Search tips
Search criteria 


Logo of wjgLink to Publisher's site
World J Gastroenterol. 2010 December 28; 16(48): 6068–6078.
Published online 2010 December 28. doi:  10.3748/wjg.v16.i48.6068
PMCID: PMC3012573

Heme oxygenase system in hepatic ischemia-reperfusion injury


Hepatic ischemia-reperfusion injury (IRI) limits access to transplantation. Heme oxygenase-1 (HO-1) is a powerful antioxidant enzyme which degrades free heme into biliverdin, free iron and carbon monoxide. HO-1 and its metabolites have the ability to modulate a wide variety of inflammatory disorders including hepatic IRI. Mechanisms of this protective effect include reduction of oxygen free radicals, alteration of macrophage and T cell phenotype. Further work is required to understand the physiological importance of the many actions of HO-1 identified experimentally, and to harness the protective effect of HO-1 for therapeutic potential.

Keywords: Ischemia-reperfusion injury, Heme oxygenase, Transplantation, Ischemic pre-conditioning


The rate of liver failure is increasing in the UK population[1]. Liver transplantation is an effective treatment for patients with end-stage disease, giving an average of 17-22 years of additional life[2,3]. Access to liver transplantation is limited by donor availability; several innovations, including split liver, living donor transplantation, non-heart beating donation (NHBD) and the expansion of the donor criteria, have been attempted to tackle this disparity[4].

Ischemia-reperfusion injury (IRI) causes a spectrum of early organ dysfunction after transplantation; the most severe form, termed primary non-function, may result in patient death. “Marginal organs”, including those from older donors, those affected by steatosis and those from donors with long pre-donation intensive care unit stay, may be judged to pose an excessive risk of IRI and be discarded, placing additional pressure on the already scarce donor resource[5].

Due to shifting patterns of organ donation, there is a tendency towards the increased use of marginal organs. Year on year, the mean donor age is increasing, in part due to improvements in road safety and declining numbers of traumatic deaths. Furthermore, NHBD (also termed donation after cardiac death) is becoming an increasingly important component of the donor resource[6]. Compared with the “gold standard” of heart beating donation (HBD), NHBD is associated with a decreased quantity of donated organs (2.1 organs per donor compared with 3.4 organs per HBD). Albeit in small studies, NHBD liver transplantation is also associated with a higher risk of IRI leading to elevated incidence of primary non-function[7].

Our group has previously estimated that negating the effects of IRI in HBD would lead to a 6% increase in the donor supply through recruitment of these marginal organs back into the donor pool[4]. In the current climate of rapidly increasing NHBD and increasing donor age, the imperative to better understand and avert hepatic ischemia is becoming ever stronger.


Interruption of blood flow to any tissue results in inadequate tissue oxygenation and an increase in cellular anaerobic pathways: if adequate oxygenation is not restored then disruption of cellular functions and cell death results. On reperfusion, despite restoration of adequate cellular oxygenation, there is further damage caused both by direct cytotoxicity from oxygen free radicals (OFR)[8] and by a secondary immunological assault upon the injured organ involving components of both the innate and adaptive immune system[9]. The sequence of injuries resulting from interruption then reinstatement of blood flow is termed IRI.


Heme oxygenase (HO) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal enzyme, which catalyzes the breakdown of heme to biliverdin, iron and carbon monoxide (CO)[10] (Figure (Figure1).1). Biliverdin is subsequently reduced to bilirubin by biliverdin reductase while free iron is sequestrated by ferritin. Of the three heme oxygenase isoforms (HO-1, HO-2 and HO-3), only HO-1 (also known as heat shock protein 32) is inducible[11]. HO-1 is a 32 kDa enzyme encoded by the hmox1 gene. It has been found to be upregulated during states of oxidative and cellular stress and plays an important role in maintaining oxidative/antioxidant homeostasis[12].

Figure 1
Putative mechanisms of heme oxygenase-1 antioxidant effect. HO-1: Heme oxygenase-1; BVR: Biliverdin; OFR: Oxygen free radicals; NADPH: Nicotinamide adenine dinucleotide phosphate.

Induction of HO-1 and its metabolites is protective in a large number of seemingly unrelated pathologies, including sepsis, malaria, endotoxic shock, IRI, organ transplant rejection, induction of tolerance, myocardial infarction, type 2 diabetes and obesity[13]. This spectrum of protection is attributed to multi-level mechanisms of cytoprotection and inflammatory modulation.

Polymorphism in the (GT)n microsatellite of the HMOX1 promoter is thought to be responsible for the variations seen in the human response of HO-1 to stimuli[14]. This may account for the differences in the susceptibility of individuals to certain pathologies and in the apparent longevity associated with increased HO-1 expression[15,16].

HO-1 in hepatic IRI

In the context of IRI and transplantation, HO-1 induction or supplementation with its metabolites has been shown repeatedly to be protective in both the liver and other organs.

In early experiments, HO-1 was induced by heat shock in donor livers prior to 44 h of cold ischemia preceding liver isogeneic transplantation in rats, resulting in marked improvements in recipient survival[17]. HO-1 upregulation with adenoviral HO-1 or cobalt protoporphyrin (CoPP) resulted in improved portal venous flow on ex-vivo perfusion, while on transplantation into syngeneic hosts, recipient survival doubled, histological injury was ameliorated and influx of macrophages and T cells was reduced[18]; findings confirmed in similar experiments using transplantation[19,20] and hepatic warm ischemia models[21]. More recently, targeted deletion of Bach-1, which normally suppresses HO-1 transcription, led to HO-1 upregulation and protection from myocardial ischemia[22], although these experiments have yet to be repeated in models of hepatic ischemia.

In our own laboratory we have shown that HO-1 is upregulated in Kupffer cells during ischemic preconditioning (IPC)[23], and that targeted deletion of hmox-1 resulted in aberrant Kupffer cell differentiation and susceptibility to ischemia-reperfusion insults[24]. Our work has suggested that Kupffer cells are a likely site of HO-1 action: recent work from Kupiec Weglinski’s laboratory has demonstrated that adoptive transfer of HO-1 overexpressing bone marrow-derived macrophages was capable of protecting mice from hepatic ischemic insults[25]. Developing this theme, other experiments have shown HO-1 induction with CoPP to protect mice from hepatic ischemia arising from liver transplantation: Kupffer cells recovered from HO-1-induced animals produced less tumor necrosis factor α (TNFα) and interleukin (IL)-6 under stimulation in ex vivo culture[26].

HO-1 in hepatic IPC

IPC is a surgical maneuvre in which an organ is paradoxically protected by a brief period of controlled ischemia and reperfusion immediately prior to a longer index ischemic event, which by itself would normally lead to injury[27,28]. Several small randomized controlled trials have looked at the effectiveness of IPC in both liver transplantation and resection; subsequent Cochrane reviews conclude that further trials are still required to evaluate its role in hepatic and transplant surgery[29,30].

Kanoria et al[31] demonstrated in their rodent model that the application of a hindleg tourniquet (remote IPC) led to a protective phenotype from hepatic IRI. Ischemic post-conditioning has also been described, in which injury is abrogated by modified reperfusion. This emphasizes that tissue damage from ischemia-reperfusion continues following the end of the ischemic insult[32].

