Ecto-5′ Nucleotidase (CD73)-Mediated Adenosine Generation and Signaling in Murine Cardiac Allograft Vasculopathy
Ecto-5′-nucleotidase (CD73) catalyzes the terminal phosphohydrolysis of 5′-adenosine monophosphate and is widely expressed on endothelial cells where it regulates barrier function. Because it is also expressed on lymphocytes, we hypothesized that it modulates vascular immune regulation under homeostatic conditions and dysregulation under stress conditions such as cardiac allotransplantation. In a heterotopic cardiac allotransplantation model, CD73 deficiency in either donors or recipients resulted in decreased graft survival and the development of cardiac allograft vasculopathy, suggesting a contribution of CD73 on both graft-resident and circulating cells in vasculopathy pathogenesis. Vascular perturbations incited by lack of CD73 included loss of graft barrier function and diminished graft expression of the A2B adenosine receptor (A2BAR), with a concordant exacerbation of the acute inflammatory and immune responses. The importance of CD73 in modulating endothelial–lymphocyte interaction was further demonstrated in allomismatched in vitro coculture experiments. Either genetic deletion or pharmacological blockade of CD73 increased transendothelial lymphocyte migration and inflammatory responses, suggesting that CD73 plays a critical role to suppress transendothelial leukocyte trafficking through its enzymatic activity. In addition, antagonism of A2BAR caused a significant increase in vascular leakage, and agonism of A2BAR resulted in marked prolongation of graft survival and suppression of cardiac allograft vasculopathy development. These data suggest a new paradigm in which phosphohydrolysis of adenosine monophosphate by CD73 on graft-resident or circulating cells diminishes transendothelial leukocyte trafficking and mitigates inflammatory and immune sequelae of cardiac transplantation via the A2BAR.
Ectoenzymes are membrane-bound proteins capable of regulating the extracellular milieu via enzymatically active sites that protrude from the plasma membrane. CD39 (ecto-nucleoside triphosphate diphosphohydrolase) and CD73 (ecto-5′-nucleotidase) are 2 major ectoenzymes that sequentially phosphohydrolyze adenine nucleotides, leading to adenosine generation.1 CD39 is primarily responsible for the phosphohydrolysis of ATP and ADP to AMP. CD73, which colocalizes with CD39 in membrane caveolae,2 hydrolyzes the phosphate group from AMP to generate adenosine. The adenine nucleotides and adenosine have been shown to play crucial role in many processes, including leukocyte extravasation,3 cardioprotection,4,5 and cellular immunoregulation.6 Signaling via specific adenosine receptor subtypes can mediate a variety of salutary vascular processes, ranging from suppression of vascular leakage and inflammation to promotion of vasodilation.
The experimental observation that CD39 confers a survival advantage to allografts and xenografts via thromboregulatory effects has been confirmed using cardiac transplantation models.7,8 CD73 sits at a more distal checkpoint in the adenosine generation cascade, but very little is known about CD73 as an effector limb of the immune or inflammatory response in the setting of cardiac transplantation. A number of studies have suggested that CD73-generated adenosine plays a beneficial role in modulating processes vital to successful transplantation, including endothelial permeability,9 neutrophil and leukocyte adhesion,10,11 the antithrombotic response,11 immunosuppression,6 and ischemic preconditioning.4 These actions of CD73 might occur either through dissipation of AMP, with attendant reductions in stimulation of putative AMP receptors, or dissipation of the downstream product of CD39 catalytic activity (AMP), which could theoretically increase flux of ADP through the CD39 catabolic pathway; dissipation of ADP would remove a potent stimulus to platelet aggregation and inflammation. As yet another alternative, the phosphohydrolytic actions of CD73 could drive production of adenosine, which could bind to its own cognate signaling receptor subtypes and hence affect vascular dilation and inflammation. These adenosine receptors (ARs) include A1AR, A2AAR, A2BAR, and A3AR, with each receptor having a unique tissue distribution, ligand affinity, and signal transduction pathway. The A1AR and A3AR inhibit adenylyl cyclase, whereas the A2AAR and A2BAR stimulate this effector system and therefore cAMP production.12 Little is known about the contribution of each subtype receptor to the events surrounding cardiac transplantation.
