Interleukin-6 Mediates the Platelet Abnormalities and Thrombogenesis Associated with Experimental Colitis
2013; Elsevier BV; Volume: 183; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2013.03.014
ISSN1525-2191
AutoresElena Y. Senchenkova, Shunsuke Komoto, Janice Russell, Lidiana D. Almeida-Paula, Li-Sue Yan, Songlin Zhang, D. Neil Granger,
Tópico(s)Autoimmune and Inflammatory Disorders
ResumoClinical studies and animal experimentation have shown that colonic inflammation is associated with an increased number and reactivity of platelets, coagulation abnormalities, and enhanced thrombus formation. The objective of this study was to define the contribution of IL-6 to the thrombocytosis, exaggerated agonist-induced platelet aggregation, and enhanced extra-intestinal thrombosis that occur during experimental colitis. The number of mature and immature platelets, platelet life span, thrombin-induced platelet aggregation response, and light/dye-induced thrombus formation in cremaster muscle arterioles were measured in wild-type (WT) and IL-6–deficient (IL-6−/−) mice with dextran sodium sulfate (DSS)-induced colitis. DSS colitis in WT mice was associated with thrombocytosis with an elevated number of both mature and immature platelets and no change in platelet life span. The thrombocytosis response was absent in IL-6−/− mice. DSS treatment also enhanced the platelet aggregation response to thrombin and accelerated thrombus development in WT mice, but not in IL-6−/− mice. Exogenous IL-6 administered to WT mice elicited a dose-dependent enhancement of thrombus formation. These findings indicate that IL-6 mediates the thrombocytosis, platelet hyperreactivity, and accelerated thrombus development associated with experimental colitis. The IL-6–dependent colitis-induced thrombocytosis appears to result from an enhancement of thrombopoiesis because platelet life span is unchanged. Clinical studies and animal experimentation have shown that colonic inflammation is associated with an increased number and reactivity of platelets, coagulation abnormalities, and enhanced thrombus formation. The objective of this study was to define the contribution of IL-6 to the thrombocytosis, exaggerated agonist-induced platelet aggregation, and enhanced extra-intestinal thrombosis that occur during experimental colitis. The number of mature and immature platelets, platelet life span, thrombin-induced platelet aggregation response, and light/dye-induced thrombus formation in cremaster muscle arterioles were measured in wild-type (WT) and IL-6–deficient (IL-6−/−) mice with dextran sodium sulfate (DSS)-induced colitis. DSS colitis in WT mice was associated with thrombocytosis with an elevated number of both mature and immature platelets and no change in platelet life span. The thrombocytosis response was absent in IL-6−/− mice. DSS treatment also enhanced the platelet aggregation response to thrombin and accelerated thrombus development in WT mice, but not in IL-6−/− mice. Exogenous IL-6 administered to WT mice elicited a dose-dependent enhancement of thrombus formation. These findings indicate that IL-6 mediates the thrombocytosis, platelet hyperreactivity, and accelerated thrombus development associated with experimental colitis. The IL-6–dependent colitis-induced thrombocytosis appears to result from an enhancement of thrombopoiesis because platelet life span is unchanged. Inflammatory bowel diseases (IBDs) are associated with an increased risk of thrombus formation both within the inflamed bowel and in extra-intestinal tissues, such as lung and skeletal muscles.1Yoshida H. Granger D.N. Inflammatory bowel disease: a paradigm for the link between coagulation and inflammation.Inflamm Bowel Dis. 2009; 15: 1245-1255Crossref PubMed Scopus (112) Google Scholar, 2Larsen S. Bendtzen K. Nielsen O.H. Extraintestinal manifestations of inflammatory bowel disease: epidemiology, diagnosis, and management.Ann Med. 2010; 42: 97-114Crossref PubMed Scopus (220) Google Scholar The accelerated thrombus development during colonic inflammation is evidenced in large arteries and veins, as well as in the microvasculature.2Larsen S. Bendtzen K. Nielsen O.H. Extraintestinal manifestations of inflammatory bowel disease: epidemiology, diagnosis, and management.Ann Med. 2010; 42: 97-114Crossref PubMed Scopus (220) Google Scholar, 3Danese S. Papa A. Saibeni S. Repici A. Malesci A. Vecchi M. Inflammation and coagulation in inflammatory bowel disease: the clot thickens.Am J Gastroenterol. 