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Bilirubin modulates leukocyte recruitment to sites of inflammation

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2017, PhD, University of Cincinnati, Medicine: Pathobiology and Molecular Medicine.
Background: Bilirubin is the principal end-product of heme catabolism. While generally thought to be little more than a metabolic by-product, there is accumulating epidemiological evidence that higher serum bilirubin levels are associated with a lower incidence of inflammatory disorders such as inflammatory bowel and cardiovascular disease. However, the mechanism(s) by which bilirubin may exert an anti-inflammatory effect remains poorly understood. The transendothelial migration of immune cells to sites of inflammation is a highly-ordered, multi-step process that is initiated when endothelial cells become activated to express adhesion molecules, including Vascular Cell Adhesion Molecule 1 (VCAM-1) and Intercellular Adhesion Molecule 1 (ICAM-1), on their luminal surface. The specific binding of leukocyte integrins to VCAM-1 and/or ICAM-1 triggers endothelial signaling cascades that result in the intracellular generation of superoxide and hydrogen peroxide. These reactive oxygen species (ROS) induce reorganization of the actin cytoskeleton, promoting leukocyte transmigration. There are many disease states in which VCAM-1 and ICAM-1 are believed to play an essential pathogenic role in mediating leukocyte trafficking. As bilirubin is a potent, chain-breaking antioxidant, our central hypothesis is that it exerts an anti-inflammatory effect by disrupting adhesion molecule-mediated leukocyte migration through the scavenging of ROS signaling intermediaries. Aim: To validate the key role played by VCAM-1 and ICAM-1 in the pathogenesis of inflammatory bowel disease and cardiovascular disease and, to elucidate the molecular mechanisms underlying the ameliorating effect of bilirubin on these disorders. Methods: In vitro analyses of the mechanisms by which bilirubin impedes the transmigration of human leukocyte cell lines across monolayers of isolated human umbilical vein endothelial cells (HUVEC) were performed using Boyden chamber assay and confocal microscopy to assess adhesion molecule-stimulated ROS generation. Murine models of inflammatory bowel disease (DSS-induced colitis) and cardiovascular disease (LDL receptor-deficient mice; Ldlr-/-) were employed to investigate the effect of bilirubin on local and systemic inflammation (immunohistochemistry, immunoassay), and tissue oxidation (immunofluorescence). Results & Conclusions: Bilirubin, at physiological concentrations (= 20 µM), was found to dose-dependently block THP-1 (monocyte) and Jurkat (lymphocyte) migration across tumor necrosis factor a-activated HUVEC monolayers, without altering leukocyte binding or cytokine/ chemokine production. Bilirubin also effectively abolished endothelial ROS generation induced by the crosslinking of VCAM-1 or ICAM-1, as validated by treatment with blocking antibodies and with specific inhibitors of VCAM-1 and ICAM-1 signaling. Bilirubin, when administered to mice with DSS-induced colitis, abrogated disease activity, specifically reducing eosinophil, lymphocyte and monocyte infiltration into the colon. Ldlr-/- mice maintained on a high-fat diet showed a marked reduction in atherosclerotic plaque formation when treated with bilirubin, without changes in circulating cholesterol, triglyceride, or cytokine/chemokine levels. Aortic roots from bilirubin-treated animals manifested reduced lipid and collagen deposition, diminished numbers of macrophages, lymphocytes and smooth muscle cells, and decreased protein oxidation, without altered VCAM-1 or ICAM-1 expression. These data support that bilirubin ameliorates inflammatory responses by inhibiting VCAM-1- and ICAM-1-mediated leukocyte migration through the scavenging of ROS signaling intermediaries, suggesting a potential mechanism for the anti-inflammatory effects of bilirubin.
Stephen Zucker, M.D. (Committee Chair)
George Deepe, M.D. (Committee Member)
David Hui, Ph.D. (Committee Member)
Alex Lentsch, Ph.D. (Committee Member)
Florence Rothenberg, M.D. (Committee Member)
162 p.

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Citations

  • Vogel, M. E. (2017). Bilirubin modulates leukocyte recruitment to sites of inflammation [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1490353646818353

    APA Style (7th edition)

  • Vogel, Megan. Bilirubin modulates leukocyte recruitment to sites of inflammation. 2017. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1490353646818353.

    MLA Style (8th edition)

  • Vogel, Megan. "Bilirubin modulates leukocyte recruitment to sites of inflammation." Doctoral dissertation, University of Cincinnati, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1490353646818353

    Chicago Manual of Style (17th edition)