In the present work, studies examined the role of CD73 on development of cardiac allograft vasculopathy (CAV), the major impediment to the long-term survival of human cardiac allografts. CAV is a rapidly progressive form of atherosclerosis that often leads to reduced blood flow and ischemia of distal tissues. Histologically, CAV is identified by a combination of proliferative myoblasts, macrophages, and T lymphocytes, leading to the formation of a neointima. The mechanism for CAV development is considered to be multifactorial and likely includes both immunologic and nonimmunologic triggers.13 CD73, which sits at an interface position between immune modulator and vascular homeostatic mediator, is an excellent target to consider for involvement in (or protection against) CAV development. Although intimal proliferation mechanisms may differ, a recent study has shown that vascular neointimal formation is increased in CD73-deficient mice after carotid artery injury. In contrast, reconstitution of wild-type mice with CD73-deficient bone marrow did not exacerbate neointimal formation in the artery injury model, indicating that CD73 produced by resident nonhematopoietic cells, rather than by circulating cells, plays an active role in mitigating neointimal hyperplasia.14 However, because CD73 is expressed by leukocytes, as well as by tissue-resident cells including endothelial cells, we hypothesized that CD73 expressed on both local and circulating cells could contribute to preserving vascular homeostasis after cardiac transplantation, at least in part, by modulating the transit of leukocytes across inflamed endothelium.15 The experiments herein examine a role for CD73 and specific adenosine receptor subtypes in modulating leukocyte trafficking and ultimately, rejection and CAV following cardiac allotransplantation.
Materials and Methods
All experiments were performed according to the protocols approved by the University of Michigan Committee on Use and Care of Animals in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Experimental protocols are described in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
CD73-deficient mice (CD73−/−) of C57BL/6 (H-2b) background, a generous gift of Dr Linda F. Thompson, have been described previously.9 CD73+/+ littermates were used as the wild-type control. B10A (H-2a) and CBA/J (H-2k) mice were purchased from The Jackson Laboratories (Bar Harbor, Me).
Completely allomismatched murine heterotopic cardiac transplantation was performed for the present study, as we have described previously in detail.13 Two groups were used to study donor sources of CD73 (CD73+/+ or CD73−/− donors into recipients) and another 2 groups were used to study recipient sources (donors into CD73+/+ or CD73−/− recipients).
In Vitro Experiments
T lymphocytes were purified from splenocytes of CD73+/+ and CD73−/− mice (H-2b). The BALB/c (H-2d)–derived endothelial cell line bEnd.3 was obtained from American Type Culture Collection (Manassas, Va).
Database management and statistical analysis were performed with the Statview version 5.0 software (SAS institute Inc, Cary, NC). All values are expressed as means±SEM. Kaplan–Meier analysis was performed to evaluate graft survival, and survival differences were compared by a log-rank test. Comparisons among groups were performed with an unpaired Student t test or 1-way ANOVA where appropriate. Values of P<0.05 were considered statistically significant.
CD73 Deficiency in Either Donor or Recipient Mice Shortens Cardiac Allograft Survival
To observe the relationship between cardiac allograft survival and CD73 expression in donor and recipient cells, completely allomismatched heterotopic cardiac transplantation was performed using CD73−/− mice as either donors or recipients. CD73+/+ donor allografts survived between 13 and 20 days (16.3±1.0 days) after transplantation, whereas CD73−/− donor allografts survived for 10.5±0.6 days. CD73+/+ recipient graft survival ranged from 14 to 18 days (16.0±0.6 days), whereas all CD73−/− recipients acutely rejected the donor hearts in less than 14 days (9.0±0.7 days). CD73 deficiency in donors or recipients significantly decreased cardiac allograft survival (P=0.0013, P=0.0005, respectively; Figure 1A).