The mechanisms behind IPC are poorly understood. Numerous candidate molecules, including nitric oxide, adenosine, protein kinase C, tyrosine kinase and mitogen-activated-protein kinase, have been identified as potential mediators of protection[33]. Given that IPC can act remotely, it may involve an immunomodulatory mechanism.

HO-1 is upregulated following IPC and may be responsible for the observed protection[23]. HO-1 is also upregulated in sites distant to the site of preconditioning. In a remote preconditioning model, renal ischemic insults led to cardiac HO-1 induction[34]. Likewise, HO-1 induction occurred in the liver following four 10 min episodes of femoral artery occlusion, and protection afforded by remote preconditioning was lost when HO-1 was inhibited with SnPP[35].

Given that HO-1 is upregulated in IPC and is known to be powerfully protective when upregulated, it is likely that HO-1 has a role in IPC together with other up- and downstream molecules.

Mechanisms of HO-1-mediated cytoprotection

As described above, it is now beyond doubt that HO-1 induction is protective in the context of hepatic ischemia and transplantation. HO-1’s cytoprotective effects can be credited to a combination of removal of toxic metabolites (heme), and production of protective second messenger molecules in the forms of biliverdin (and subsequently bilirubin), CO, and free iron (which induces ferritin)[36]. HO-1 may also have other mechanisms of protection, independent of its enzyme activity[37].

Catabolysis of free heme

Heme consists of an iron atom contained within a porphyrin ring: heme moieties are usually contained within the “heme pockets” of hemoproteins. Although the most abundant hemoproteins are hemoglobin and myoglobin, heme moieties are contained within many other ubiquitous enzymes, for example iNOS and the mitochondrial electron transfer chain.

Under conditions of oxidative stress, hemoproteins may be oxidized, leading to the release of free heme which causes cellular injury by multiple mechanisms. Firstly, the central iron ion of the heme moiety catalyzes the production of free radicals by Fenton chemistry as follows: Step 1: Fe2+ + H2O2 → Fe3+ + OH· + OH-; Step 2: Fe3+ + H2O2 → Fe2+ + OOH· + H+.

Heme-dependent free radical generation can cause direct cytotoxicity, including damage to the cytoskeleton, lipid bilayer, intermediary metabolic enzymes and DNA[38,39], while circulating heme can cause LDL oxidation leading to endothelial cell toxicity[40]. Heme sensitizes cells to pro-death signals including TNFα and Fas, an effect abrogated by treatment with antioxidants, implying dependence upon free radical production[36].

HO-1 reduces free heme concentrations by two mechanisms. Firstly, catabolysis of heme into metabolites biliverdin, iron and CO directly removes free heme. Secondly, binding of CO to hemoglobin, forming carboxyhemoglobin, prevents its oxidation to methemoglobin and ensuing release of free heme moieties[36].

Some authors have argued strongly that neutralization of free heme during cellular injury can account for a large proportion of the protection offered by HO-1. In experimental malaria, it was found that C57/Bl6J mice had reduced HO-1 induction compared with BALB/c animals, and consequently had higher levels of circulating free heme and greater disease severity. Administering exogenous heme to BALB/c animals caused worsening of their disease severity whereas conversely, CO treatment of C57Bl6J mice was protective[41]. In the same model, HO-1 protected animals from hepatic failure induced by overwhelming circulating heme[42].

Hepatic HO-1 expression is focused in Kupffer cells. This cell type is highly adapted to detect hemolysis: the hemoglobin-haptoglobin complex receptor CD163 is able to induce HO-1 in an IL-10-dependent manner[43,44].

Production of biliverdin

Biliverdin is produced by catalysis of the heme porphyrin ring and is almost immediately converted by biliverdin reductase to unconjugated bilirubin. This, in turn, is glucuronidated to render it water soluble. Bilirubin is a powerful antioxidant believed to be responsible for much of the antioxidant activity of serum[45]. At micromolar concentrations it is capable of scavenging large volumes of reactive oxygen species (ROS), protecting cells from high concentrations of peroxide, with enhanced efficacy in hypoxic conditions, making this effect particularly relevant in the context of ischemia[46]. Bilirubin’s extraordinary antioxidant capacity may arise from an active cycle in which biliverdin reductase undertakes NADPH-dependent reduction of biliverdin to bilirubin: in turn bilirubin is oxidized by free radicals back to biliverdin, before enzymatic reduction back to bilirubin. Although controversial[47], this cycling mechanism has been proposed as a highly adapted mechanism through which cells and whole organisms maintain redox status[45].

Bilirubin’s effects may extend beyond its antioxidant action. It has been identified as an endogenous ligand of the aryl hydrocarbon receptor (AHR), present on many immune cell populations[48]. In some experiments AHR ligands have suppressed T cell proliferation through expansion of T regulatory (Treg) cells[49], although expansion of either Treg or their polar opposite, Th17 cells, has been shown depending upon which experimental AHR ligand is used[50].

Patients with persistent hyperbilirubinemia (Gilbert’s syndrome) have been observed to have significantly lower incidence of atherosclerotic disease in several studies, an effect attributed to enhanced antioxidant potential of serum[51]. The effect of chronic hyperbilirubinemia upon T cell phenotype is unclear, although immunosuppression has been reported in patients with cholestatic jaundice[52].

Work using ex vivo liver perfusion models in which explanted livers were stored for 16[53] or 24 h[54] at 4°C in University of Wisconsin solution, prior to mechanical perfusion, found that addition of low concentrations of bilirubin to the graft perfusate mimicked the protective effect of heme oxygenase induction. Graft function was improved in terms of portal venous flow and bile production, and hepatocellular injury was ameliorated in terms of histological injury scoring and transaminase release. Biliverdin has also been shown to have an immunomodulatory effect. In an MHC-mismatched cardiac allograft model, twice or three times daily injections of biliverdin for 2 wk increased graft survival, and led to complete allograft tolerance in 50% of animals[55].

To test whether bilirubin was protective in the clinical setting, our group hypothesized that hyperbilirubinemia would protect transplant recipients from IRI, in which case there would be an inverse correlation between pre-operative bilirubin and post-operative transaminase measurements. In a small retrospective study, no relationship was observed, although given the heterogeneity of the study population an effect could not be ruled out[56].

Production of Fe2+

Paradoxically for an antioxidant enzyme, one of HO-1’s reaction products, free iron, is a powerful oxidant. Free iron catalyzes the generation of OFR by Fenton chemistry in a manner comparable to free heme as described above. However, it is more effectively neutralized than when contained within a heme ring, being actively exported from the cell[57] and chelated by iron-binding proteins, including ferritin. Ferritin sequesters Fe2+ by oxidation and placement of iron ions within a “core” in which they are unable to catalyze Fenton reactions[58]. HO-1 induction leads to increased ferritin expression[59], through a mechanism dependent upon the production of free iron[60,61].

Since other HO-1 metabolites had been shown to be capable of substituting for HO-1 induction in protecting animals from IRI, Berberat et al[62] hypothesized that ferritin overexpression could also confer protection. Using an ex vivo perfusion model of hepatic IRI, this group identified that transfection with adenoviral heavy chain ferritin conferred protection in terms of bile flow, portal blood flow, histological injury scoring and transaminase release, and improved survival of syngeneic liver recipients.

