extraintestinal thrombosis associated with murine colitis.Gastroenterology. 2008; 135: 882-888Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 19Rumbaut RE Slaff DW Burns AR Microvascular thrombosis models in venules and arterioles in vivo.Microcirculation. 2005; 12: 259-274Crossref PubMed Scopus (79) Google Scholar the time required for the initiation (onset) and termination (cessation) of thrombus formation is much longer in arterioles than in venules. Although no differences were noted in either the time of onset or time to flow cessation between venules of control and colitic mice, both variables were significantly reduced (consistent with accelerated thrombosis) in cremaster arterioles of colitic mice. The accelerated thrombus formation in arterioles of DSS colitic mice is consistent with previous reports.3Anthoni C Russell J Wood KC Stokes KY Vowinkel T Kirchhofer D Granger DN Tissue factor: a mediator of inflammatory cell recruitment, tissue injury, and thrombus formation in experimental colitis.J Exp Med. 2007; 204: 1595-1601Crossref PubMed Scopus (72) Google Scholar, 6Yoshida H Russell J Stokes KY Yilmaz CE Esmon CT Granger DN Role of the protein C pathway in the extraintestinal thrombosis associated with murine colitis.Gastroenterology. 2008; 135: 882-888Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar The figure also illustrates that a similar pattern of enhanced thrombosis in cremaster arterioles is evidenced in an immune model of colitis that results from the transfer of CD4+ T-cells from IL-10−/− mice into immunodeficient RAG-1−/− mice. These results indicate that the enhanced extra-intestinal thrombosis observed during colonic inflammation is not unique to the DSS model. Figure 2 summarizes the changes detected in the number of rolling and firmly adherent leukocytes in venules of cremaster muscle in mice with DSS-induced colonic inflammation. Although no significant differences in wall shear rate were noted between venules of control (724.1 ± 68.5 seconds−1) and colitic (714.82 ± 78.9 seconds−1) mice, a marked increase in the number of rolling and adherent leukocytes was detected in venules of colitic mice. IL-1β immunoblockade significantly reduced the total number of both rolling and adherent leukocytes increased by DSS colitis. These results suggest that the cremaster microcirculation assumes an inflammatory phenotype during DSS colitis and that IL-1β contributes to this response. Mice rendered neutropenic with anti-neutrophil serum were studied to determine whether the leukocyte recruitment noted in the cremaster muscle of DSS-treated mice contributes to the enhanced thrombosis response. A comparison of the time of onset (2.0 ± 0.5 minutes versus 3.3 ± 0.7 minutes; untreated versus anti-neutrophil serum-treated) and the time to flow cessation (15.4 ± 1.6 minutes versus 16.2 ± 0.9 minutes) between DSS and neutropenic DSS mice revealed no statistically significant differences in the thrombosis responses to light/dye injury, suggesting that neutrophils do not mediate the extra-intestinal thrombosis observed in DSS mice. Figure 3 summarizes the IL-1β concentrations detected in colonic and skeletal muscle tissue of control and DSS colitic mice. The inflamed colon exhibited a large (∼18-fold) increase in IL-1β concentration, compared with normal colon. Although plasma IL-1β tended to be higher in DSS colitic mice compared with controls (2.06 ± 1.49 versus 0.56 ± 0.39 pg/ml), this difference did not reach statistical significance (P = 0.31). Although IL-1β concentration measured in skeletal muscle of control mice was largely undetectable (1.34 ± 0.67 pg/ml), a significantly elevated concentration (21.32 ± 5.5 pg/g) was detected in skeletal muscle tissue of DSS colitic mice. Figure 4A compares the thrombosis responses to light/dye injury in arterioles of cremaster muscle between control WT mice on normal drinking water to a similar group of mice receiving an intrascrotal injection of saline. These findings demonstrate that the intrascrotal injection protocol per se does not alter the thrombosis responses to light/dye injury. Figure 4B compares the thrombosis responses to light/dye injury in arterioles of control WT, DSS-treated WT, and control WT receiving an intrascrotal injection of either 0.2, 1.0, or 5.0 μg/kg of IL-1β. Although the lowest dose (0.2 μg/kg) of IL-1β did not alter thrombosis, the higher doses significantly accelerated thrombosis, as manifested by a reduction in both the time of onset and time to flow cessation. However, the highest dose (5.0 μg/kg) of IL-1β did not produce a greater response than noted with 1.0 μg/kg. Even with the higher doses of IL-1β, the magnitude of the prothrombotic effect on the time to flow cessation did not equal that elicited by DSS treatment. Figure 5 compares the thrombosis responses to light/dye injury in cremaster
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