CD73 Deficiency Increases Graft Permeability Following Ischemia/Reperfusion Injury
Based on previous findings indicating that CD73 is critical for control of vascular leakage,9 we evaluated graft permeability in the ischemia/reperfusion (I/R) phase after transplantation. The permeability in cardiac allografts at 4 hours after transplantation was significantly increased in all cases in which CD73 was deficient either in the donor or the recipient (Figure 1B). These data indicate that there is an important role for CD73 in circulating cells, as well as cells resident in or surrounding the cardiac graft. Because the early inflammatory response during reperfusion is initiated by neutrophil infiltration into the allograft,16 we next evaluated the extent of the neutrophil infiltration using immunohistochemical Ly6G staining for direct neutrophil detection and a myeloperoxidase (MPO) activity assay. Compared with experiments in which CD73 was present in either donors or recipients, both the number of graft-infiltrating Ly6G-positive cells and the intragraft MPO activity were significantly increased in grafts involving CD73−/− mice (donors or recipients) (Figure 1C through 1E).
CD73 Deficiency Accelerates Acute Graft Rejection
At day 7 posttransplantation, we examined the histology of cardiac allografts to evaluate the acute alloimmune response (Figure 2A). Infiltration of mononuclear or polymorphonuclear cells with associated cardiomyocyte damage was greater and more diffuse, and the parenchymal rejection scores were significantly higher, in allografts involving CD73−/− versus CD73+/+ donors or recipients (Figure 2B). The numbers of infiltrating CD4-, CD8-, and CD11b-positive cells were significantly increased in experiments involving the transplantation of CD73−/− donors or recipients (Figure 2C through 2E).
CD73 Deficiency Increases Graft Expression of Cytokines, Chemokines, and Adhesion Molecules
At day 7 posttransplantation, we examined whether CD73 expression could modulate the mRNA expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, RANTES (regulated on activation normal T cell expressed and secreted), intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 in cardiac allografts. Compared with the CD73+/+ transplantations, mRNA expression of each of the above genes was significantly increased in the grafts involving CD73−/− donors or recipients (Figure 3).
CD73 Deficiency Aggravates Cardiac Allograft Vasculopathy and Graft Tolerance
To evaluate the severity of CAV development, we examined the histology of cardiac allografts at day 60 posttransplantation using elastin-stained tissue sections (Figure 4A). Compared with CD73+/+ transplantations, the severity of luminal occlusion in the graft coronary arteries involving CD73−/− donors or recipients was significantly increased (75.9±5.4 versus 46.6±2.5% and 70.5±6.5 versus 46.1±2.0%; P=0.0006 and P=0.0051, respectively; Figure 4B). Next, we investigated the impact of CD73 expression on humoral immunity in chronic rejection. CD73 deficiency in donors or recipients resulted in significantly higher levels of donor-reactive alloantibodies in the chronic rejection phase than in transplants between CD73+/+ donors and recipients (Figure 4C). To further assess the effect of CD73 expression on recipient antidonor cellular immune responsiveness, we evaluated cell proliferation of recipient lymphocytes using an ex vivo 1-way mixed lymphocyte reaction. The cell proliferation was significantly amplified in the transplantation of CD73−/− donors or recipients (Figure 4D).
Intragraft Expression of CD73 As It Relates to Adenosine Receptor Expression
After cardiac transplantation, recipient circulating cells infiltrate into allografts, thereby promoting the graft injury during the I/R phase and the phases of acute and chronic rejection. To elucidate the impact of CD73 expression in cardiac allografts, we measured mRNA and protein levels of CD73 in allografts at 4 hours, 7 days and 60 days after transplantation (Figure 5A, 5C, and 5D). In CD73+/+ donors or recipients, CD73 mRNA expression in cardiac allografts was markedly upregulated at 4 hours posttransplantation, upregulated but attenuated at day 7, and finally downregulated at day 60 posttransplantation. CD73−/− donors or recipients had lower levels of CD73 mRNA in all phases posttransplantation. Because extracellular adenosine produced by CD73 can signal through any of 4 ARs (A1AR, A2AAR, A2BAR, or A3AR), we next measured mRNA and protein levels of each AR in each phase after transplantation (Figure 5B through 5D). Intragraft A2BAR expression was upregulated in all groups at 4 hours and 7 days after transplantation, although CD73−/− donors or recipient groups had significantly lower levels of upregulation when compared with the CD73+/+ groups. At day 7 posttransplantation, intragraft A3AR expression was significantly upregulated in all groups, though CD73−/− donor or recipient groups showed significantly more upregulation of A3AR, compared with CD73+/+ groups. Although intragraft A2AAR mRNA expression was significantly downregulated throughout posttransplantation, there was no significant difference between the CD73+/+ and CD73−/− groups.
