The Injury Hypothesis, as published by us in Transplantation in 1994, holds that the reactive oxygen species (ROS)-mediated reperfusion injury to an allograft (in addition to its degree of foreignness) initiates and induces the adaptive alloimmune response (acute rejection) predominantly through activation of antigen-presenting cells. Furthermore, the ROS-induced injury contributes to the development of alloatherosclerosis of donor organ vessels (chronic rejection) through endothelial injury-induced proliferation of smooth muscle cells (1, 2). A role for injury up-regulated costimulatory molecules on antigen-presenting cells, as suggested by the coincident expression of adhesion molecules, was also noted (3) (Fig. 1).
;)
FIGURE 1.:
The Injury Hypothesis: (A) Oxidative stress to the brain-dead donor organism, (B) generation of reactive oxygen species (ROS) during reperfusion of the allograft, and (C) chronic risk factors in the recipient represent acute and chronic injurious events to the donor organ inducing acute rejection and contributing to chronic rejection. By activation of donor/recipient Toll-like receptor (TLR)-bearing dendritic cells (DCs) and donor TLR-bearing vascular cells of innate immunity by putative endogenous ligands of TLRs, these events lead to initiation of adaptive alloimmunity and contribute to the development of alloatherosclerosis. The hypothesis was originally established in 1994 (blue), extended and refined in 2002 (red), and again modified in this overview (pink). PELs, putative endogenous ligands of TLRs.
The concept emerged from a clinical trial in cyclosporine-treated kidney transplant recipients. We found that intraoperative treatment of postischemic reperfusion injury to allografts with the free radical scavenger superoxide dismutase significantly reduced the incidence of acute rejection episodes and early immune-mediated graft loss, and, in addition, remarkably improved the long-term graft outcome. To our knowledge, these reports were the first to suggest that a nonspecific tissue injury to allografts leads to T-cell alloactivation and, thus, induces alloimmune responsiveness. The concept nicely fitted in Matzinger’s “Danger Hypothesis,” which holds that the primary driving force of the immune system is the need to detect and protect against danger; danger, however, equals tissue destruction. The immune system is turned on only when cellular damage occurs (4).
In 1999, the Injury Hypothesis was extended by explicitly addressing dendritic cells (DCs) as the major target of reperfusion injury: This allograft injury probably contributes to an up-regulated activity on three levels of DC and T-cell interaction: (1) presentation of allogeneic peptide and major histocompatibility complex (MHC) antigens, (2) costimulation, and (3) adhesion. A role of a mitigated reperfusion injury for future trials of successful allotolerance induction was also noted (5, 6).
With the rediscovery of innate immunity in the mid-1990s (7–12), it became clear that our early clinical observations and their interpretation reflected events of the innate immune system. Consequently, in 2002, we reviewed data in support of the concept that ROS-mediated injury to allografts activates the innate immune system of both the donor and the recipient (13–16). We discussed the possibility that ROS-mediated intracellular appearance of non-native oxidized proteins induces the chaperoning activity of heat shock proteins (HSPs), which, in turn, interact with and activate Toll-like receptor (TLR)-bearing cells of the innate immune system, in particular DCs representing the bridge to adaptive alloimmunity and vascular cells contributing to alloatherogenesis (Fig. 1). In view of such mechanisms, the early and late fate of an allograft is predominantly determined by its exposure to a variety of different stressful factors causing graft damage. Oxidative stress to allografts seems to be the most important stress factor, whereas chronic conditions of the recipient such as hypertension, hyperlipidemia, viral infections, and the daily administration of organ-toxic immunosuppressive agents are supposed to mediate continuous but milder stress.