2007; 102: 174-186Crossref PubMed Scopus (312) Google Scholar The prothrombogenic phenotype in IBD is often accompanied by increases in the blood count (reactive thrombocytosis)4Harries A.D. Beeching N.J. Rogerson S.J. Nye F.J. The platelet count as a simple measure to distinguish inflammatory bowel disease from infective diarrhoea.J Infect. 1991; 22: 247-250Abstract Full Text PDF PubMed Scopus (44) Google Scholar and reactivity of platelets3Danese S. Papa A. Saibeni S. Repici A. Malesci A. Vecchi M. Inflammation and coagulation in inflammatory bowel disease: the clot thickens.Am J Gastroenterol. 2007; 102: 174-186Crossref PubMed Scopus (312) Google Scholar and by an imbalance between procoagulant and anticoagulant mechanisms.3Danese S. Papa A. Saibeni S. Repici A. Malesci A. Vecchi M. Inflammation and coagulation in inflammatory bowel disease: the clot thickens.Am J Gastroenterol. 2007; 102: 174-186Crossref PubMed Scopus (312) Google Scholar Although a 50% to 100% increase in blood platelet count is commonly reported for patients with active IBD5Collins C.E. Rampton D.S. Rogers J. Williams N.S. Platelet aggregation and neutrophil sequestration in the mesenteric circulation in inflammatory bowel disease.Eur J Gastroenterol Hepatol. 1997; 9: 1213-1217PubMed Google Scholar, 6Heits F. Stahl M. Ludwig D. Stange E.F. Jelkmann W. Elevated serum thrombopoietin and interleukin-6 concentrations in thrombocytosis associated with inflammatory bowel disease.J Interferon Cytokine Res. 1999; 19: 757-760Crossref PubMed Scopus (90) Google Scholar, 7Webberley M.J. Hart M.T. Melikian V. Thromboembolism in inflammatory bowel disease: role of platelets.Gut. 1993; 34: 247-251Crossref PubMed Scopus (215) Google Scholar and the association of thrombocytosis with active IBD has led to the proposal that platelet count is a useful marker of disease activity,8Harries A.D. Fitzsimons E. Fifield R. Dew M.J. Rhoades J. Platelet count: a simple measure of activity in Crohn's disease.Br Med J (Clin Res Ed). 1983; 286: 1476Crossref PubMed Scopus (91) Google Scholar it remains unclear whether the elevated platelet count is mechanistically linked to the enhanced platelet reactivity and accelerated thrombus development in IBD. Such a link appears tenable because accelerated thrombopoiesis would result in an increased percentage of circulating platelets that are young and more hemostatically active than older platelets.9Ault K.A. Knowles C. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation.Exp Hematol. 1995; 23: 996-1001PubMed Google Scholar, 10Thompson C.B. Jakubowski J.A. Quinn P.G. Deykin D. Valeri C.R. Platelet size and age determine platelet function independently.Blood. 1984; 63: 1372-1375PubMed Google Scholar Enhanced thrombus formation and the other extra-intestinal manifestations (eg, platelet abnormalities, inflammation of eyes, joints, liver) of IBD2Larsen S. Bendtzen K. Nielsen O.H. Extraintestinal manifestations of inflammatory bowel disease: epidemiology, diagnosis, and management.Ann Med. 2010; 42: 97-114Crossref PubMed Scopus (220) Google Scholar, 11Irving P.M. Pasi K.J. Rampton D.S. Thrombosis and inflammatory bowel disease.Clin Gastroenterol Hepatol. 2005; 3: 617-628Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar suggest that cellular (eg, activated leukocytes or platelets) and/or chemical signals are liberated from the inflamed bowel to elicit these responses in distant vascular beds and hematopoietic tissue. Several pro-inflammatory cytokines [eg, IL-1β, tumor necrosis factor-α (TNF-α), IL-6] produced and released by the inflamed colon in human and experimental IBD12Alex P. Zachos N.C. Nguyen T. Gonzales L. Chen T.E. Conklin L.S. Centola M. Li X. Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis.Inflamm Bowel Dis. 2009; 15: 341-352Crossref PubMed Scopus (582) Google Scholar, 13Reinecker H.C. Steffen M. Witthoeft T. Pflueger I. Schreiber S. MacDermott R.P. Raedler A. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease.Clin Exp Immunol. 1993; 94: 174-181Crossref PubMed Scopus (802) Google Scholar have been shown to target components of the coagulation cascade and to alter platelet function in a manner that would predispose the vasculature to thrombus development.14Yoshida H. Russell J. Senchenkova E.Y. Almeida Paula L.D. Granger D.N. Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis.Am J Pathol. 2010; 177: 2774-2781Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 15Yoshida H. Yilmaz C.E. Granger D.N. Role of tumor necrosis factor-alpha in the extraintestinal thrombosis associated with colonic inflammation.Inflamm Bowel Dis. 2011; 17: 2217-2223Crossref PubMed Scopus (42) Google Scholar, 16Levi M. van der Poll T. Two-way interactions between inflammation and coagulation.Trends Cardiovasc Med. 2005; 15: 254-259Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar Of the cytokines studied to date, only IL-6 exhibits the ability to mediate all of the platelet responses that are characteristic of colonic inflammation, that is, thrombocytosis,17Ishibashi T. Kimura H. Shikama Y. Uchida T. Kariyone S. Hirano T. Kishimoto T. Takatsuki F. Akiyama Y. Interleukin-6 is a potent thrombopoietic factor in vivo in mice.Blood. 1989; 74: 1241-1244Crossref PubMed Google Scholar platelet hyperreactivity,18Burstein S.A. Effects of interleukin 6 on megakaryocytes and on canine platelet function.Stem Cells. 1994; 12: 386-393Crossref PubMed Scopus (44) Google Scholar, 19Peng J. Friese P. George J.N. Dale G.L. Burstein S.A. Alteration of platelet function in dogs mediated by interleukin-6.Blood. 1994; 83: 398-403PubMed Google Scholar and accelerated thrombus formation.20Mutlu G.M. Green D. Bellmeyer A. Baker C.M. Burgess Z. Rajamannan N. Christman J.W. Foiles N. Kamp D.W. Ghio A.J. Chandel N.S. Dean D.A. Sznajder J.I. Budinger G.R. Ambient particulate matter accelerates coagulation via an IL-6-dependent pathway.J Clin Invest. 2007; 117: 2952-2961Crossref PubMed Scopus (248) Google Scholar For example, animals treated with IL-6 exhibit an increased platelet count, and the platelets are more sensitive to activation by thrombin and other platelet agonists.18Burstein S.A. Effects of interleukin 6 on megakaryocytes and on canine platelet function.Stem Cells. 1994; 12: 386-393Crossref PubMed Scopus (44) Google Scholar IL-6 has been shown to act directly on megakaryocytes to increase platelet production,18Burstein S.A. Effects of interleukin 6 on megakaryocytes and on canine platelet function.Stem Cells. 1994; 12: 386-393Crossref PubMed Scopus (44) Google Scholar and it acts on hepatocytes to increase the production and release of thrombopoietin (TPO), a potent stimulant of platelet production.6Heits F. Stahl M. Ludwig D. Stange E.F. Jelkmann W. Elevated serum thrombopoietin and interleukin-6 concentrations in thrombocytosis associated with inflammatory bowel disease.J Interferon Cytokine Res. 1999; 19: 757-760Crossref PubMed Scopus (90) Google Scholar Although IL-6 has been implicated in the thrombocytosis of IBD on the basis of clinical reports that describe an association between the elevated plasma concentrations of TPO and IL-6 with blood platelet count,6Heits F. Stahl M. Ludwig D. Stange E.F. Jelkmann W. Elevated serum thrombopoietin and interleukin-6 concentrations in thrombocytosis associated with inflammatory bowel disease.J Interferon Cytokine Res. 1999; 19: 757-760Crossref PubMed Scopus (90) Google Scholar the contribution of IL-6 to colitis-induced thrombocytosis has not been directly addressed in either the clinical or experimental setting. The overall aim of this study was to assess the contribution of IL-6 to the platelet abnormalities and accelerated extra-intestinal thrombosis that are observed in a murine model of colonic inflammation. We evaluated the role of IL-6 in mediating the increased platelet count, whether IL-6 is responsible