Ferritin induction has been observed in retinal[63] and cardiac IPC[64] in a manner dependent upon an iron signal[65]: although there are no published studies of ferritin expression in hepatic preconditioning, it would be reasonable to hypothesize that similar results would be obtained.

Although ferritin is capable of protecting cells and organs from oxidative stress, it is worth noting that its induction is unlikely to be the only mechanism of cellular protection by HO-1. Sheftel et al[61] compared heme and sodium arsenite (a non-heme inducer of HO-1) in vitro, and found sodium arsenite to confer protection through HO-1 induction without parallel ferritin induction.

Production of CO

CO is best known for its toxicity, causing death at atmospheric concentrations of 500-1000 ppm, however, at lower doses CO has been shown to have important cytoprotective and immunomodulatory functions: it is released by HO-1 during the catabolysis of heme and can substitute for the protective effect of HO-1.

CO cannot have a specific receptor since it binds only to transition metals and is not thought to be capable of direct interaction with amino acids. Therefore, its pharmacology as a signaling molecule is necessarily novel, and subject to ongoing debate. An important hypothesis is that CO effects must be mediated by interactions with proteins which contain transition metal cores, for example in heme rings contained within a range of enzymes including soluble guanylate cyclase (sGC), cytochromes, hemoglobin, myoglobin and nitric oxide synthase[66]. It has been suggested that CO protects from oxidative stress by preventing hemoprotein oxidation and subsequent release of heme rings[36], diminishing production of free radicals and subsequent apoptosis[67]. Others have suggested that the protection may be mediated by antagonism of respiratory chain enzymes, reducing cellular oxygen requirements[66], by inducing vasodilatation via sGC[68] or by opening calcium-sensitive ion channels[69].

Using an ex-vivo liver perfusion model, Amersi et al[70] demonstrated enhanced portal blood flow and bile production, and amelioration of hepatic IRI, in terms of histological injury score and transaminase release when perfusate was supplemented with CO at 300 ppm. Blockade of heme oxygenase using zinc protoporphyrin (ZnPP) did not obliterate the protective effect, indicating that CO was capable of substituting for HO-1 function. Inhibitor studies have demonstrated that the CO effect was independent of iNOS and cGMP but dependent upon p38 MAPK.

In a cardiac transplantation model, no hearts stored at 4°C for 24 h prior to implantation into syngeneic recipients functioned after implantation, whereas 5 out of 6 grafts survived when HO-1 was induced with CoPP, an effect lost with HO-1 inhibition. Administration of 400 ppm CO to the heart donor during cold storage resulted in survival equivalent to that achieved with HO-1 induction[71]. In xenotransplant models, HO-1 inhibition with tin protoporphyrin (SnPP) caused rejection, whereas this effect was overcome by recipient CO inhalation[72].

Although the finding that CO can be protective at low doses is scientifically exciting, the potential therapeutic use of inhaled CO can be limited by practical difficulties, both in control of dosage, and spillage of the gas into the environment around the patient. For this reason, Motterlini has developed transition metal carbonyl “CO releasing compounds” (CORMs). Of these (CORM-A1, CORM-2, CORM-3), the most commonly used has been CORM-3 [tricarbonylchloro(glycinato)ruthenium(II)], which releases CO when dissolved in saline but not water[73]. To control for the presence of the ruthenium compound, a CO depleted substance (iCORM3) can be prepared by dissolving CORM-3 in PBS prior to the experiment.

Using a coronary occlusion model in which mice were subjected to 30 min of ischemia followed by 24 h of reperfusion, investigators administered CORM-3 during the first hour of reperfusion, which halved the area of myocardial infarct compared to iCORM control[74]. In a further study, the same group compared the effects of CORM-3 with a late-phase IPC protocol. Animals received either IPC or intravenous CORM-3 infusion lasting 60 min, with appropriate controls. Animals then underwent 30 min coronary ischemic insults 24 to 120 h later. The reperfusion phase lasted for 24 h post-operatively before animals were killed and hearts examined for infarct size. Both IPC and CORM-3 infusion resulted in cardioprotection at between 24 and 72 h compared with appropriate control groups[75].

In the context of transplantation, the potential for using CO to enhance graft function has been explored in various transplantation models. In the kidney, several studies have successfully protected grafts using storage media supplemented with CORM-3/CORM-A1[76] or CORM-2[77] for extended periods of cold ischemia prior to isogenic transplantation. In a vascular allograft model, donor HO-1 induction or CORM treatment reduced subsequent CD8 influx and intimal hyperplasia lasting until animals were sacrificed at 6 wk[78].

In the liver, CO persufflation of University of Wisconsin (UW) storage solution improved subsequent graft function[79], a result replicated using CORM-3 supplemented UW[80,81] after cold ischemic times of 48 h. Use of CO treatment in experimental liver transplant recipients has also been explored. Tomiyama et al[82] transplanted livers from wild-type donors into E-GFP transgenic rat recipients who received inhaled CO prior to and for the 24 h post transplantation. Grafts within CO-treated recipients were found to have reduced numbers of infiltrating CD45+ host leucocytes, while purified donor CD68+ Kupffer cells produced less IL-6 and TNFα: primary cultured ex vivo Kupffer cells from CO-treated animals secreted less IL-6 and TNFα in response to lipopolysaccharide (LPS) stimulus.

CO clearly has powerful antioxidant and anti-inflammatory effects in a variety of systems. For this reason, despite the difficulties presented by administering it in its inhaled form, phase 2 clinical trials are underway in renal transplantation ( identifier NCT00531856), chronic obstructive pulmonary disease (NCT00094406) and post-operative intestinal ileus (NCT01050712). Further development of CORMs should make CO therapies more convenient, thereby widening their potential clinical applications: on the basis of the preclinical data presented above such applications would be expected to include hepatic IRI and transplantation.

Other mechanisms of HO-1-mediated cytoprotection

The bulk of data exploring mechanisms of HO-1-induced cytoprotection supports the concept that it is its antioxidant function which protects cells and animals from injury. Two separate pieces of data suggest that other mechanisms should be considered. Firstly, Ponka’s laboratory have demonstrated that the amounts of intracellular heme available for degradation may be insufficient to account for HO-1’s powerful antioxidant effects[61]. Secondly, Dennery’s laboratory has shown HO-1 translocation to the nucleus after cellular hypoxia or heme treatment, suggesting a role in transcriptional regulation[37]. Developing this work, this group has published research using a reporter system with catabolically inactive HO-1 which suggests HO-1 may have a forward-acting positive effect upon its own transcription[83], although as yet there has been no work demonstrating a protective effect of catabolically inactive HO-1.


It is likely that HO-1 protects organs from IRI by modulation of both direct cellular injury and both the innate and adaptive immune systems[84]. Deficiency of HO-1 in both humans and mice is associated with a pro-inflammatory phenotype[85,86].