There are other approaches to the issue of injury in relation to the description of allograft deterioration such as the concept of Halloran (17) and Gourishankar and Halloran (18). This new view defines (late) allograft failure as a composite phenotype reflecting the total burden of stressful injuries mainly mediated by five groups of risk factors: (1) donor age; (2) brain death; (3) preservation, implantation, ischemia-reperfusion; (4) immune-mediated injury: rejection; and (5) posttransplant stresses and systemic stresses in the recipient environment. It is interesting that there are remarkable similarities between Halloran’s concept and our hypothesis. Many of those risk conditions in the five groups (e.g., brain death and reperfusion as covered in this article) are associated with the generation of ROS or expression of HSPs, thereby potentially activating innate immune events.
Advanced donor age as a detrimental factor for graft survival may be explained on the basis of increased intracellular oxidative stress during ageing because of decreased antioxidative capacity and increased free-radical leakage from mitochondria. The oxidative stress may contribute to activation of innate signaling pathways aggravating or leading to age-related changes such as graft fibrosis and atherosclerosis (14, 16). This explanation is in agreement with Lane’s Double-Agent Theory proposing that ageing is a function of increasing intracellular oxidative stress, mainly as the result of continuous mitochondrial leakage during ageing, which produces a genetic response through proinflammatory redox-sensitive transcription factors such as nuclear factor (NF)-κB, leading to chronic inflammation characteristic of old age (19). According to this interpretation, ageing per se might mirror continuously ongoing events of innate immunity.
Similarly, immune-mediated processes such as rejection have been shown to induce a heat-shock response that may perpetuate adaptive responses through activation of innate events in terms of a vicious circle (20, 21).
As reviewed elsewhere, continuous posttransplant systemic stresses such as hypertension, hyperlipidemia, viral infections, and the use of calcineurin inhibitors, are associated with the generation of ROS or production of HSPs (16). Again, HSPs may activate TLR-bearing cells of innate immunity (e.g., intragraft vascular cells, macrophages, and monocytes) resulting in ongoing intragraft fibrosis and intimal thickening of arteries. Likewise, diabetes mellitus is associated with oxidative stress and might per se represent a disease of innate immunity (22, 23). Finally, preliminary evidence indicates that renal diseases in general reflect events of innate immunity (24).
INNATE IMMUNITY
The innate immune system evolved to protect the host as a rapid first line of defense against invading pathogens. It is now well established that the activation of adaptive responses requires direction from the innate immune system. The signals for activation are largely provided by DCs. In particular, the family of innate immune signaling receptors, known as the TLRs, has proven, in addition to other pattern recognition receptors, to be essential in the detection and signaling of infection (25–28).
The mammalian TLRs comprise a family of germline-encoded transmembrane receptors that recognize conserved bacterial, viral, fungal, and protozoal molecular structures called pathogen-associated molecular patterns (PAMPs). In 1998, TLR4 was shown to be involved in the recognition of lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria (29). Subsequently until today, 11 TLRs have been cloned in mammals, and each receptor seems to be involved in the recognition of a unique set of PAMPs that act as exogenous ligands of TLRs (27, 28, 30–33) (Table 1).
TABLE 1:
Toll-like receptors and their exogenous ligands (PAMPs) and putative endogenous ligands
Of utmost importance for the validity of the Injury Hypothesis is the early observation that danger signaling stress proteins (=HSPs), known to be released in a host as a result of any tissue injury, act as putative endogenous ligands of TLRs and, by this interaction, are able to activate cells of the innate immune system. Originally, HSPs were described as intracellular molecular chaperones of aberrantly folded, nonnative, denatured proteins (=altered self-proteins) primarily involved in cytoprotection and adaptation for cell survival in response to stressful stimuli (in terms of a first line of host defense). However, in recent years, new functions of HSPs have been revealed. On more intense stress (e.g., associated with cell necrosis), HSPs may act independently from their intracellular function by being extracellularly released as molecular chaperones in a cytokine-like manner and may be regarded as chaperokines. The chaperokine activity of HSPs is mediated in part by interaction with TLRs leading to the activation of the innate immune system (the second line of host defense). Ultimately, the adaptive immune response, succeeding innate immunity, represents the third line of defense, in fact, the most sophisticated defense system (15, 34).