Genetic Deletion or Pharmacological Blockade of CD73 Promotes Activation of Endothelial Cells and T Lymphocytes In Vitro
To further elucidate the effects of CD73 on interactions between endothelial cells and T lymphocytes found in cardiac allografts, we performed an allomismatched coculture of endothelial cells (H-2d) and lymphocytes (H-2b; CD73+/+ or CD73−/−) with or without α1β-methylene ADP (APCP) in vitro. First, we evaluated the contribution of T lymphocytes to endothelial cells on the simple coculture experiments. After a 72-hour coculture, mRNA expressions of endothelial cell TNF-α and VCAM-1 were significantly upregulated in the coculture with CD73−/− T lymphocytes as compared with CD73+/+ T lymphocytes (P=0.0195 and P=0.0270, respectively; Figure 6A). The addition of APCP significantly enhanced these upregulations in the coculture with CD73−/− T lymphocytes (TNF-α and VCAM-1; P=0.0436 and P=0.0329, respectively; Figure 6A). Next, we evaluated the contribution of endothelial cells to T lymphocytes using transmigration coculture experiments. After a 24-hour coculture, the number of T lymphocytes that had transmigrated into endothelial cells significantly increased in CD73−/− T lymphocytes compared with CD73+/+ T lymphocytes (P=0.0004; Figure 6B), and APCP significantly enhanced the transmigration in the coculture of CD73−/− T lymphocytes (P=0.0353; Figure 6B). IFN-γ mRNA expression in the posttransmigrated T lymphocytes was significantly upregulated in all experimental groups when compared with pretransmigrated T lymphocytes, and the IFN-γ mRNA upregulation was significantly higher in the coculture of CD73−/− T lymphocytes compared with the coculture of CD73+/+ T lymphocytes (P=0.0042; Figure 6C). There was no significant enhancement of the IFN-γ mRNA upregulation when APCP was added to the coculture of CD73−/− T lymphocytes.
Influence of Exogenous Adenosine Receptor Modulators on Cardiac Transplantation
To further evaluate the CD73-mediated contribution of specific AR subtypes during allograft rejection or CAV, we performed heterotopic cardiac transplantation using AR modulators given intraperitoneally. First, we examined which AR is acutely responsible for increased vascular leakage in the murine heterotopic cardiac transplantation model. At 4 hours after transplantation, graft permeability tended to increase for each AR antagonist applied (A2B, MRS1754>A2A, SCH58261 > A1, DPCPX>A3, MRS1191), although only the A2BAR antagonist MRS1754 caused a statistically significant increase in vascular leakage (P<0.0001 versus nontreatment control by ANOVA; Figure 7A). Based on the results of this graft permeability assay, we selected A2AR agonists (A2A, CGS21680; A2B, N-ethylcarboxamidoadenosine [NECA]) to establish their potential effects on cardiac allograft rejection or vasculopathy. Both CGS21680 and NECA treatments significantly increased graft survival compared with nontreatment controls, although the survival in NECA-treated recipients was significantly longer than that in CGS21680-treated recipients. When CD73−/− donors or recipients were studied, NECA treatment significantly increased graft survival compared with wild-type nontreatment controls, whereas there was no significant increase in survival between CGS21680 treatment and wild-type nontreatment (Figure 7B). The next set of experiments was designed to measure the role of the A2BAR in CAV. In both CD73+/+ and CD73−/− donors or recipients, the severity of luminal occlusion at day 30 posttransplantation was significantly attenuated by NECA treatment, compared with wild-type nontreatment controls (Figure 7C and 7D). Taken together, these data suggest that A2BAR strongly contributes to CD73-mediated allograft protection in murine heterotopic cardiac transplantation.