According to Matzinger’s “Danger Model” of the initiation of immune responses to injured organs, injured parenchymal cells release “Danger-Associated Molecular Patterns” (DAMPs) which stimulate antigen presenting cell (APC) activation through pattern recognition receptors[87,88]. Activated APCs subsequently activate and recruit immune effector cells through the secretion of pro-inflammatory mediators (cytokines, chemokines and adhesion molecules) and antigen presentation[9], resulting in the activation and recruitment of T cells and neutrophils (Figure (Figure2).2). This recruitment of leucocytes into the tissue is further facilitated by increased endothelial permeability and adhesion molecule expression resulting from endothelial activation.

Figure 2
Heme oxygenase-1-mediated suppression of the inflammatory response in hepatic ischemia-reperfusion injury. HMGB1: High Mobility Group Box-1; TLR: Toll-like receptor; DAMPs: Danger-associated molecular patterns; TNFα: Tumor necrosis factor α; ...

Danger-associated molecular patterns

Hypoxic cellular injury can be regarded as the initiating event in a cascade of immunological activation. “Danger-Associated Molecular Patterns” or “alarmins” spilt by dying cells act on tissue resident immune populations via various molecular sensors including the toll-like receptor (TLR) family and purinergic receptors. A prototypic “alarmin”, High Mobility Group Box-1 (HMGB1) is released passively from injured cells, a signal amplified by active secretion from surrounding viable immune and non-immune cells[89]. Several authors have published studies demonstrating that circulating HMGB1 levels rise after experimental hepatic ischemia and that anti-HMGB1 neutralizing antibodies are capable of lowering circulating TNFα and IL-6 levels, and ameliorating injury[90-92]. Human clinical studies have confirmed the synthesis of HMGB1 during early reperfusion after liver transplantation, an effect exaggerated in steatotic livers, and with levels correlating with injury severity as measured by peak ALT[93]. Recombinant HMGB1 has been found to worsen the severity of experimental hepatic ischemic injury, an effect neutralized by targeted deletion of its receptor TLR-4[92].

HO-1 is capable of modulating the HMGB1 alarmin system. In sepsis[94] and acute lung injury[95] models, macrophage LPS-mediated HMGB1 release was suppressed by HO-1 induction by CORMs resulting in improved survival. Oxidative stress can initiate macrophage HMGB1 synthesis which occurs in a dose response relationship with peroxide stress in vitro[96]. It is therefore unclear whether HO-1’s modulation of HMGB1 secretion arises simply by reducing the extent of parenchymal cell injury or whether it is dependant upon another mechanism.

Free heme is another potential DAMP, being released from damaged cells and acting on TLRs[97]. Heme has also been shown to induce neutrophil chemotaxis using in vitro transmigration assays and after intraperitoneal injection in vivo[98]. By metabolising free heme, HO-1 induction would be expected to reduce this immune stimulus, further contributing to the possible mechanisms of protection from injury.

Intriguingly, HO-1 and CO also modulate TLR signaling upon which many DAMPs converge[99]. CO treatment of cultured RAW264.7 macrophages was found to reduce TLR4 signaling by reducing TLR4/Myd88 interactions and movement of TLR4 receptors to the cell surface[100].

It is likely that much of the immune modulation offered by HO-1 is owed to its antioxidant action which quietens the initial immune stimulus by reducing cellular injury and spillage of DAMPs.

Kupffer cells

Hepatic tissue resident macrophages (Kupffer cells) are the first immune cells to be activated by IRI, being acted upon by DAMPs released from surrounding parenchymal cells as well as being subject themselves to cellular hypoxia. Subsequently, they coordinate an appropriate influx of other immune cells by secretion of chemokines and cytokines. Kupffer cells have the ability to harm surrounding parenchymal cells by secretion of pro-inflammatory cytokines including TNFα, IL-6 and IL-1, which may be directly toxic[101]. On the other hand, Kupffer cells may protect the tissues by secretion of anti-inflammatory cytokines including IL-10. Furthermore, as the principle HO-1-expressing cells of the liver[24] they may secrete diffusible CO which may act in a paracrine manner upon surrounding cells to protect them from oxidative stress.

In our work, we have identified that HO-1 acts as a powerful switch on resting macrophage differentiation. We found HO-1-deficient Kupffer cells in vivo and bone marrow-derived monocytes (BMDMs) in vitro to differentiate down a Ly6c+ MARCO+ F4/80- pathway associated with macrophage inflammatory protein (MIP)-1 responsive pro-inflammatory monocytes[102]. HO-1 deficiency was associated with marked susceptibility to hepatic IRI measured in terms of ALT release and histological injury score[24]. Parallel work elsewhere using an in vitro migration assay has demonstrated enhanced migration of Hmox-1-/- BMDMs towards MIP-1α[95]. In an experimental liver transplant model, inhaled CO conferred protection from injury and reduced secretion of pro-inflammatory cytokines from recovered CD68+ cells cultured ex vivo[82], reducing the subsequent influx of CD45+ leucocytes. In vitro and in vivo, low dose CO downregulates the production of macrophage pro-inflammatory cytokines [TNFα, IL1β and MIP-1β (CCL4)] and increases the expression of the anti-inflammatory cytokine IL10[103].

Data from injury models showing modulation of pro-inflammatory cytokine secretion by HO-1 induction is subject to the criticism that the changes observed are merely the downstream effects of parenchymal cellular protection, and suppression of DAMPs. However, data from our laboratory has shown HO-1 effects upon the resting state of macrophage differentiation implying that HO-1 may modulate immune responses themselves.

Adaptive immune system

There is growing evidence from a variety of animal IRI models (T cell-deficient nude rats, severe combined immunodeficiency mice, RAG 1-/- mice, CD4+/CD8+-/- mice and CD4+ depleted mice) that there is a significant decrease in biochemical and histopathological evidence of injury in the absence of T cells (reviewed by Linfert et al[9]). More specifically, Khandoga et al[104] demonstrated it was a CD4+ rather than CD8+ T cell-dependent phenomenon; depletion of CD4+ T cells leads to a significant reduction in the observed injury. This influx of CD4+ cells on reperfusion is rapid and may determine the mode of neutrophil activation and subsequently the extent of the observed tissue damage[105,106]. In a series of elegant adoptive transfer experiments with CD4+ cells in a renal model, Burne et al[107] found IRI to be an interferon-γ-dependent process; this may imply the importance of the T helper 1 lineage in the pathology of IRI.

HO-1 has a number of immunomodulatory effects on effector and Treg cells. HO-1 and CO appear to inhibit T cell proliferation and activation through the suppression of IL-2 secretion[108]. HO-1 induction has also been shown to induce the apoptosis of activated T helper cells through activation-induced cell death[109].

Treg cells suppress the activation of the immune system and inhibit the activation of autoreactive T cells[110]. In clinical studies, Treg/Th17 “imbalance” has been associated with acute coronary syndromes[111]. HO-1 is induced by FoxP3: inhibition of HO-1 function in vivo reduced the suppressive ability of naturally occurring Tregs[112]. Furthermore, studies from Fritz Bach’s group have shown tolerogenesis to require HO-1[109]. Recently, however, these studies have been challenged by showing that Hmox-1-/- mice have normal numbers of Treg cells[113] and that HO-1 is not necessary or sufficient for T reg function[114]. These controversies may be resolved by work from the Agarwal group which showed that although Hmox-1-/- mice had normal (or elevated) numbers of circulating Treg cells, their suppressive function was dependant upon HO-1 expression by antigen-presenting cells[115].