for the appearance of immature platelets, and if the life span of platelets is altered in an IL-6–dependent manner in experimental IBD. The role of IL-6 in colitis-induced platelet hyperreactivity to thrombin was also examined as well as the contribution of membrane-bound and soluble IL-6 receptors to thrombin-mediated aggregation of platelets from colitic mice. Finally, the contribution of IL-6 to colitis-enhanced extra-intestinal thrombosis was evaluated with immunoblockade or genetic deficiency of IL-6. Our findings indicate that IL-6 plays a major role in the enhanced production of highly reactive platelets and accelerated thrombus development in extra-intestinal tissue during colonic inflammation. Male C57BL/6J mice and IL-6–deficient (IL-6−/−) mice (B6.129S6-Il6tm1Kopf, C57BL/6J background) were purchased from The Jackson Laboratory (Bar Harbor, ME). The 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. Acute colitis was induced with 3% dextran sodium sulfate (DSS; 40,000 molecular weight; MP Biomedicals, Solon, OH) dissolved in filter-purified drinking water for 7 days, as previously described.14Yoshida H. Russell J. Senchenkova E.Y. Almeida Paula L.D. Granger D.N. Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis.Am J Pathol. 2010; 177: 2774-2781Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Yoshida H. Russell J. Stokes K.Y. Yilmaz C.E. Esmon C.T. Granger D.N. 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 (19) Google Scholar Control mice received regular drinking water without DSS. Histological examination was performed on samples of distal colon, which were fixed in 10% formalin before embedding in paraffin and staining with hematoxylin and eosin. The histological examination was performed in a blinded manner (S.Z.) with the use of a previously validated scoring system,22Dieleman L.A. Palmen M.J. Akol H. Bloemena E. Pena A.S. Meuwissen S.G. Van Rees E.P. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.Clin Exp Immunol. 1998; 114: 385-391Crossref PubMed Scopus (915) Google Scholar in which severity of inflammation (0 to 3 scale), depth of injury (0 to 3 scale), and crypt damage (0 to 4 scale) are multiplied by an integer from 1 through 4, representing the percentage of involvement of the colonic wall (1, 0% to 25%; 2, 26% to 50%; 3, 51% to 75%; 4, 76% to 100%), for a maximum possible score of 40. Flow cytometry was used to quantify the relative numbers and life spans of mature and immature platelets in blood of control mice and colitic mice. The in vivo biotinylation method of Ault et al9Ault K.A. Knowles C. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation.Exp Hematol. 1995; 23: 996-1001PubMed Google Scholar was used to measure platelet life span. After intravenous administration (1.2 mg dissolved in 300 μL of saline), biotin (Sulfo-NHS-LC-biotin; ProteoChem, Denver, CO) covalently binds to free amino groups on the surface of all blood cells. Biotin-positive cells were detected in tail vein blood samples (10 to 15 μL, drawn daily over 5 days) with the use of flow cytometry by binding with streptavidin conjugated with phycoerythrin (eBioscience, San Diego, CA). Platelets were distinguished from other biotin-positive blood cells by staining with CD41-allophycocyanin (isotype control rat IgG1, κ; eBioscience). Newly released (immature) platelets are biotin negative, whereas mature platelets remain biotin positive for the remainder of their life span. Hence, the entire platelet population becomes biotin negative as the platelet population ages. Young reticulated platelets were identified by staining with thiazole orange (TO; Sigma-Aldrich, St. Louis, MO), which binds to nucleic acids (DNA and RNA). Fresh blood samples were incubated with 1 μg/mL TO (dissolved in PBS) for 15 minutes at room temperature.23Matic G.B. Chapman E.S. Zaiss M. Rothe G. Schmitz G. Whole blood analysis of reticulated platelets: improvements of detection and assay stability.Cytometry. 