CD73 effects the terminal phosphohydrolysis of AMP, which in turn generates adenosine. In the present experiments, the use of CD73−/− mice as either donors or recipients of heterotopic cardiac allografts allowed us to demonstrate the critical roles that CD73 plays in allograft survival and CAV prevention. Comparisons of CD73−/− mice with CD73+/+ mice showed less rejection and diminished vasculopathy when CD73 was present. These experiments indicate that CD73 promotes graft barrier function, suppresses the inflammatory response, and dampens alloeffector immune responses including the trafficking of leukocytes across allogeneic endothelium. These results could be attributed to the dissipation by CD73 of AMP, resulting in reduced stimulation of putative AMP receptors or by removing the terminal CD39 reaction product (AMP), thereby accelerating ADP catabolism and reducing the procoagulant and proinflammatory effects of ADP. However, it is also quite likely that generation of adenosine as a byproduct of AMP phosphohydrolysis could participate in these salutary vascular effects.
CD73 contributes in a major way to local adenosine concentrations especially at the vascular intimal surface where it is generated. Adenosine in the local vascular microenvironment is known to suppress inflammation, promote vasodilation, and inhibit vascular leakage, each action dependent on the receptor subtype to which it predominantly binds. Use of specific adenosine receptor agonists and antagonists in the present experiments allow us to conclude that the predominant vascular effects of CD73 in murine cardiac allotransplantation are mediated via the A2BAR. When an A2AAR agonist was given to recipients, graft survival was slightly prolonged (ie, rejection diminished) regardless of CD73 genotype. Interestingly, we have shown that intragraft A2AAR mRNA expression was significantly downregulated, possibly suggesting that it might have less involvement in neointimal formation after cardiac allotransplantation. When an A2BAR agonist was given to recipients, graft survival was markedly prolonged (ie, rejection diminished) regardless of CD73 genotype. These acute rejection experiments, indicating a dominant immune suppressive role mediated via the A2BAR, led us to investigate the effects of chronic A2BAR stimulation on development of CAV. Chronic A2BAR stimulation resulted in a marked suppression of CAV development, and this rescue occurred regardless of whether CD73 was itself absent from the donor or recipient genotype. Taken together, these data clearly demonstrate an antirejection and anti-CAV role for CD73, which is likely to be mediated proximately by the local generation of adenosine and its actions predominantly via the A2BAR.
The microvascular endothelium is a dynamic barrier that regulates the exchange of fluid, solutes, and cells between the vessel lumen and tissues. I/R injury triggers an endothelial barrier dysfunction, characterized by neutrophil infiltration and increased graft permeability, that plays a significant role in the pathophysiology of CAV development in cardiac transplantation. In the present study, a lack of CD73 in donors or recipients attenuated A2BAR expression and promoted an inflammatory cascade involving enhanced graft permeability, neutrophil infiltration, and subsequent MPO release in cardiac allografts during the I/R phase. Previous studies have also shown that CD73-mediated activation of the A2BAR is critical for the maintenance and regulation of endothelial barrier function during hypoxia.4,17 During episodes of inflammation, transendothelial neutrophil infiltration into the tissues has the potential to disturb the barrier function via limitation of the A2BAR activation.17,18 Interestingly, we observed a positive correlation between CD73 and A2BAR mRNA expression in cardiac allografts at 4 hours after transplantation (data not shown).
A2BAR expression in cardiac allografts is still upregulated in the acute rejection phase and, to a lesser degree, in the I/R phase in CD73-deficient transplantations. It has been reported recently that inflammatory cytokines such as IL-1, TNF-α, and IFN-γ modulate A2BAR expression and function on microvascular endothelial cells19 and that the A2BAR protects against vascular lesion formation via regulation of inflammatory cytokines, chemokines, and adhesion molecules.20,21 We have shown here that CD73 expression in donors or recipients plays an important role in regulating those inflammatory factors in the acute rejection phase of cardiac transplantation. Therefore, our results indicate that activation of A2BAR via CD73-generated adenosine modifies the production of inflammatory molecules. Such interactions could be an important mechanism for dampening endothelial activation and the inflammatory response in the acute allograft rejection.
