Ischaemic/reperfusion injury: Role of infliximab

Abstract

Ischaemia/reperfusion (I/R) injury is an underlying complex interrelated patho-physiological process which effects the outcome of many clinical situations, in particular transplantation. Tumor necrosis factor (TNF)-α is a pleiotropic inflammatory cytokine; a trimeric protein encoded within the major histocompatibility complex which plays a pivotal role in this disease process. This review is based at looking into an update, particularly the new insights in the mechanisms of action of TNF antagonist such as infliximab. Infliximab may thus play a dual role in the field of transplantation where it might not only down regulate the I/R injury, it may also have a beneficial role in the reduction of acute rejection.

Key Words: Infliximab; Ischaemia/reperfusion injury; Tumor necrosis factor-α

BACKGROUND

Transplantation, in particular renal is the best modality for treating end stage disease[1]. Due to a lack of suitable organs for transplantation from traditional sources[1], there is a renewed interest into other alternatives such as live donors, extended criteria donors (ECD) and donation after cardiac death donors (DCD) (non-heart beating donors[2]). The long term function and survival of DCD’s are comparable to heart beating donors, hence making the ongoing research and development important[3,4]. Though results are encouraging, delayed graft function (DGF) and primary non function are a significant problem as a consequence of prolonged warm ischaemic insult during renal organ retrievals[4-8]. DGF leads to significant service related burdens such as prolonged haemodialysis and psychological impact on the patient[9]. Early graft injury in addition is associated with an increase in acute rejection (AR) and chronic allograft nephropathy[10,11]. The underlying pathophysiology of early graft injury is thought to be a complex interrelated sequence of events called ischaemic/reperfusion (I/R) injury. This review is aimed to assess and construct a concise evidence based literature document about the possible role of infliximab and its effects on I/R injury and use in transplantation, particularly ECD and DCD’s.

SEARCH STRATEGY

MEDLINE (PubMed - 1966-2010), The Cochrane Central Register of Controlled Trials, EMBASE (1974-2009) the Database of Abstracts of Reviews of Effects, Health Technology Assessment Database and by attended relevant meetings, hand searched pertinent journals to identify relevant studies of all randomizes trials, meta analysis and case series. The search strategy included: (1) ischaemia reperfusion injury; (2) infliximab; (3) cytokines in I/R injury; and (4) tumor necrosis factor (TNF). No other search restrictions were applied and all related reference articles were reviewed.

I/R INJURY

I/R injury involves ATP breakdown product accumulation in an ischaemic environment, which following reperfusion are converted to xanthine and superoxide anion by xanthine oxidase, an isoform of xanthine dehydrogenase. The conversion of dehyrogenase to oxide is under the influence of a calcium dependent protease which is activated by ischaemia[12,13]. This commences a cascade of free radical formation, causing direct injury to lipids, proteins, DNA and initiating pro-inflammatory, apoptotic and complement pathways[12]. Depletion of ATP in this process leads to cell membrane instability via the incapacitated sodium/potassium (Na⁺/K⁺) pump[14] and thereby intracellular calcium accumulation which inhibits mitochondrial function and acts as a secondary messenger in apoptotic pathways[15].

Apoptosis, a morphological form of programmed cell death has dual role in renal injury. On one hand it serves as a healing mechanism related to the resolution of inflammation[16-18], while on the other hand it leads to accelerated apoptosis causing cell deletion and graft injury[16,19-22]. Although different signals initiate apoptosis, the patho-physiological process of apoptosis is surprisingly similar even in different cell types, suggesting that the final stage of apoptotic death is highly conserved[16,23]. Two phases of the apoptosis process have been described[23] The initiation phase involves death factors/death receptors or mitochondrial dysfunction. Death receptors are members of TNF super-family from which the TNF-α, TNF receptor 1 (TNF-R1) and Fas CD95/APO-1 play major roles.

The ischaemic proximal tubule epithelial cells generate a number of mediators that potentiate the tubule-interstitial inflammatory response. These include TNF-α, interleukin (IL)-1, IL-α, IL-β, IL-6 which are pivotal factors in IRI process for native and transplanted kidneys. TNF is a homotrimeric cytokine produced by numerous cell types including monocytes and macrophages that play an important role in pathogenesis of the inflammatory response. TNF-α and other cytokines expression can be regulated at different levels. The two principal mechanisms identified are a transcriptional and a post-transcriptional regulation triggered by different transcription factors and signalling cascades activated by a variety of stimuli ranging from cell damaging physical factors to mitogens and cytokines.