In the context of hepatic ischemia, further work is needed firstly to establish which T cell populations are responsible for IRI and IPC, and secondly to resolve the controversy concerning the role of HO-1 in the expansion of different T cell subsets.


Severe IRI is associated with significant influx of neutrophils[9]. Their recruitment from the vascular compartment into the liver is mediated by the expression of chemokines, cytokines and adhesion molecules[9]. Accumulation of activated neutrophils directly injures hepatocytes and the vascular epithelium through the release of proteases and the generation of ROS[116]. The initial recruitment of neutrophils appears to be a CD4+-dependent process[105]; continued neutrophil recruitment and activation may be dependent on the neutrophil production of IL-17A, which is important in a positive feedback mechanism[117]. As described above, heme is also capable of initiating neutrophil chemotaxis[98].

Although neutrophil influx is enhanced in Hmox-1-/- animals after ischemia-reperfusion insults[24], it is unclear whether this is due to a direct effect upon neutrophils or merely as a result of reduced stimulation due to upstream effects.


A wealth of data has now proven that HO-1, and its metabolites iron, bilirubin and CO, protect the liver from IRI. HO-1 appears to ameliorate ischemic injury through synergistic actions at many levels in the danger pathway. Powerful effects have been identified upon neutralization of OFR, and apparently at every step of the ensuing danger pathway. What remains is to understand the true physiological importance of each of these effects, and to attempt to separate apparent immunomodulation from suppression of upstream danger signals. Further understanding of the detailed pathophysiology of IRI and the mechanisms underlying IPC would also be invaluable in identifying the full range of potential therapeutic targets.

The protective effect of HO-1 upregulation in IRI was first discovered over a decade ago, and is proven beyond doubt in preclinical studies. Work to date has highlighted multiple potential therapeutic strategies ranging from CORM infusions to cell therapies with HO-1-transduced macrophages. In light of the severity of the many clinical problems caused by IRI including the liver donor shortage, there is now some urgency to translate this body of scientific knowledge into viable treatments which will benefit patients.


Supported by The Maurice Wohl Fellowship from the Royal College of Surgeons of Edinburgh and a Research Training Fellowship from The Wellcome Trust (to Richards JA); Tenovus Scotland and The Peel Medical Research Trust to support his current work (to Richards JA); A Clinician Scientist Fellowship from the Academy of Medical Sciences and the Health Foundation (to Devey LR)

Peer reviewer: Chi-Hin Cho, B.Pharm, PhD, Professor of Pharmacology and Chairman, Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China