1998; 34: 229-234Crossref PubMed Scopus (86) Google Scholar Formaldehyde (1%; Polyscience Inc., Warrington, PA) was used for cell fixation. An analysis of the staining pattern of platelets (CD41+) (detected over several days after TO and biotin administration) for TO+ and streptavidin-phycoerythrin+ was used to show the numbers and life spans of both mature and immature platelets, as previously described.9Ault K.A. Knowles C. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation.Exp Hematol. 1995; 23: 996-1001PubMed Google Scholar, 24Robinson M. MacHin S. Mackie I. Harrison P. In vivo biotinylation studies: specificity of labelling of reticulated platelets by thiazole orange and mepacrine.Br J Haematol. 2000; 108: 859-864Crossref PubMed Scopus (61) Google Scholar The three-color analysis was performed on an LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FACSDiva software version 6.1.3 (BD Biosciences), with the gate set around CD41-allophycocyanin+ cells, and 10,000 to 20,000 events were collected. The absolute blood platelet count was determined manually with the use of a hemocytometer. Platelet life span and immature and mature platelet counts were determined in the following groups of mice: i) WT-control (n = 5), ii) WT-DSS (n = 7), iii) IL-6−/−-DSS (n = 5), and iv) IL-6−/− bone marrow chimera (IL-6−/−⇒WT) chimera-DSS (n = 5). Arterial blood from control and colitic mice was drawn into a syringe that contained acid citrate dextrose with 1 mmol/L EDTA and apyrase (grade VII; Sigma-Aldrich). Sample processing was started immediately after blood collection. Aggregation was induced with thrombin 0.63 U/mL (EC50), and platelet aggregation velocity was measured as previously described.14Yoshida H. Russell J. Senchenkova E.Y. Almeida Paula L.D. Granger D.N. Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis.Am J Pathol. 2010; 177: 2774-2781Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 25Mindukshev I. Gambaryan S. Kehrer L. Schuetz C. Kobsar A. Rukoyatkina N. Nikolaev V.O. Krivchenko A. Watson S.P. Walter U. Geiger J. Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors.Clin Chem Lab Med. 2012; 50: 1253-1263Crossref PubMed Google Scholar, 26Gavins F.N. Russell J. Senchenkova E.L. De Almeida Paula L. Damazo A.S. Esmon C.T. Kirchhofer D. Hebbel R.P. Granger D.N. Mechanisms of enhanced thrombus formation in cerebral microvessels of mice expressing hemoglobin-S.Blood. 2011; 117: 4125-4133Crossref PubMed Scopus (47) Google Scholar Platelet aggregation velocity was measured from platelet-rich plasma derived from the following groups of mice: i) WT-control (n = 7), ii) WT-DSS (n = 7), iii) IL-6−/−-DSS mice (n = 7), iv) IL-6−/−⇒WT chimera-DSS (n = 6), v) WT-DSS mice treated with a rat anti–IL-6rα (CD126) blocking antibody (Angio-Proteomic, Boston, MA; 100 μg/mouse in 0.2 mL of normal saline administered 24 hour before the experiment; n = 5), and vi) WT-DSS mice treated with an anti-gp130 blocking antibody (R&D System Inc., Minneapolis, MN; 20 μg/mouse in 0.2 normal saline administered 24 hours before the experiment; n = 6). The mouse cremaster muscle was prepared for microscopic observation as previously described.14Yoshida H. Russell J. Senchenkova E.Y. Almeida Paula L.D. Granger D.N. Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis.Am J Pathol. 2010; 177: 2774-2781Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 27Rumbaut R.E. Randhawa J.K. Smith C.W. Burns A.R. 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 (42) Google Scholar Light and fluorescent microscopic images from an upright microscope (BX51WI; Olympus, Tokyo, Japan) with a 40× water immersion objective lens (LUMPlan FI/IR 40×/0.80×; Olympus) were projected onto a monitor (Trinitron PVM-2030; Sony, Tokyo, Japan) through a color video camera (VK-C150; Hitachi, Tokyo, Japan) or a charge-coupled device video camera (XC-77; Hamamatsu, Hamamatsu City, Japan), respectively. The images were recorded with a DVD recorder (SR-MV50; JVC, Wayne, NJ). A video timer (Time-Date Generator WJ-810; Panasonic, Osaka, Japan) was connected to the monitor to record time and date. The diameters of the selected cremaster muscle microvessels were measured by video analysis software (ImageJ software version 1.37; NIH, Bethesda, MD) on a personal