Putative endogenous ligands of TLRs were first described in studies with HSPs including HSP60, HSP70, and gp96. The immunostimulatory and inflammatory activity of these molecules are mediated by TLRs, such as TLR2 and TLR4 (7, 11, 35–39). Most of these studies were performed using recombinant HSP preparations from which LPS is notoriously difficult to remove. Consequently, contradictory reports were published by raising the question whether the activation of TLR4 was really induced by these endogenous ligands or by an exogenous ligand in the form of a tightly bound LPS contaminant (40). Nevertheless, ostensibly, HSPs could engage TLRs on release from necrotic cells and thereby induce an inflammatory response (28). Subsequent studies revealed additional injury-induced molecules that are able to interact with TLR2 or TLR4 including the inducible host antibiotic β-defensin and extracellular matrix components, such as fibronectin, hyaluronan fragments, heparan sulfate, and extravascular fibrin deposits, which are early and persistent hallmarks of inflammation accompanying injury. Nucleic chromatin seems to be another endogenous ligand that signals through TLR9 (41–46) (Table 1). It has been proposed that exogenous and endogenous ligands of TLRs might not be mutually exclusive groups of molecules. Many of them might be part of an evolutionarily ancient alert system in which the hydrophobic portions of biologic molecules operate, when exposed, as universal damage-associated molecular patterns to initiate repair, remodeling, and immunity (47).
It is now well recognized that both exogenous and putative endogenous ligands of TLRs are able to induce activation and maturation of DCs. In addition to secretion of cytokines and chemokines, as well as presenting peptides in the frame of MHC molecules to T cells, mature DCs provide T cells with the required “second signal” by expressing costimulatory molecules on their surface (25, 28, 48, 49). Ligation of DC-TLRs regulates the adaptive immune response in two different ways: In addition to controlling the expression of costimulatory molecules on DCs, TLR triggering in DCs contributes to T-helper (Th)-cell activation by overcoming their suppression mediated by regulatory CD4+ CD25+ T cells (50). In fact, most studies have shown that TLRs control the activation of antigen-specific Th1 but not Th2 adaptive immune responses (25, 51). Vice versa and logically, lack of exogenous and endogenous ligands of TLRs implies lack of TLR ligation and, consequentially, absence of maturation. Notably, however, immature DCs are incapable of initiating or priming T-cell responses but rather can contribute to the expansion and differentiation of regulatory T cells. Immature DCs, therefore, are able to prolong allograft survival and seem to be tolerogenic by bringing about antigen-specific unresponsiveness or central and peripheral tolerance by mechanisms of deletion or suppression (52, 53). Thus, the nature and intensity of an underlying injury may influence this DC-mediated dichotomy by inducing distinct development stages of DCs. Full-scale injury results in the maturation of DCs, enabling them to induce adaptive immunity and rapidly polarize the immune response to Th1 or Th2 types and improve T-cell memory. Minimal injury or even lack of injury may leave them in a semi-mature or immature state resulting in the induction of only weak adaptive responses or tolerance. In fact, we vaguely proposed the possibility of allotolerance induction through avoidance and minimization of the reperfusion injury to allografts already in the 1990s (5, 6).
Intracellular signaling pathways in cells of innate immunity are elicited from the Toll/interleukin (IL)-1 receptor (TIR) domain, which is conserved among the cytoplasmic regions of TLRs (Fig. 2). Both TLR and IL-1R interact with an adaptor protein, myeloid differentiation marker 88 (MyD88). On stimulation, MyD88 recruits a death domain-containing serine/threonine kinase, the IL-1R associated kinase (IRAK). IRAK is activated by phosphorylation and then associates with tumor necrosis factor receptor-associated factor 6 (TRAF6) leading to activation of two distinct signaling pathways to ultimately induce the master transcription factors NF-κB (=early-phase activation) and activator protein-1. The canonic signaling pathway for TLRs after PAMP ligation involves the interaction of MyD88 with the TLR, IRAK, and TRAF6 cascade ultimately leading to the production of proinflammatory cytokines (27, 28, 54–56). Recent observations indicate that the specificity of the MyD88-dependent signaling pathways through all TLRs is provided by a TIR-associated protein (TIRAP), the second TIR domain-containing adaptor molecule that together with MyD88 takes part in TLR4-mediated proinflammatory cytokine production (28, 57).