S- Editor Shi ZF L- Editor Logan S E- Editor Zheng XM


1. Leon DA, McCambridge J. Liver cirrhosis mortality rates in Britain from 1950 to 2002: an analysis of routine data. Lancet. 2006;367:52–56. [PubMed]
2. Schnitzler MA, Whiting JF, Brennan DC, Lentine KL, Desai NM, Chapman W, Abbott KC, Kalo Z. The life-years saved by a deceased organ donor. Am J Transplant. 2005;5:2289–2296. [PubMed]
3. Barber K, Blackwell J, Collett D, Neuberger J. Life expectancy of adult liver allograft recipients in the UK. Gut. 2007;56:279–282. [PMC free article] [PubMed]
4. Devey LR, Friend PJ, Forsythe JL, Mumford LL, Wigmore SJ. The use of marginal heart beating donor livers for transplantation in the United kingdom. Transplantation. 2007;84:70–74. [PubMed]
5. Briceño J, Solórzano G, Pera C. A proposal for scoring marginal liver grafts. Transpl Int. 2000;13 Suppl 1:S249–S252. [PubMed]
6. Devey L, Wigmore SJ. Non-heart-beating organ donation. Br J Surg. 2009;96:833–835. [PubMed]
7. Abt PL, Desai NM, Crawford MD, Forman LM, Markmann JW, Olthoff KM, Markmann JF. Survival following liver transplantation from non-heart-beating donors. Ann Surg. 2004;239:87–92. [PubMed]
8. Parks DA, Granger DN. Ischemia-induced vascular changes: role of xanthine oxidase and hydroxyl radicals. Am J Physiol. 1983;245:G285–G289. [PubMed]
9. Linfert D, Chowdhry T, Rabb H. Lymphocytes and ischemia-reperfusion injury. Transplant Rev (Orlando) 2009;23:1–10. [PMC free article] [PubMed]
10. Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA. 1968;61:748–755. [PubMed]
11. Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem. 1986;261:411–419. [PubMed]
12. Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA. 1989;86:99–103. [PubMed]
13. Soares MP, Bach FH. Heme oxygenase-1: from biology to therapeutic potential. Trends Mol Med. 2009;15:50–58. [PubMed]
14. Kimpara T, Takeda A, Watanabe K, Itoyama Y, Ikawa S, Watanabe M, Arai H, Sasaki H, Higuchi S, Okita N, et al. Microsatellite polymorphism in the human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer and Parkinson disease. Hum Genet. 1997;100:145–147. [PubMed]
15. Exner M, Minar E, Wagner O, Schillinger M. The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic Biol Med. 2004;37:1097–1104. [PubMed]
16. Yamaya M, Nakayama K, Ebihara S, Hirai H, Higuchi S, Sasaki H. Relationship between microsatellite polymorphism in the haem oxygenase-1 gene promoter and longevity of the normal Japanese population. J Med Genet. 2003;40:146–148. [PMC free article] [PubMed]
17. Redaelli CA, Tian YH, Schaffner T, Ledermann M, Baer HU, Dufour JF. Extended preservation of rat liver graft by induction of heme oxygenase-1. Hepatology. 2002;35:1082–1092. [PubMed]
18. Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD, Zhao D, Zaky J, Melinek J, Lassman CR, et al. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest. 1999;104:1631–1639. [PMC free article] [PubMed]
19. Coito AJ, Buelow R, Shen XD, Amersi F, Moore C, Volk HD, Busuttil RW, Kupiec-Weglinski JW. Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury. Transplantation. 2002;74:96–102. [PubMed]
20. Kato H, Amersi F, Buelow R, Melinek J, Coito AJ, Ke B, Busuttil RW, Kupiec-Weglinski JW. Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation. Am J Transplant. 2001;1:121–128. [PubMed]
21. Ke B, Shen XD, Lassman CR, Gao F, Busuttil RW, Kupiec-Weglinski JW. Cytoprotective and antiapoptotic effects of IL-13 in hepatic cold ischemia/reperfusion injury are heme oxygenase-1 dependent. Am J Transplant. 2003;3:1076–1082. [PubMed]
22. Yano Y, Ozono R, Oishi Y, Kambe M, Yoshizumi M, Ishida T, Omura S, Oshima T, Igarashi K. Genetic ablation of the transcription repressor Bach1 leads to myocardial protection against ischemia/reperfusion in mice. Genes Cells. 2006;11:791–803. [PubMed]
23. Patel A, van de Poll MC, Greve JW, Buurman WA, Fearon KC, McNally SJ, Harrison EM, Ross JA, Garden OJ, Dejong CH, et al. Early stress protein gene expression in a human model of ischemic preconditioning. Transplantation. 2004;78:1479–1487. [PubMed]
24. Devey L, Ferenbach D, Mohr E, Sangster K, Bellamy CO, Hughes J, Wigmore SJ. Tissue-resident macrophages protect the liver from ischemia reperfusion injury via a heme oxygenase-1-dependent mechanism. Mol Ther. 2009;17:65–72. [PubMed]
25. Ke B, Shen XD, Gao F, Ji H, Qiao B, Zhai Y, Farmer DG, Busuttil RW, Kupiec-Weglinski JW. Adoptive transfer of ex vivo HO-1 modified bone marrow-derived macrophages prevents liver ischemia and reperfusion injury. Mol Ther. 2010;18:1019–1025. [PubMed]
26. Zeng Z, Huang HF, Chen MQ, Song F, Zhang YJ. Heme oxygenase-1 protects donor livers from ischemia/reperfusion injury: the role of Kupffer cells. World J Gastroenterol. 2010;16:1285–1292. [PMC free article] [PubMed]
27. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124–1136. [PubMed]
28. Clavien PA, Selzner M, Rüdiger HA, Graf R, Kadry Z, Rousson V, Jochum W. A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning. Ann Surg. 2003;238:843–850; discussion 851-852. [PubMed]
29. Gurusamy KS, Kumar Y, Sharma D, Davidson BR. Ischaemic preconditioning for liver transplantation. Cochrane Database Syst Rev. 2008:CD006315. [PubMed]
30. Gurusamy KS, Kumar Y, Pamecha V, Sharma D, Davidson BR. Ischaemic pre-conditioning for elective liver resections performed under vascular occlusion. Cochrane Database Syst Rev. 2009:CD007629. [PubMed]
31. Kanoria S, Jalan R, Davies NA, Seifalian AM, Williams R, Davidson BR. Remote ischaemic preconditioning of the hind limb reduces experimental liver warm ischaemia-reperfusion injury. Br J Surg. 2006;93:762–768. [PubMed]
32. Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: new strategies for cardioprotection. Diabetes Obes Metab. 2008;10:451–459. [PubMed]
33. Koti RS, Seifalian AM, Davidson BR. Protection of the liver by ischemic preconditioning: a review of mechanisms and clinical applications. Dig Surg. 2003;20:383–396. [PubMed]
34. Raju VS, Maines MD. Renal ischemia/reperfusion up-regulates heme oxygenase-1 (HSP32) expression and increases cGMP in rat heart. J Pharmacol Exp Ther. 1996;277:1814–1822. [PubMed]
35. Lai IR, Chang KJ, Chen CF, Tsai HW. Transient limb ischemia induces remote preconditioning in liver among rats: the protective role of heme oxygenase-1. Transplantation. 2006;81:1311–1317. [PubMed]
36. Gozzelino R, Jeney V, Soares MP. Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol. 2010;50:323–354. [PubMed]
37. Lin Q, Weis S, Yang G, Weng YH, Helston R, Rish K, Smith A, Bordner J, Polte T, Gaunitz F, et al. Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress. J Biol Chem. 2007;282:20621–20633. [PubMed]
38. Ryter SW, Tyrrell RM. The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties. Free Radic Biol Med. 2000;28:289–309. [PubMed]
39. Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, Figdor CG. Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev. 2003;55:551–571. [PubMed]
40. Jeney V, Balla J, Yachie A, Varga Z, Vercellotti GM, Eaton JW, Balla G. Pro-oxidant and cytotoxic effects of circulating heme. Blood. 2002;100:879–887. [PubMed]
41. Pamplona A, Ferreira A, Balla J, Jeney V, Balla G, Epiphanio S, Chora A, Rodrigues CD, Gregoire IP, Cunha-Rodrigues M, et al. Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria. Nat Med. 2007;13:703–710. [PubMed]
42. Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, Larsen R, Rebelo S, Penido C, Smith NR, Coutinho A, et al. Heme oxygenase-1 affords protection against noncerebral forms of severe malaria. Proc Natl Acad Sci USA. 2009;106:15837–15842. [PubMed]
