Liver ischaemia and reperfusion injury

Liver ischaemia and reperfusion injury (IRI), a continuum of processes that culminates in hepatocellular damage, is responsible for up to 10 per cent of early organ failures after transplantation and may cause an increased incidence of both acute and chronic rejection1. IRI also contributes to the shortage of organs available for transplantation because of the greater susceptibility of extended-criteria donor livers to the ischaemic insult. Hepatic steatosis, a common finding in donor livers, accentuates IRI and may contribute to poor organ function2. Although minimizing the adverse effects of IRI may increase the number of patients who might successfully undergo transplantation, at present there is no specific treatment available to prevent this form of organ damage. A better understanding of the mechanisms of IRI seems essential for the design of therapeutic strategies to improve the outcome of transplantation and allow success with marginal donor livers. Platelet–leucocyte–endothelial cell interactions play a central role in IRI3. The accumulation of leucocytes at sites of tissue injury is mediated by a number of cell adhesion molecules, including the selectin family (P-, E- and L-selectins). P-selectin (CD62), stored in the Weibel–Palade bodies of endothelial cells and the α granules of platelets, is expressed rapidly in the early phase of reperfusion; it initiates the adhesion–rolling of leucocytes and then recruits them to the injured site. The blockade of P-selectin, and P-selectin glycoprotein ligand (PSGL) 1 interactions in vivo with a recombinant soluble form of PSGL-1 (rPSGL-Ig) or specific monoclonal antibody, protects the liver from IRI4. Decreased portal resistance, increased bile production and reduced polymorphonuclear leucocyte infiltration have been demonstrated in rat liver models of ex vivo cold ischaemia followed by reperfusion. In the rat orthotopic liver transplant model, rPSGL-Ig treatment markedly increases animal survival in normal and steatotic liver situations in association with decreased hepatocellular damage and ameliorated histological features of IRI, reduced intrahepatic leucocyte infiltration, and inhibited expression of genes encoding proinflammatory cytokines (tumour necrosis factor (TNF) α, interleukin (IL) 1β, IL-6, interferon (IFN) γ, IL-2). These experimental data have paved the way for a recently initiated clinical trial at University of California, Los Angeles of P-selectin–PSGL-1 blockade (YSPSL™; Y's Therapeutics, Burlingame, California, USA) in liver transplant recipients. The haem oxygenases (HOs) are ubiquitous enzymes that catalyse the rate-limiting steps in the oxidative degradation of haem into biliverdin, carbon monoxide and free iron5. The HO inducible isoform, HO-1, is among the most critical of the cytoprotective enzymes activated during cellular stress, having antioxidant and anti-inflammatory functions, modulating the cell cycle and maintaining the microcirculation. Upregulation of HO-1 protects against cellular stress following inflammation, radiation or ischaemia by preventing the deleterious effects of haem. Given the multifactorial cytoprotective properties of the HO system, its use as a strategy to prevent IRI in transplant recipients has been studied extensively6. Indeed, HO-1 induction in the donor liver, by pharmacological means or genetic engineering, maintains tissue architecture, preserves organ function and leads to prolonged survival following transplantation7. Orthotopic liver transplants in animals overexpressing HO-1 (primarily by infiltrating macrophages) exhibit less macrophage infiltration and depressed inducible nitric oxide synthase expression. Interestingly, local expression of antiapoptotic B cell lymphoma-2 (Bcl-2) and Bcl-2-associated anthanogene 1 (Bag-1) increases in well functioning transplants, whereas caspase 3 protein expression is markedly diminished. Moreover, HO-1 downstream mediators (carbon monoxide and biliverdin) also exert striking cytoprotection against IRI with the modulation of proapoptotic and antiapoptotic pathways8. As IRI develops in the absence of exogenous antigens, the innate immunity, and the sentinel Toll-like receptor (TLR) system in particular, might be involved in its initiation1. Livers in TLR4 (but not TLR2) knockout mice are resistant to IRI and its associated intrahepatic inflammation9. Furthermore, the myeloid differentiation primary response gene 88 (MyD88)-independent signalling pathway mediated by IFN regulatory factor (IRF) 3 downstream of TLR4 activation is critical, as mice deficient in IRF-3 but not MyD88 are protected from IRI. MyD88-dependent signalling in the TLR4 activation pathway leads to direct nuclear factor κB activation and induction of proinflammatory cytokines, whereas IRF-3-mediated signalling induces type 1 IFN and IFN inducible genes, such as that encoding chemokine (C-X-C motif) ligand (CXCL) 10. The induction of CXCL10 during the course of IRI is rapid, restricted to the ischaemic lobe and specific, without affecting the other two (monokine induced by IFN-gamma and IFN-gamma-inducible α-chemoattractant) chemokine (C-X-C motif) receptor 3 ligands10. Not surprisingly, livers in CXCL10 knockout mice are fully protected from IRI. Interestingly, although intrahepatic induction of proinflammatory genes, such TNF-α, IL-1 and IL-6, is reduced, expression of the immune regulatory gene IL-10 remains intact, indicating an altered immune response to IRI in the absence of CXCL10. However, neutralization of IL-10 in CXCL10 knockout mice restores intrahepatic inflammation and hepatocellular damage. Thus, the balance between CXCL10 and IL-10 determines the ischaemia–reperfusion-triggered liver innate immune response. Targeting of CXCL10, with resultant downregulation of proinflammatory and sparing immune regulatory responses, may provide a novel and effective means of reducing hepatic IRI. During IRI portal vein occlusion results in congestion of the intestinal wall, leading to the release of gut-derived molecules, including endotoxin, into the bloodstream. Thus, lipopolysaccharide (LPS) is an obvious candidate for TLR4 activation, consistent with its role as an initiating factor in liver IRI. However, IRI may also develop in animals under aseptic conditions and so, although gut-derived LPS certainly contributes to IRI, it might not actually trigger the intrahepatic inflammation cascade that culminates in tissue damage. The relevance of endotoxin to the development of liver IRI has been addressed recently directly by using LPS neutralizing recombinant bactericidal/permeability-increasing protein (rBPI21)11. IRI develops despite in vivo neutralization of endotoxin and endogenous TLR4 ligands generated during IRI trigger local inflammation, leading to the hepatocellular damage. One may conclude that primary non-specific IRI to the donor organ induces events in two distinct immunological defence systems in the transplant recipient. First, there is a broadly directed innate TLR-dependent host defence (normally directed against infection). This results in a definite adaptive host defence, which leads to a specific graft injury (rejection). Effective control of these two interlocked pathways is critical for long-term transplant acceptance.