FIGURE 2.:
Tentative oversimplified scheme of injury-induced putative endogenous ligands binding to TLR. Subsequent TLR-triggered signaling cascades lead to maturation and activation of cells of innate immunity preceding adaptive alloimmunity and contributing to alloatherosclerosis. TLRs (e.g., TLR4) may signal through a myeloid differentiation marker 88 (MyD88)-interleukin (IL)-1 receptor-associated kinase (IRAK)-tumor necrosis factor receptor-associated factor 6 (TRAF) pathway (specified by the second adaptor molecule TIR-associated protein; TIRAP) to induce early-phase activation of redox-sensitive nuclear factor (NF)-κB (released from degraded IκB) by activation of IκB kinase-complex (IKK) and redox-sensitive activator protein-1 by activation of the MAPkinase-cascade (ERK-JnK-p38). MyD88-independent pathways leading to activation of late-phase activation of NF-κB by TRAF6 and to IFN-β promoter by TRAF family member-associated NF-κB activator-binding kinase 1 (TBK)/interferon regulatory factor 3 (IRF3), and associated with maturation of DCs and induction of IFN-inducible genes, may use the two adaptor molecules TIR-domain containing adaptor inducing IFN-β (TRIF) and TRIF-related adaptor molecule (TRAM). Only reports from the literature with relevance to the relationship among reperfusion injury, innate immunity, and organ transplantation are cited in this diagram (
27, 28, 54–64).
However, other sets of recent experimental studies showed that TLR signaling is composed of other pathways, the MyD88-independent pathways, which are associated with the induction of interferon (IFN)-inducible genes and maturation of DCs (27, 28, 58, 59). New insights into the molecular mechanisms of these pathways have recently been published (Fig. 2). Thus, another TIR domain-containing adaptor molecule, the TIR-domain containing adaptor inducing IFN-β (TRIF) (also called TICAM-1) has been identified, which is crucial in the MyD88-independent pathways in signaling downstream to TLR3 and TLR4. TRIF associates with TRAF6 and TRAF family member-associated NF-κB activator-binding kinase 1 (TBK 1) and is obviously involved in the activation of two distinct transcription factors: late-phase activation of NF-κB and as activation of interferon regulatory factor 3 (IRF3), which operates as a transcription factor essential for the production of IFN-β promoter and, thus, for the antiviral immune response. In addition, a fourth TIR domain-containing adaptor was identified with the discovery of TRIF-related adaptor molecule (TRAM), which specifically and separately mediates the MyD88-independent pathway of TLR4 signaling. As does TRIF, TRAM activates IFN regulatory factor IRF3, IRF7, and NF-κB signaling pathways. The TLR3- and TLR4-restricted use of these IRF-inducing adaptor molecules induces not only the cytokines, costimulatory molecules, and antimicrobial peptides that are induced by all TLRs, but also antiviral type 1 IFNs and specific chemokines including IFN-γ inducible protein-10 and RANTES (60–64).
LPS-TLR4 signaling, in contrast with all other PAMPs-TLRs interactions, requires both the MyD88-dependent and the TRIF-dependent pathways, that is, all four adaptor molecules, TRAM, TRIF, MyD88, and TIR-associated protein. Thus, the combinatorial use of TRAM, TRIF, MyD88, and TIR-associated protein may allow a specific tailoring of the immune response to the pathogens that activate TLR4 (63). In analogy, an endogenous ligand-TLR4 interaction may allow a specific tailoring of the alloimmune response according to the nature and intensity of allograft injuries that activate TLR4.
