Novel critical role of Toll-like receptor 4 in lung ischemia-reperfusion injury and edema.
ABSTRACT Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)- and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-kappaB was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemia-reperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability.
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ABSTRACT: The endothelial cell (EC) lining of the pulmonary vasculature forms a semipermeable barrier between the blood and the interstitium of the lung. Disruption of this barrier occurs during inflammatory disease states such as acute lung injury and acute respiratory distress syndrome and results in the movement of fluid and macromolecules into the interstitium and pulmonary air spaces. These processes significantly contribute to the high morbidity and mortality of patients afflicted with acute lung injury. The critical importance of pulmonary vascular barrier function is shown by the balance between competing EC contractile forces, which generate centripetal tension, and adhesive cell-cell and cell-matrix tethering forces, which regulate cell shape. Both competing forces in this model are intimately linked through the endothelial cytoskeleton, a complex network of actin microfilaments, microtubules, and intermediate filaments, which combine to regulate shape change and transduce signals within and between EC. A key EC contractile event in several models of agonist-induced barrier dysfunction is the phosphorylation of regulatory myosin light chains catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase and/or through the activity of the Rho/Rho kinase pathway. Intercellular contacts along the endothelial monolayer consist primarily of two types of complexes (adherens junctions and tight junctions), which link to the actin cytoskeleton to provide both mechanical stability and transduction of extracellular signals into the cell. Focal adhesions provide additional adhesive forces in barrier regulation by forming a critical bridge for bidirectional signal transduction between the actin cytoskeleton and the cell-matrix interface. Increasingly, the effects of mechanical forces such as shear stress and ventilator-induced stretch on EC barrier function are being recognized. The critical role of the endothelial cytoskeleton in integrating these multiple aspects of pulmonary vascular permeability provides a fertile area for the development of clinically important barrier-modulating therapies.Journal of Applied Physiology 11/2001; 91(4):1487-500. · 3.75 Impact Factor
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ABSTRACT: There is growing evidence of a role of the immune system in the pathophysiology of ischemia-reperfusion (I/R) injury, but the influence of I/R injury on innate immunity is still undetermined. Sprague-Dawley rats were used. I/R injury was induced by clamping both renal arteries for 45 min, and the rats were killed 1, 3, 5, and 7 days later. Activation of innate immunity was evaluated in terms of the expression of toll-like receptor (TLR) 2 or TLR4 mRNAs and protein, by the level of the TLR ligand (heat shock protein [HSP] 70), and maturation of dendritic cells by double-label immunohistochemistry of dendritic cells for major histocompatibility complex (MHC) class II antigen. I/R injury increased TLR2 and TLR4 mRNA and protein expression, and they were mainly observed on renal tubular cells. I/R injury also produced endogenous TLR ligand (HSP70) on renal tubular cells. I/R injury increased not only the numbers of dendritic cells but also the production of MHC class II antigen in dendritic cells, suggesting maturation of these cells. Activation of innate immunity was observed at day 1, peaked at days 3 to 5 after I/R injury, and thereafter gradually decreased. I/R injury rapidly activates the innate immune response.Transplantation 06/2005; 79(10):1370-7. · 4.00 Impact Factor
Article: Toll-like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion.[show abstract] [hide abstract]
ABSTRACT: Ischemia/reperfusion (I/R) injury leads to graft dysfunction and may contribute to alloimmune responses posttransplantation. The molecular mechanisms of cold I/R injury are only partially characterized but may involve toll-like receptor (TLR)-4 activation by endogenous ligands. We tested the hypothesis that TLR4 mediates the early inflammatory response in the setting of cold I/R in a murine cardiac transplant model. Syngeneic heart transplants were performed in mutant mice deficient in TLR4 signaling (C3H/HeJ) and wild-type mice (C3H/HeOuJ). Transplants were also performed between the strains (mutant hearts into wild-type recipients and the converse). Donor hearts were subjected to 2 hr of cold ischemia. The grafts were retrieved at 3 and 24 hr after reperfusion. Serum samples were collected for cytokine analysis. Reverse-transcription polymerase chain reaction and histologic analysis were used to assess intra-graft inflammation. After transplant, serum tumor necrosis factor (TNF), interleukin (IL)-6, JE/monocyte chemotractant protein (MCP)-1, IL-1beta, and troponin I levels, as well as intragraft TNF, IL-1beta, IL-6, early growth response (EGR)-1, intercellular adhesion molecule (ICAM)-1, and inducible nitric oxide synthase (iNOS) mRNA levels, were significantly lower in the mutant-->mutant group compared to the wild-type-->wild-type group (P< or =0.05). Intermediate levels of serum IL-6, JE/MCP-1, as well as intragraft TNF, IL-1beta, IL-6, and ICAM-1 mRNA were observed after transplants in the mutant-->wild-type and wild-type-->mutant groups. Immunohistochemistry revealed less myocardial nuclear factor-kappaB nuclear translocation at and less neutrophil infiltration in the mutant-->mutant group compared to the wild-type-->wild-type group. These findings demonstrate that TLR4 signaling is central to both the systemic and intragraft inflammatory responses that occur after cold I/R in the setting of organ transplantation and that TLR4 signaling on both donor and recipient cells contributes to this response.Transplantation 12/2007; 84(10):1279-87. · 4.00 Impact Factor