computer (G4 Macintosh; Apple, Cupertino City, CA). Fluorescein isothiocyanate-dextran (5%) 10 mL/kg (150,000 molecular weight; Sigma Chemicals, St. Louis, MO) was infused via a cannulated jugular vein. Photoactivation of fluorescein isothiocyanate-dextran (excitation, 495 nm; emission, 519 nm) within the selected microvessels was achieved by epi-illumination with a 175-W xenon lamp (Lambda LS, Sutter, CA) and a fluorescein filter cube (HQ-FITC; Chroma Technology, Bellows Falls, VT). The excitation power density was measured daily (ILT 1700 Radiometer, SED033 detector; International Light Technologies, Peabody, MA) and maintained within 1% of 0.77 W/cm2, as previously described.27Rumbaut R.E. Randhawa J.K. Smith C.W. Burns A.R. 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 (42) Google Scholar, 28Rumbaut R.E. Slaff D.W. Burns A.R. Microvascular thrombosis models in venules and arterioles in vivo.Microcirculation. 2005; 12: 259-274Crossref PubMed Scopus (81) Google Scholar Second- or third-order arterioles with a diameter of 30 to 50 μm, a minimum length of 100 μm, and a wall shear rate >500/s were randomly selected in the mouse cremaster muscle to study thrombus formation. Epi-illumination was continuously applied to the vessels, and thrombus formation was quantified by determining the time of onset of platelet deposition/aggregation (onset time) and the time required for complete cessation for >60 seconds (cessation time). Epi-illumination was discontinued once blood flow ceased in the vessel under study. The results from each arteriole were averaged from two to three thrombi produced in each mouse. The light/dye method was used to monitor thrombus formation in the following experimental groups: i) control WT mice receiving an intrascrotal injection of 0.2 mL of saline at 5 hours before vessel photoactivation (n = 7); ii) control WT mice injected intrascrotally with recombinant mouse IL-6 (R&D Systems, Minneapolis, MN) at a concentration of either 10 (n = 5), 100 (n = 5), or 500 ng (n = 5) per mouse (dissolved in 0.2 mL of normal saline) at 5 hours before photoactivation; iii) WT mice on water (n = 10); iv) DSS-treated WT mice (WT-DSS) (n = 10); v) anti–IL-6 antibody (BD Biosciences) treated (100 μg/mouse administered 24 hours before photoactivation) WT-DSS mice (n = 8); and iv) DSS-treated IL-6−/− mice (n = 9). In some mice, bone marrow from IL-6−/− donor mice was transplanted into WT recipients to produce IL-6−/−⇒WT chimeras, wherein only circulating blood cells are IL-6 deficient. IL-6–deficient BM cells (5 × 106) were transferred into the irradiated recipient mice, as previously described.29Stokes K.Y. Calahan L. Russell J.M. Gurwara S. Granger D.N. Role of platelets in hypercholesterolemia-induced leukocyte recruitment and arteriolar dysfunction.Microcirculation. 2006; 13: 377-388Crossref PubMed Scopus (29) Google Scholar Bone marrow chimerization was detected after 8 weeks, and only mice with >90% chimerization were used. Data were analyzed with standard statistical analysis, that is, one-way analysis of variance, Fisher's post hoc test, Neuman-Keuls multicomparison test for >2 groups, or a Student's t-test for only two groups of animals. All values are reported as means ± SEM with more than five mice per group. A well-characterized response to human IBD is thrombocytosis.4Harries A.D. Beeching N.J. Rogerson S.J. Nye F.J. The platelet count as a simple measure to distinguish inflammatory bowel disease from infective diarrhoea.J Infect. 1991; 22: 247-250Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 8Harries A.D. Fitzsimons E. Fifield R. Dew M.J. Rhoades J. Platelet count: a simple measure of activity in Crohn's disease.Br Med J (Clin Res Ed). 