BRAIN DEATH, REPERFUSION INJURY, AND ALLOIMMUNITY
Although it has been shown in a mice model that vigorous allograft rejection can occur in the absence of injurious danger (65), there are accumulating experimental and clinical data in support of the notion that the injury to the donor organ contributes or leads to the activation of the innate immune response preceding the adaptive alloimmune response. As described in more detail elsewhere, one has to discriminate between an injury to potential organs occurring already in the deceased brain-dead donor and injuries to these organs occurring during or after transplantation in the recipient (15, 16).
In experimental studies in rats, molecular mechanisms of organ damage occurring during the phase of donor brain death has been extensively elucidated by Tilney’s group in Boston (66). The central catastrophic injury not only evokes an upsurge of catecholamines with resultant peripheral tissue vasoconstriction and ischemia but also promotes release of hormones and inflammatory mediators such as cytokines, chemokines, and adhesion molecules detected in kidney, heart, and peripheral blood. Oxidative stress may induce this condition or be the result of it. In fact, there is recent convincing clinical evidence suggesting that donor kidneys are already exposed to oxidative stress in brain-dead organisms (67). Plausibly, innate immature DCs in those inflamed organs of brain dead-donors mature and are activated by expecting subsequent engagement with T cells once implanted in the recipient (=direct allorecognition-alloactivation).
Postischemic reperfusion of the donor organ in the recipient induces a second burst of injury associated with activation of (1) intragraft- residing donor DCs and (2) recipient DCs entering the graft, followed by travel of now matured DCs from the graft into the secondary lymphoid tissue where they interact with T cells (=direct/indirect allorecognition-alloactivation). There is growing evidence in support of the notion that the reperfusion injury to allografts activates TLR4-bearing cells of innate immunity resulting in subsequent activation of adaptive alloimmunity: First, numerous investigators have studied a variety of experimental models to show a role for ROS in postischemic reperfusion of organ transplants (3). With the electron paramagnetic resonance-spectroscopy technology, our group directly demonstrated the generation of the toxic hydroxyl radicals in the venous blood of cold-stored, reperfused human renal allografts removed from deceased donors (68).
In addition, there are numerous experimental studies clearly showing the induction by ROS-mediated reperfusion injury of putative endogenous ligands of TLRs in different organs and species, such as HSPs, heparan sulfate, hyaluronic acid, and fibronectin (15, 69–71). By using a specific anti-HSP70 monoclonal antibody (72), our group recently showed the expression of inducible HSP70 in cold-stored human renal allografts removed from brain-dead donors, which was dramatically up-regulated after reperfusion in the recipients (73) (Fig. 3).
FIGURE 3.:
Immunohistochemical demonstration of expression of inducible heat shock protein (HSP)70 in a cadaveric human kidney (cold ischemia time: 18 hr) before (A) and 60 min after (B) reperfusion in the recipient. Inducible HSP70 is remarkably up-regulated after reperfusion. The demonstration was performed with the use of the monoclonal antibody HSP6B3 (GSF; noncommercially available), which is specific for inducible HSP70, in a dilution of 1:150 at room temperature for 1 hr. As a detection system, alkaline phosphatase and antialkaline phosphatase from the rat (Fa.Dako, Hamburg, Germany) is used by incubating with linking antibody and staining with fast red. Further properties (e.g., specificity) of the monoclonal antibodies are described in detail in References
72 and
73.
From studies in knockout mice, there is now first experimental evidence showing that full-scale reperfusion-induced organ injury is, at least in part, mediated by activation of TLR4-bearing cells. An experimental model of liver reperfusion injury in TLR4-, MyD88-, and IRF3-deficient mice showed that full-scale liver reperfusion injury is initiated and induced by activation of TLR4. The study showed that the reperfusion injury activates TLR4 through an MyD88-independent, but IRF3-dependent, pathway to induce the chemokine inducible protein-10, which may then be responsible for the recruitment and activation of T cells (74). Similar results were obtained in studies in another TLR4-deficient mouse model of hepatic ischemia-reperfusion injury (75). A murine model of myocardial ischemia-reperfusion injury (two strains of TLR4-deficient mice) showed that TLR4-deficient mice sustain smaller infarctions and exhibit less inflammation after myocardial reperfusion injury (76). Notably, apart from defined ROS-mediated injury, there are obviously various sources of injury that might activate the innate immune system as has recently been shown in other injury models including severe hemorrhage-induced acute lung injury (77, 78).