Upon activation by their cognate ligands, TNF-α, Fas and TNF-R1 recruit an intracellular death complex consisting of adapter proteins and procaspases. The death complex then activates apical caspases, mainly caspase-8, which subsequently activates downstream effector caspases; caspase-3. In the alternative initiation pathway, cellular stress triggers release of cytochrome c to bind Apaf-1, which in turn activates caspase-9. Here onwards both the pathways converge because caspase-9 also activates effector caspases. Caspases (14 different members) are a class of proteases contributing to cell injury and execution of the death programme[24-26]. As all pro-forms of caspases contain both recognition and cleavage sites implying their activation occurs either autocatalytically or by other capases. Thus Caspase-3 activation is by two major pathways, either mediated by death receptors (caspase-8) or by mitochondria (caspase-9)[27,28] during the execution phase of apoptosis dismantle the cells by sequential activation and cleavage of key proteins. Caspase-3 is major execution enzyme acting upstream of DNA fragmentation[27-29] and can also be activated via endoplasmic reticulum pathways (caspase-12)[30]. Previous studies demonstrate the increase of caspases in I/R injury in various organs[31,32].

The pathophysiology of I/R injury has been investigated by a large number of in vivo and in vitro studies. Methods described to attenuate this process include removal and inhibition of leucocytes, inhibition of classical and alternative complement pathways, inhibition of platelets, down regulation of endothelial cell adhesion molecules, inhibition of cytokines (TNF and IL-1)[33-35], inhibition of free radical forming enzymes, free radical chelation and antiapoptic agents[32,36-39]. Post ischaemia protection is possible because genes are up regulated after ischaemia, allowing a window of opportunity for intervention[39]. Gene transfer technology, with RNA interference, allows specific silencing of genes by delivering highly homologus RNA into the cell[40].

ROLE OF TNF-α IN I/R INJURY

TNF-α is a central regulator of inflammation, and thus TNF-α antagonists may be effective in treating inflammatory disorders of which TNF-α plays an important pathogenetic role[41]. TNF-α is a pleiotropic inflammatory cytokine; a trimeric protein encoded within the major histocompatibility complex. It is evident that this mediator is the prototypic member of a gene superfamily that regulates essential biologic functions such as immune response, cell proliferation, survival, differentiation and apoptosis[41]. These biological activities include beneficial effects in immune response against several pathogens and in organogenesis of lymphoid structures as well as host damaging effects in sepsis, cachexia, autoimmune and inflammatory diseases[42].

TNF-α is primarily produced by immune cells such as monocytes and macrophages, but other cell types are also capable of releasing this cytokine, including acinar cells. It is initially synthesized as a 26 kDa cell surface associated molecule anchored by an N-terminal hydrophobic domain. This membrane-bound form of TNF-α possesses biological activity. A specific matrix metalloproteinase protein, called TNF-converting enzyme, cleaves the 26 kDa form into a soluble 17 kDa form[41] which self-assembles in non covalent bound homotrimers[43], an important feature for the cross-linking and the activation of TNF receptors.

TNF-α and its specific receptors TNFR1/TNFR2 are the major members of a gene superfamily of ligand and receptors that regulates essential biologic functions. Receptor activation by TNF family ligands causes recruitment of various adaptor proteins with subsequent activation of downstream signalling pathways. TNFR superfamily can be classified in three major groups according to specific intracellular sequences and to signalling adaptors recruited. The first group include receptors, such as TNFR1 (p55 or 55-kDa TNFR), Fas, where they share a highly conserved sequence of about 80 amino acids in the cytoplasmic region called the death domain. Activation of these receptors leads to homotypic interactions with adaptor proteins containing death domains such as Fas-associated death domain (FADD) and TNFR-associated death domain (TRADD). The latter signalling pathway requires an interaction between TRADD and FADD, which in turn interact with caspase-8. Though Recruitment of TRADD can also trigger downstream events related to inflammation through further adaptor proteins including TNF receptor associated factors, receptor interacting protein and mitogen activated kinase-activating death domain[41].

A strong link has been established between TNF-α production and oxidative stress during the IRI process[33,34]. Thus inflammatory mediators such as the cytokine, TNF-α is thought to have a central role in the pathophysiology of renal injury[44]. TNF-α is consistently up-regulated in response to renal ischaemic injury, induced by the activation of p38 mitogen-activated protein kinase via enhanced tyrosine phosphorylation[33,34,45,46]. It is also known to induce other mediators of inflammation and tissue injury such as IL-1, IL-2, interferon-γ, adhesion molecules (ICAM-1, VCAM-1) which exacerbate the injury process[47]. Activation of TNF-α may induce apoptosis, cell death as well as inflammation[41]. TNF-α is implicated in the pathogenesis of different renal diseases and can promote renal dysfunction by direct cytotoxicity, vasoconstriction, and inflammatory cells recruitment[33,48-50]. Up-regulation of mRNA and protein levels of TNF occurs at a whole-organ level within minutes to hours of the onset of I/ R Injury[33]. To date FR167653, an inhibitor of TNF-α has been shown to improve effects of warm ischaemia in a porcine model[35].