43. Schaer CA, Schoedon G, Imhof A, Kurrer MO, Schaer DJ. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin. Circ Res. 2006;99:943–950. [PubMed]
44. Philippidis P, Mason JC, Evans BJ, Nadra I, Taylor KM, Haskard DO, Landis RC. Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis: antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circ Res. 2004;94:119–126. [PubMed]
45. Baranano DE, Rao M, Ferris CD, Snyder SH. Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci USA. 2002;99:16093–16098. [PubMed]
46. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 1987;235:1043–1046. [PubMed]
47. Maghzal GJ, Leck MC, Collinson E, Li C, Stocker R. Limited role for the bilirubin-biliverdin redox amplification cycle in the cellular antioxidant protection by biliverdin reductase. J Biol Chem. 2009;284:29251–29259. [PMC free article] [PubMed]
48. Bock KW, Köhle C. The mammalian aryl hydrocarbon (Ah) receptor: from mediator of dioxin toxicity toward physiological functions in skin and liver. Biol Chem. 2009;390:1225–1235. [PubMed]
49. Kerkvliet NI. AHR-mediated immunomodulation: the role of altered gene transcription. Biochem Pharmacol. 2009;77:746–760. [PMC free article] [PubMed]
50. Quintana FJ, Basso AS, Iglesias AH, Korn T, Farez MF, Bettelli E, Caccamo M, Oukka M, Weiner HL. Control of T(reg) and T(H)17 cell differentiation by the aryl hydrocarbon receptor. Nature. 2008;453:65–71. [PubMed]
51. Vítek L, Jirsa M, Brodanová M, Kalab M, Marecek Z, Danzig V, Novotný L, Kotal P. Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels. Atherosclerosis. 2002;160:449–456. [PubMed]
52. Wu JF, Chiang BL, Chen HL, Lai HS, Chang MH, Ni YH. Impaired T-lymphocyte proliferation function in biliary atresia patients with chronic cholestatic jaundice after a Kasai operation. Pediatr Res. 2006;60:602–606. [PubMed]
53. Kato Y, Shimazu M, Kondo M, Uchida K, Kumamoto Y, Wakabayashi G, Kitajima M, Suematsu M. Bilirubin rinse: A simple protectant against the rat liver graft injury mimicking heme oxygenase-1 preconditioning. Hepatology. 2003;38:364–373. [PubMed]
54. Fondevila C, Shen XD, Tsuchiyashi S, Yamashita K, Csizmadia E, Lassman C, Busuttil RW, Kupiec-Weglinski JW, Bach FH. Biliverdin therapy protects rat livers from ischemia and reperfusion injury. Hepatology. 2004;40:1333–1341. [PubMed]
55. Yamashita K, McDaid J, Ollinger R, Tsui TY, Berberat PO, Usheva A, Csizmadia E, Smith RN, Soares MP, Bach FH. Biliverdin, a natural product of heme catabolism, induces tolerance to cardiac allografts. FASEB J. 2004;18:765–767. [PubMed]
56. Manzinate F, McDaid J, Devey L, Gunson B, Wigmore SJ. Pretransplant bilirubin concentration does not correlate with early reperfusion injury following liver transplantation. Transplantation. 2007;83:103–104. [PubMed]
57. Ferris CD, Jaffrey SR, Sawa A, Takahashi M, Brady SD, Barrow RK, Tysoe SA, Wolosker H, Barañano DE, Doré S, et al. Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol. 1999;1:152–157. [PubMed]
58. Harrison PM, Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta. 1996;1275:161–203. [PubMed]
59. Vile GF, Basu-Modak S, Waltner C, Tyrrell RM. Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts. Proc Natl Acad Sci USA. 1994;91:2607–2610. [PubMed]
60. Bornman L, Baladi S, Richard MJ, Tyrrell RM, Polla BS. Differential regulation and expression of stress proteins and ferritin in human monocytes. J Cell Physiol. 1999;178:1–8. [PubMed]
61. Sheftel AD, Kim SF, Ponka P. Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism. J Biol Chem. 2007;282:10480–10486. [PubMed]
62. Berberat PO, Katori M, Kaczmarek E, Anselmo D, Lassman C, Ke B, Shen X, Busuttil RW, Yamashita K, Csizmadia E, et al. Heavy chain ferritin acts as an antiapoptotic gene that protects livers from ischemia reperfusion injury. FASEB J. 2003;17:1724–1726. [PubMed]
63. Obolensky A, Berenshtein E, Konijn AM, Banin E, Chevion M. Ischemic preconditioning of the rat retina: protective role of ferritin. Free Radic Biol Med. 2008;44:1286–1294. [PubMed]
64. Berenshtein E, Vaisman B, Goldberg-Langerman C, Kitrossky N, Konijn AM, Chevion M. Roles of ferritin and iron in ischemic preconditioning of the heart. Mol Cell Biochem. 2002;234-235:283–292. [PubMed]
65. Chevion M, Leibowitz S, Aye NN, Novogrodsky O, Singer A, Avizemer O, Bulvik B, Konijn AM, Berenshtein E. Heart protection by ischemic preconditioning: a novel pathway initiated by iron and mediated by ferritin. J Mol Cell Cardiol. 2008;45:839–845. [PubMed]
66. Boczkowski J, Poderoso JJ, Motterlini R. CO-metal interaction: Vital signaling from a lethal gas. Trends Biochem Sci. 2006;31:614–621. [PubMed]
67. Wang X, Wang Y, Kim HP, Nakahira K, Ryter SW, Choi AM. Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation. J Biol Chem. 2007;282:1718–1726. [PubMed]
68. Kim HP, Ryter SW, Choi AM. CO as a cellular signaling molecule. Annu Rev Pharmacol Toxicol. 2006;46:411–449. [PubMed]
69. Williams SE, Wootton P, Mason HS, Bould J, Iles DE, Riccardi D, Peers C, Kemp PJ. Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel. Science. 2004;306:2093–2097. [PubMed]
70. Amersi F, Shen XD, Anselmo D, Melinek J, Iyer S, Southard DJ, Katori M, Volk HD, Busuttil RW, Buelow R, et al. Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway. Hepatology. 2002;35:815–823. [PubMed]
71. Akamatsu Y, Haga M, Tyagi S, Yamashita K, Graça-Souza AV, Ollinger R, Czismadia E, May GA, Ifedigbo E, Otterbein LE, et al. Heme oxygenase-1-derived carbon monoxide protects hearts from transplant associated ischemia reperfusion injury. FASEB J. 2004;18:771–772. [PubMed]
72. Sato K, Balla J, Otterbein L, Smith RN, Brouard S, Lin Y, Csizmadia E, Sevigny J, Robson SC, Vercellotti G, et al. Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J Immunol. 2001;166:4185–4194. [PubMed]
73. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, Green CJ. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ Res. 2002;90:E17–E24. [PubMed]
74. Guo Y, Stein AB, Wu WJ, Tan W, Zhu X, Li QH, Dawn B, Motterlini R, Bolli R. Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol. 2004;286:H1649–H1653. [PMC free article] [PubMed]
75. Stein AB, Guo Y, Tan W, Wu WJ, Zhu X, Li Q, Luo C, Dawn B, Johnson TR, Motterlini R, et al. Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction. J Mol Cell Cardiol. 2005;38:127–134. [PMC free article] [PubMed]
76. Sandouka A, Fuller BJ, Mann BE, Green CJ, Foresti R, Motterlini R. Treatment with CO-RMs during cold storage improves renal function at reperfusion. Kidney Int. 2006;69:239–247. [PubMed]
77. Yoshida J, Ozaki KS, Nalesnik MA, Ueki S, Castillo-Rama M, Faleo G, Ezzelarab M, Nakao A, Ekser B, Echeverri GJ, et al. Ex vivo application of carbon monoxide in UW solution prevents transplant-induced renal ischemia/reperfusion injury in pigs. Am J Transplant. 2010;10:763–772. [PMC free article] [PubMed]
78. Clarke HM, Shrivastava S, Motterlini R, Sawle P, Chen D, Dorling A. Donor HO-1 expression inhibits intimal hyperplasia in unmanipulated graft recipients: a potential role for CD8+ T-cell modulation by carbon monoxide. Transplantation. 2009;88:653–661. [PubMed]
79. Koetting M, Leuvenink H, Dombrowski F, Minor T. Gaseous persufflation with carbon monoxide during ischemia protects the isolated liver and enhances energetic recovery. Cryobiology. 2010;61:33–37. [PubMed]
80. Dolores PM, Rodriguez JV, Mamprin ME, Fuller BJ, Mann BE, Motterlini R, Guibert EE. Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation. Cryobiology. 2009:Epub ahead of print. [PubMed]