The Injury Hypothesis asserts that allografting in mice with deficiencies in signaling molecules of cells of innate immunity or in mice with treated ROS-mediated reperfusion injury should lead to prolongation of allograft survival. In fact, the first reports of such studies have recently been published. In experiments in mice with targeted deletion of universal TLR signal adaptor protein MyD88 minor antigen-mismatched (HY-mismatched) allograft rejection did not occur in the absence of MyD88 signaling, indicating that, at least in this system, innate immunity is linked to the initiation of the adaptive alloimmune response. Notably, the experimental setting used in this study showed that the MyD88-dependent skin allograft rejection was TLR-driven and not induced by IL-1 or IL-18, which can also signal through MyD88. Furthermore, the study demonstrated that the inability to reject these allografts results from a reduced number of mature DCs in draining lymph nodes, leading to an impaired generation of antigraft-reactive T cells and impaired Th1 but not Th2 adaptive response (79). However, in similar experiments in these MyD88-deficient mice performed by the same group, fully MHC-mismatched skin and cardiac allografts were promptly rejected, emphasizing the importance of MyD88-independent pathways in innate recognition of fully MHC-mismatched allografts. In addition, however, this study provided evidence showing that priming of naïve recipient T cells by allogeneic DCs and activation of Th1 immune responses were controlled by and dependent on MyD88. But again, in agreement with prior studies, Th2 immune responses remained intact in the absence of MyD88 (80). Repetition of such experiments in mice with other innate signaling deficiencies (e.g., in TRIF-, TRAF6-, and IRF3-deficient mice) is warranted, thereby also investigating a potential role of reperfusion injury for Th2 responses.
In this context is the first clinical study suggesting an impact of recipient TLR4 polymorphism on acute lung allograft rejection. In this study, recipients with TLR4 Asp299Gly and Thr399Ile polymorphism showed a statistically significant reduction in incidences of acute rejection episodes at 6 months after lung transplantation compared with wild-type. In regard to their observation, the authors suggest an activation of innate immunity through TLR4 in lung transplant recipients that contributes to the development of acute rejection (81).
Treatment of the reperfusion injury, for example, by using cytoprotective heme oxygenase (HO)-1 (HO-1=HSP32), has been documented as a successful therapeutic strategy in a number of transplantation models. The overall effect of HO-1 is to remove a pro-oxidant (heme) while generating a putative antioxidant (bilirubin), carbon monoxide, and iron ions; thus, HO-1 may be regarded as an antioxidant (82, 83). In addition, interference with allograft reperfusion by giving a single intravenous dose of 10 mg/kg of the new free radical scavenger (MCI-186/edaravone) during transplantation of hearts from C57BL/10 mice into CBA mice induced a significant prolongation of graft survival (mean survival time: 78 days compared with 8 days in controls). In this study, the transfer of cells from permanent cardiac allograft-bearing CBA mice into untreated CAB mice recipients of heart transplants again significantly prolonged the survival of fully allogeneic cardiac grafts, suggesting a kind of Treg cell-mediated tolerant state (84).
