Laboratory Investigation (2006) 86, 9–22 & 2006 USCAP, Inc All rights reserved 0023-6837/06 $30.00 www.laboratoryinvestigation.org Lipopolysaccharide signaling in endothelial cells Shauna M Dauphinee1,2 and Aly Karsan1,2,3,4 1Department of Medical Biophysics, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; 2Experimental Medicine Program, University of British Columbia, Vancouver, British Columbia, Canada; 3Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, British Columbia, Canada and 4Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada Sepsis is the systemic immune response to severe bacterial infection. The innate immune recognition of bacterial and viral products is mediated by a family of transmembrane receptors known as Toll-like receptors (TLRs). In endothelial cells, exposure to lipopolysaccharide (LPS), a major cell wall constituent of Gram- negative bacteria, results in endothelial activation through a receptor complex consisting of TLR4, CD14 and MD2. Recruitment of the adaptor protein myeloid differentiation factor (MyD88) initiates an MyD88-dependent pathway that culminates in the early activation of nuclear factor-jB (NF-jB) and the mitogen-activated protein kinases. In parallel, a MyD88-independent pathway results in a late-phase activation of NF-jB. The outcome is the production of various proinflammatory mediators and ultimately cellular injury, leading to the various vascular sequelae of sepsis. This review will focus on the signaling pathways initiated by LPS binding to the TLR4 receptor in endothelial cells and the coordinated regulation of this pathway. Laboratory Investigation (2006) 86, 9–22. doi:10.1038/labinvest.3700366; published online 7 November 2005 Keywords: lipopolysaccharide; Toll-like receptors; innate immunity; endothelial cell; inflammation Sepsis is the leading cause of mortality in critically Endothelial dysfunction in sepsis ill patients.1 The development of sepsis occurs as a result of a systemic inflammatory response to a The endothelium plays a major role in the patho- severe bacterial infection.2 Under normal condi- genesis of sepsis. Endothelial cells line the inner wall of blood vessels, lying at the interface between tions, a controlled cellular response to bacterial 4 products protects the host from infection. In circulating blood and the surrounding tissue. sepsis, hyperactivation of the immune response Although these cells are potentially highly suscep- leads to the excessive production of various tible to injury given that they are the first cells proinflammatory cytokines and cellular injury.2 exposed to invading pathogens circulating in the bloodstream, endothelial cells have a remarkable In mammals, the innate immune system is the 4 first line of host defense involved in detecting capacity to protect themselves. The endothelium the wide variety of invading microbial pathogens.3 serves a multitude of functions that help to maintain Receptors of the innate immune system are activated organ homeostasis, including vasoregulation, selec- tive vascular permeability and providing an anti- by microbial components such as lipopolysacchar- 5 ide (LPS) (also known as endotoxin), which is a coagulant surface. However, during infection, the key molecule involved in the initiation of the normal physiological functions of the endothelium sepsis syndrome.3 are perturbed, contributing to the organ failure characteristic of sepsis.6 Although there is debate on the extent of vascular dysfunction that is due to the direct effects of LPS on the endothelium, relative to the effects that are secondary to the release of Correspondence: Dr A Karsan, MD, Department of Medical inflammatory mediators, such as tumor necrosis Biophysics, British Columbia Cancer Research Centre, 675 West factor a (TNFa), interleukin-1b (IL-1b), interferons 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. (IFNs) and others, from macrophages and immune E-mail: [email protected] 7 Funding: CIHR Grant # MOP-64409 cells, this review will concentrate on the direct Received 1 September 2005; accepted 2 October 2005; published cellular and molecular effects of LPS on the online 7 November 2005 endothelium. LPS signaling in endothelial cells SM Dauphinee and A Karsan 10 Vasoregulation facilitate a strong association with cell-adhesion molecules (CAMs) on the surface of cytokine- The endothelium secretes a diversity of paracrine 19 8 stimulated endothelial cells (ICAM-1, VCAM-1). agents that mediate vascular tone. Vasodilating LPS directly increases expression of E-selectin and compounds such as nitric oxide (NO) and prostacy- integrin counter receptors,20 and the upregulation of clin increase the diameter of the intravascular space this adhesion