81. Pizarro MD, Rodriguez JV, Mamprin ME, Fuller BJ, Mann BE, Motterlini R, Guibert EE. Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation. Cryobiology. 2009;58:248–255. [PubMed]
82. Tomiyama K, Ikeda A, Ueki S, Nakao A, Stolz DB, Koike Y, Afrazi A, Gandhi C, Tokita D, Geller DA, et al. Inhibition of Kupffer cell-mediated early proinflammatory response with carbon monoxide in transplant-induced hepatic ischemia/reperfusion injury in rats. Hepatology. 2008;48:1608–1620. [PubMed]
83. Lin QS, Weis S, Yang G, Zhuang T, Abate A, Dennery PA. Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress. Free Radic Biol Med. 2008;44:847–855. [PubMed]
84. Soares MP, Marguti I, Cunha A, Larsen R. Immunoregulatory effects of HO-1: how does it work? Curr Opin Pharmacol. 2009;9:482–489. [PubMed]
85. Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci USA. 1997;94:10925–10930. [PubMed]
86. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, Koizumi S. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest. 1999;103:129–135. [PMC free article] [PubMed]
87. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol. 1994;12:991–1045. [PubMed]
88. Matzinger P. Friendly and dangerous signals: is the tissue in control? Nat Immunol. 2007;8:11–13. [PubMed]
89. Xu H, Su Z, Wu J, Yang M, Penninger JM, Martin CM, Kvietys PR, Rui T. The alarmin cytokine, high mobility group box 1, is produced by viable cardiomyocytes and mediates the lipopolysaccharide-induced myocardial dysfunction via a TLR4/phosphatidylinositol 3-kinase gamma pathway. J Immunol. 2010;184:1492–1498. [PubMed]
90. Watanabe T, Kubota S, Nagaya M, Ozaki S, Nagafuchi H, Akashi K, Taira Y, Tsukikawa S, Oowada S, Nakano S. The role of HMGB-1 on the development of necrosis during hepatic ischemia and hepatic ischemia/reperfusion injury in mice. J Surg Res. 2005;124:59–66. [PubMed]
91. Pardo M, Budick-Harmelin N, Tirosh B, Tirosh O. Antioxidant defense in hepatic ischemia-reperfusion injury is regulated by damage-associated molecular pattern signal molecules. Free Radic Biol Med. 2008;45:1073–1083. [PubMed]
92. Tsung A, Klune JR, Zhang X, Jeyabalan G, Cao Z, Peng X, Stolz DB, Geller DA, Rosengart MR, Billiar TR. HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med. 2007;204:2913–2923. [PMC free article] [PubMed]
93. Ilmakunnas M, Tukiainen EM, Rouhiainen A, Rauvala H, Arola J, Nordin A, Mäkisalo H, Höckerstedt K, Isoniemi H. High mobility group box 1 protein as a marker of hepatocellular injury in human liver transplantation. Liver Transpl. 2008;14:1517–1525. [PubMed]
94. Tsoyi K, Lee TY, Lee YS, Kim HJ, Seo HG, Lee JH, Chang KC. Heme-oxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo. Mol Pharmacol. 2009;76:173–182. [PubMed]
95. Takamiya R, Hung CC, Hall SR, Fukunaga K, Nagaishi T, Maeno T, Owen C, Macias AA, Fredenburgh LE, Ishizaka A, et al. High-mobility group box 1 contributes to lethality of endotoxemia in heme oxygenase-1-deficient mice. Am J Respir Cell Mol Biol. 2009;41:129–135. [PMC free article] [PubMed]
96. Tang D, Shi Y, Kang R, Li T, Xiao W, Wang H, Xiao X. Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1. J Leukoc Biol. 2007;81:741–747. [PMC free article] [PubMed]
97. Figueiredo RT, Fernandez PL, Mourao-Sa DS, Porto BN, Dutra FF, Alves LS, Oliveira MF, Oliveira PL, Graça-Souza AV, Bozza MT. Characterization of heme as activator of Toll-like receptor 4. J Biol Chem. 2007;282:20221–20229. [PubMed]
98. Porto BN, Alves LS, Fernández PL, Dutra TP, Figueiredo RT, Graça-Souza AV, Bozza MT. Heme induces neutrophil migration and reactive oxygen species generation through signaling pathways characteristic of chemotactic receptors. J Biol Chem. 2007;282:24430–24436. [PubMed]
99. Wang XM, Kim HP, Nakahira K, Ryter SW, Choi AM. The heme oxygenase-1/carbon monoxide pathway suppresses TLR4 signaling by regulating the interaction of TLR4 with caveolin-1. J Immunol. 2009;182:3809–3818. [PubMed]
100. Nakahira K, Kim HP, Geng XH, Nakao A, Wang X, Murase N, Drain PF, Wang X, Sasidhar M, Nabel EG, et al. Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts. J Exp Med. 2006;203:2377–2389. [PMC free article] [PubMed]
101. Suzuki S, Nakamura S, Sakaguchi T, Ochiai H, Konno H, Baba S, Baba S. Alteration of reticuloendothelial phagocytic function and tumor necrosis factor-alpha production after total hepatic ischemia. Transplantation. 1997;64:821–827. [PubMed]
102. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003;19:71–82. [PubMed]
103. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, Wysk M, Davis RJ, Flavell RA, Choi AM. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med. 2000;6:422–428. [PubMed]
104. Khandoga A, Hanschen M, Kessler JS, Krombach F. CD4+ T cells contribute to postischemic liver injury in mice by interacting with sinusoidal endothelium and platelets. Hepatology. 2006;43:306–315. [PubMed]
105. Zwacka RM, Zhang Y, Halldorson J, Schlossberg H, Dudus L, Engelhardt JF. CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver. J Clin Invest. 1997;100:279–289. [PMC free article] [PubMed]
106. Caldwell CC, Okaya T, Martignoni A, Husted T, Schuster R, Lentsch AB. Divergent functions of CD4+ T lymphocytes in acute liver inflammation and injury after ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol. 2005;289:G969–G976. [PubMed]
107. Burne MJ, Daniels F, El Ghandour A, Mauiyyedi S, Colvin RB, O'Donnell MP, Rabb H. Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure. J Clin Invest. 2001;108:1283–1290. [PMC free article] [PubMed]
108. Pae HO, Oh GS, Choi BM, Chae SC, Kim YM, Chung KR, Chung HT. Carbon monoxide produced by heme oxygenase-1 suppresses T cell proliferation via inhibition of IL-2 production. J Immunol. 2004;172:4744–4751. [PubMed]
109. Yamashita K, Ollinger R, McDaid J, Sakahama H, Wang H, Tyagi S, Csizmadia E, Smith NR, Soares MP, Bach FH. Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs. FASEB J. 2006;20:776–778. [PubMed]
110. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–1164. [PubMed]
111. Cheng X, Yu X, Ding YJ, Fu QQ, Xie JJ, Tang TT, Yao R, Chen Y, Liao YH. The Th17/Treg imbalance in patients with acute coronary syndrome. Clin Immunol. 2008;127:89–97. [PubMed]
112. Choi BM, Pae HO, Jeong YR, Kim YM, Chung HT. Critical role of heme oxygenase-1 in Foxp3-mediated immune suppression. Biochem Biophys Res Commun. 2005;327:1066–1071. [PubMed]
113. Zelenay S, Chora A, Soares MP, Demengeot J. Heme oxygenase-1 is not required for mouse regulatory T cell development and function. Int Immunol. 2007;19:11–18. [PubMed]
114. Biburger M, Theiner G, Schädle M, Schuler G, Tiegs G. Pivotal Advance: Heme oxygenase 1 expression by human CD4+ T cells is not sufficient for their development of immunoregulatory capacity. J Leukoc Biol. 2010;87:193–202. [PubMed]
115. George JF, Braun A, Brusko TM, Joseph R, Bolisetty S, Wasserfall CH, Atkinson MA, Agarwal A, Kapturczak MH. Suppression by CD4+CD25+ regulatory T cells is dependent on expression of heme oxygenase-1 in antigen-presenting cells. Am J Pathol. 2008;173:154–160. [PubMed]
116. Jaeschke H, Smith CW. Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol. 1997;61:647–653. [PubMed]
117. Li L, Huang L, Vergis AL, Ye H, Bajwa A, Narayan V, Strieter RM, Rosin DL, Okusa MD. IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. J Clin Invest. 2010;120:331–342. [PMC free article] [PubMed]

Articles from World Journal of Gastroenterology are provided here courtesy of Baishideng Publishing Group Inc