INJURY AND ALLOATHEROSCLEROSIS
Our current understanding of the vascular biology of atherogenesis and its clinical manifestations suggest a pathophysiology that is much more complex than mere lipid storage. Recent advances support the modern notion of atherosclerosis as an inflammatory and autoimmune process that promotes lesion development from initiation through progression and, ultimately, to the point of acute thrombotic complications and clinical events. In particular, cellular and humoral immunity to HSP60 seems to be the initiating mechanism in the earliest stages of atherosclerosis (85, 86). Consequently, atherosclerosis of allograft arteries concomitant with interstitial fibrosis, as characteristic and dominant features of chronic allograft dysfunction, may be regarded as processes induced by inflammatory, “isoimmune,” and alloimmune responses, the last events obviously being responsible for the rapid development of alloatherosclerosis compared with “native” atherosclerosis. As a common root of these three responses, innate mechanisms can be discussed. Indeed, there is growing evidence suggesting that atherogenesis reflects an event of innate immunity in which exogenous and putative endogenous ligands of TLRs, located within the vessel wall, interact with TLR-bearing vascular cells and lead to local inflammatory processes through cytokine and chemokine secretion and adhesion molecule overexpression (16, 86–88).
As reviewed earlier, acute and chronic risk factors for the development of chronic allograft dysfunction (known to simultaneously be risk factors for the development of atherosclerosis) evoke overexpression of endogenous ligands in the form of HSPs in endothelial cells, macrophages, and smooth muscle cells. These factors include oxidative stress (reperfusion injury, application of calcineurin inhibitors, and smoking), hypertension (oxidative and biomechanical stress to the vessel wall), hyperlipidemia (oxidized low density lipoprotein), and viral infections (16). Thus, HSPs seem to have a general role in the response of the arterial wall to stressful injuries and may serve, in their function as endogenous ligands, as inducers of alloatherosclerosis and native atherosclerosis by interacting with TLRs. In fact, in addition to the detection of HSPs and TLRs in human atherosclerotic lesions, mouse knockout studies and epidemiologic studies of human TLR4 polymorphisms have recently demonstrated that TLR4-triggered innate pathways may play a role in the initiation and progression of atherosclerosis. LPS-induced formation of intimal lesions was reduced in TLR4-deficient mice by 60% compared with that in wild-type mice. In another atherosclerotic mouse model, TLR4 activation was shown to stimulate plaque formation. Studies in both ApoE-deficient and MyD88-deficient mice showed that a cholesterol-rich diet markedly reduced aortic atherosclerosis compared with controls (89). Another similar study in hyperlipidemic, MyD88-deficient mice confirmed these findings (90). Obviously, these observations link elevated serum lipid levels to activation of innate signaling pathways. Recently, the first clinical studies assumed a role of TLR4 in the progression of atherosclerotic disease. Thus, it could be shown that human beings with the Asp299GlyTLR4 polymorphism had a lower risk of carotid atherosclerosis and less intima/media thickness in the common carotid artery (91). In addition, the TLR4 polymorphism was also associated with a higher efficacy of statin therapy (92).
CONCLUSION
The road from allograft injury to two clinically and pathohistologically divergent processes, acute and chronic transplant rejection, as proposed by us on the basis of clinical observations 10 years ago, has meanwhile been slowly but steadily paved with fitting “hypothesis-confirming molecular stone, particularly derived from recent studies in brain-dead rats and knockout mice. Growing evidence is now available to support the notion that TLR4 is involved in injury-induced and injury-mediated adaptive alloimmunity and alloatherogenesis. Nevertheless, we envisage only the beginning of a new research era in organ transplantation, this time related to mechanisms of innate immunity. The exact role of TLRs in organ transplantation remains poorly understood, and further investigations are needed. Likewise, other intracellular-based recognition systems such as NOD and PKR proteins, may also contribute to the initiation of adaptive alloimmune responsiveness. The discovery of adaptor proteins and other signaling molecules required for the activation of cells of innate immunity represents a fascinating future tool for the development of new therapeutic strategies in regard to improving immunosuppression and inducing allotolerance. In the situation of human organ transplantation, it is time to follow our SOD trial and start clinical trials to minimize ROS-mediated allograft injury. Treatment of oxidative stress to donor organs in the brain-dead organism and during postischemic reperfusion in the recipient using novel antioxidants (e.g., edaravone and M40403/M40419) seems urgently warranted.
Acknowledgment
The author thanks Ms. Susanne Martin for her dedicated and excellent secretarial assistance.
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