molecule expression requires the by relaxing adjacent smooth muscle cells within the 9 nuclear localization of nuclear factor-kappa B (NF- vessel wall. These mediators counteract vasocon- kB).21,22 In septic patients, elevated levels of endo- strictors such as angiotensin to maintain a balance 23 10 thelial selectins correlate with poor prognosis. in vascular resistance. Upon exposure to LPS, Furthermore, mice deficient for endothelial selec- endothelial cells upregulate the expression of tins show increased survival in an animal model of inducible NO synthase (NOS2), which leads to an sepsis.24 increase in the production of NO.11 The use of pharmacological NOS inhibitors in patients with septic shock increases blood pressure and restores Hemostasis vascular resistance.12 Furthermore, NOS2-deficient mice do not experience the impaired vasoconstric- Under normal physiological conditions, the en- tion associated with endotoxemia.13 This excessive dothelium is maintained in an anticoagulatory state production of vasoactive mediators, such as NO, by the action of thrombin on the surface of the results in the impaired vasoconstriction and asso- endothelial cell.25 Thrombin, bound to thrombomo- ciated hypotension seen in patients with sepsis.14 dulin, cleaves protein C into its active form, activated protein C, which inhibits the activated coagulation factors, Va and VIIIa.26 During sepsis, Vascular Permeability the endothelium shifts from an anticoagulant sur- face to a procoagulant surface by reduced expression Endothelial cells serve as a selective barrier for the of anticoagulatory molecules such as thrombo- exchange of fluid and macromolecules from the modulin, thereby shifting the action of thrombin vascular compartment to the tissue.5 An increase in towards the cleavage of fibrinogen and the genera- vascular permeability is mediated by retraction of tion of fibrin clots.27,28 Furthermore, LPS may the endothelium and phosphorylation of the light directly induce the prothrombotic state by upregu- chain of nonmuscle myosin.15 This phosphorylation lating the endothelial expression of tissue factor event induces a conformational change in the through an NF-kB-dependent mechanism.29 These myosin light chain that promotes the interaction of changes contribute to the disseminated intravascu- actin and myosin, thereby supporting the contractile lar coagulation characteristic of sepsis. state.15 A recombinant form of LPS derived from the horseshoe crab, Limulus polyphemus, that is designed to neutralize endotoxin action, has been Toll-like receptor (TLR)4 signaling shown to prevent LPS-induced actin depolymeriza- LPS is a key component of the cell wall of Gram- tion, thereby preventing an LPS-induced increase in 30 endothelial permeability.16 LPS also directly con- negative bacteria. It is composed of three structural tributes to endothelial barrier dysfunction through a elements: a core oligosaccharide, an O-specific caspase-mediated cleavage of junctional proteins chain made up of repeating sequences of polysac- involved in regulating transport of material between charides and a lipid A component, which is the vascular space and tissue.17 responsible for the proinflammatory properties of LPS.30 The binding of LPS to the surface of endothelial cells results in endothelial activation, Leukocyte Recruitment and Adhesion as demonstrated by the expression of proinflamma- tory cytokines and adhesion molecules, and, in During the healing process, circulating leukocytes some cases, endothelial apoptosis.31 LPS also acti- are recruited to the endothelium, where they adhere vates monocytes and macrophages to stimulate the and traverse between the endothelial cells to enter production of proinflammatory mediators, which in the site of inflammation.18 The initial step in this turn modulate endothelial function. Collectively, process involves recruitment of the leukocyte to the this initiates a parallel cascade of events that endothelial surface through the association between contribute to the clinical manifestations of sepsis. selectin molecules, which are found on the surface The TLRs are a family of pattern recognition of both the circulating leukocytes (L-selectin) and receptors that are classified on the basis of homology the endothelium (E- and P-selectin), and sialylated, of the cytoplasmic domain with that of the inter- carbohydrate moieties on the contacting cell.18 The leukin-1 receptor (IL-1R) family, which is known as initial selectin-mediated rolling followed by tighter the Toll/IL-1R (TIR) domain.32 To date, there have adhesion to the endothelium is also mediated by the been 12 TLRs identified in mice33 and 10 TLRs in integrin family. Integrins are heterodimeric