1983; 286: 1476Crossref PubMed Scopus (91) Google Scholar, 30Morowitz D.A. Allen L.W. Kirsner J.B. Thrombocytosis in chronic inflammatory bowel disease.Ann Intern Med. 1968; 68: 1013-1021Crossref PubMed Scopus (150) Google Scholar Figure 1A shows that DSS-induced colonic inflammation in WT mice is also associated with a significant increase in total platelet count on day 6 of DSS treatment. This response was accompanied by increases in the numbers of immature (TO+) and mature (TO−) platelets (Figure 1, B–D). These increases in total, immature, and mature platelets were not detected in either IL-6−/− mice or in IL-6−/−⇒WT bone marrow chimeras, suggesting that IL-6 derived from cells of myeloid origin mediates the thrombocytosis associated with DSS-induced colitis. A role for IL-6 in mediating DSS-induced thrombocytosis is also supported by the observation (data not shown) that WT-DSS mice treated on day 5 with an IL-6rβ neutralizing antibody (936 ± 163 × 103 platelets/μL), but not an IL-6rα antibody (1368 ± 94 × 103 platelets/μL), resulted in a significant reduction in platelet count on day 6 of DSS, compared with untreated colitic (day 6) mice (1741 ± 215 × 103 platelets/μL). The IL-6–dependent increase in immature platelets in DSS colitic mice is consistent with the known ability of this cytokine to enhance platelet production.17Ishibashi T. Kimura H. Shikama Y. Uchida T. Kariyone S. Hirano T. Kishimoto T. Takatsuki F. Akiyama Y. Interleukin-6 is a potent thrombopoietic factor in vivo in mice.Blood. 1989; 74: 1241-1244Crossref PubMed Google Scholar Because an increased platelet count can also result from an extended platelet life span, we also evaluated the influence of DSS-induced colonic inflammation on the life span of mature and immature platelets of both WT and IL-6−/− mice. As shown in Figure 2, A and B, the life span of immature (23.10 ± 0.8 hours) and mature (110 ± 5.7 hours) platelets in WT noncolitic (control) mice was not significantly altered by DSS treatment in WT, IL-6−/−, or IL-6−/−⇒WT mice. Platelets from patients with IBD are known to exhibit a hypersensitivity to aggregation in response to different agonists.31Andoh A. Yoshida T. Yagi Y. Bamba S. Hata K. Tsujikawa T. Kitoh K. Sasaki M. Fujiyama Y. Increased aggregation response of platelets in patients with inflammatory bowel disease.J Gastroenterol. 2006; 41: 47-54Crossref PubMed Scopus (41) Google Scholar This phenomenon is also evident in platelets derived from mice with DSS colitis.14Yoshida H. Russell J. Senchenkova E.Y. Almeida Paula L.D. Granger D.N. Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis.Am J Pathol. 2010; 177: 2774-2781Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Figure 3A shows the large increase in platelet aggregation velocity observed in platelet-rich plasma from DSS colitic WT mice after exposure to an EC50 dose of thrombin. This 2.5-fold increase (compared with WT controls) in platelet aggregation velocity was not evident in platelet-rich plasma derived from either IL-6−/− or IL-6−/−⇒WT mice treated with DSS, suggesting that IL-6 (derived from myeloid cells) plays an important role in the altered platelet aggregation response that accompanies DSS colitis. To further evaluate the contribution of IL-6 to colitis-induced platelet hyperaggregation, WT DSS colitic mice were treated with a blocking antibody directed against either the α or β (gp130) subunit of the IL-6 receptor (Figure 3B). The results of these experiments indicated that immunoblockade of the β-subunit of IL-6r completely prevented the exaggerated aggregation response of platelets from colitic mice to thrombin. Although blockade of the α-subunit (membrane-bound) of IL-6r also reduced the aggregation response to thrombin, this intervention was not as effective as β-subunit blockade. Measurements of platelet count in arterial blood of WT controls (992 ± 60 × 103/μL), WT-DSS mice (1741 ± 215 × 103/μL), and mice treated with the IL-6r antibodies indicated that the DSS-induced thrombocytosis was significantly blunted by treatment with the IL-6rβ monoclonal antibody (936 ± 163 × 103/μL; P < 0.01), but not with the IL-6rα monoclonal antibody (1368 ± 94 × 103/μL). Although some pro-inflammatory cytokines (eg, TNF-α, IL-1β) have been shown to promote a microvascular thrombosis response similar to experimental colitis,14Yoshida H. Russell J. Senchenkova E.Y. Almeida Paula L.D. Granger D.N. Interleukin-1beta mediates the extra-intestinal th
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