INFLIXIMAB

Infliximab is one of 3 licensed TNF antagonists. Infliximab is a chimeric antibody with a mouse variable fragment (Fv) and human antibody with immunoglobulin G1 (IgG1) and k constant regions[41,51,52], which neutralizes the biological activity of TNF by binding to the soluble and trans-membrane forms of TNF and inhibits the binding of TNF with its receptors. The structure of Infliximab is similar to that of naturally occurring antibodies[41]. Though Infliximab’s mechanism of action is not completely understood. This chimaeric monoclonal antibody, composed of a complement fixing ‘human’ IgG1 constant region (75%) and a murine derived antigen-binding variable region (25%), binds soluble TNF; however, its action is thought to depend in part on its ability to bind precursor cell-surface TNF, perhaps leading to monocyte apoptosis[41]. Two pivotal trials demonstrated the efficacy of infliximab in patients with Crohn’s disease (FDA approved)[53-55].

Infliximab has been shown to inhibit functional TNF-α activity in a variety of in vitro bioassays using human fibroblasts, endothelial cells, neutrophils, lymphocytes, and epithelial cells[56]. In vivo, infliximab is indicated for the treatment of rheumatologic, gastrointestinal, dermatologic, and chronic ocular diseases[51,57]. The role of infliximab has been shown to improve I/R injury in spinal injury models[51,58] and cardiac injury models[59,60]. Guven et al[51] demonstrated that the use of infliximab significantly reduced vascular proliferation, oedema and neuron loss following I/R injury and concluded that the agent may protect the spinal cord against injury in a rabbit I/R model. Niemann et al[59] in a porcine ventricular fibrillation cardiac arrest model showed that infliximab significantly blocked TNF-α levels at 30 min after cardiac arrest and these animals showed a significantly greater mean arterial pressure and stroke volume which was sustained throughout 3-h post resuscitation period.

Porcine TNF shares a similar structure with that of human and murine TNF and exhibits cytotoxicity to target indicator cells (PK and L929)[61] at similar concentrations[62]. Porcine TNF-α cytotoxic activity can be totally neutralized with anti-human TNF monoclonal antibody[59,63]. Porcine TNF-α receptors likewise share a structure similar to that of humans, and mice and human soluble TNF-α receptors bind porcine TNF-α[64]. Considering these characteristics, binding to and neutralization of porcine TNF-α by Infliximab would be expected and has been successfully used to improve cardiac dysfunction in a porcine model[59].

Although it is generally safe, serious complications can ensue. In addition to occasional hypersensitivity and infusion reactions, a number of deaths have been reported as a result of tuberculosis or sepsis[65]. The complication may simply be due to effective immune modulation rather than the specific drug. Infliximab has been associated with hypersensitivity reactions that include urticaria, dyspnoea and hypotension, and usually occur within 2 h of infusion. Serum sickness-like reactions were observed in some Crohn’s disease patients 3 to 12 d after therapy was reinitiated following an extended period without infliximab. Fever, rash, headache, sore throat, myalgia, polyarthralgias, hand and facial oedema and dysphagia were also associated with a marked increase in antibodies to infliximab[58,66,67].

However, the effects of infliximab in reducing renal ischaemic injury have not been clearly determined and this manipulating agent may have a potential role in DCD, ECD kidney transplantation. To date, Cau et al[35] has shown that the use of an agent FR167653, a potent inhibitor of TNF-α and IL-1β reduces the early and long term effects of WI in the their porcine ischaemic model. This effect was particularly marked against fibrosis and inflammation, which would contribute to deterioration of renal function. Bagul et al[68] have shown preliminary results of Infliximab are promising where this agent significantly improved kidney perfusion, oxygen delivery and reduced TNF-α levels in an ex-vivo model of renal transplantation and concluded that further investigation to assess infliximab’s potential to ameliorate I/R injury in renal transplantation is warranted.

In addition to this Infliximab may play a role to reduce AR as not only it may reduce DGF which has a direct link to AR[10,11], it is a potent TNF antagonist where TNF-α in itself is a Th1-type cytokine (IL-2, interferon-γ, TNF-α) which mediates cellular response[69-72]. Importantly Th1-type cytokines may down regulate Th2-type cytokines (IL-4, IL-5, IL-10) which mediate the humoral response[72]. There is a body of evidence which show cytokines are involved in allograft rejection, where Th1-type cytokines are believed to be associated with rejection while Th2 cytokines with tolerance[73,74].

CONCLUSION

The new insights into the mechanisms of action of TNF antagonist such as Infliximab need to be studied further and coupling this with the drug’s safety profile, pharmacokinetics and immunogenicity; the drug may have dual role in transplantation benefiting not only from I/R injury but also AR.