A3AR expression in cardiac allografts is also upregulated to a greater degree in CD73-deficient transplantation during the acute rejection phase. Interestingly, we observed a negative correlation between CD73 and A3AR mRNA expression (data not shown). Although much attention has focused on the effects of activating A3AR in the heart, the role played by A3AR in apoptosis remains unclear because some studies support a protective role for the receptor, whereas others indicate that it induces myocardial apoptosis.22,23 In the present study, CD73 deficiency in donors or recipients promoted apoptosis in cardiac allografts during the acute rejection phase (data not shown). The effects of A3AR activation appear to depend on the pattern of receptor activation (endogenous or exogenous) and drug administration (dose or duration), and we believe that this relationship between CD73 and A3AR in cardiac transplantation may be explained as a compensatory and protective upregulation of A3AR in response to apoptosis or to a deficiency of CD73. Further studies are needed to further elucidate this complex relationship.
Our present research supports earlier work that showed that adenosine generated by CD73 on T lymphocytes mediates immune suppression in skin allografts and in vitro experiments.6 Using histological studies, we have demonstrated that CD73 deficiency in donors or recipients correlates with intense acute rejection, as evidenced by impressive graft infiltration of both CD4- and CD8- positive T lymphocytes in the acute rejection phase following transplantation. IFN-γ, which enhances antigen presentation and promotes cellular immunity by activated macrophages, natural killer (NK) cells, and Th1 lymphocytes,24 was also significantly upregulated in CD73-deficient cardiac allografts. A critical event during the progression of acute allograft rejection is the recruitment and transmigration of alloantigen-primed CD4- and CD8- positive T lymphocytes into the graft, followed by the release of cytokines by both endothelial cells and T lymphocytes.25 Our in vitro coculture experiments demonstrated that both genetic deletion and pharmacological blockade of CD73 promote the transendothelial migration of T lymphocytes and upregulate expression of TNF-α, VCAM-1, and IFN-γ. In addition, the present studies show that CD73 deficiency in donors or recipients resulted in an increase in the production of donor-reactive alloantibodies and T-lymphocyte proliferation in the chronic rejection phase of cardiac transplantation. Taken together, these results indicate that CD73 regulates allogeneic interactions between endothelial cells and T lymphocytes and thus plays an immunomodulatory role that promotes allograft survival.
T lymphocytes may not be the only effector cells relevant to cardiac allograft rejection that are modulated by CD73. NK cells are a type of cytotoxic lymphocytes that are able to kill targets cells without prior exposure to antigen. Because their lethal effector functions are triggered without prior antigen priming, they are considered to be an integral constituent cell of the innate immune system and, hence, relevant to cardiac allograft rejection or vasculopathy. CD73 is indeed expressed by NK cells, as well as endothelial cells and other leukocytes.26 Recently, Uehara et al27 demonstrated that NK cells can promote CAV in a murine cardiac transplantation model; however, these cells do so in a milieu that requires T cells and other alloeffector mechanisms. Interestingly, the interaction between NK cells and T cells that contributes to CAV likely involves IFNs and other cytokines. In the present study, we focused on the immunologic crosstalk between endothelial cells and T cells in transplant alloresponses. Although we did not specifically evaluate NK cell activity, these cells could indeed be activated in cardiac allografts because intragraft IFN-γ mRNA expression was upregulated at 7 days posttransplantation. IFN-γ mRNA levels were significantly increased in the allografts in which CD73 was absent in either the implanted graft or the recipient compared with wild-type transplants. Therefore, NK cells in CD73-deficient recipients might contribute to CAV development. Further work would need to be performed to understand whether the biological function of CD73−/− NK cells are as same as those of CD73+/+ NK cells. Complicating the prediction even further, 1 recent article has shown that NK cells promote transplant tolerance by killing donor antigen-presenting cells.28 Further studies would be needed to understand a role of NK cells in chronic rejection, especially it relates to CD73 on NK cells or their immune targets.
Our study involved both donor (endothelial and parenchymal cells) and recipient (leukocytes) sources of CD73, thus allowing us to explore the contributions that each source makes to the overall transplant milieu. In allotransplant settings, recipient leukocytes attack donor endothelial cells, resulting in acute rejection characterized by endothelial injury and dysfunction, altered endothelial permeability, and neointimal formation (vasculopathy). The alloimmune injury induced by cross–major histocompatibility complex barrier transplantation can be a sustained and severe endothelialitis, which differs from that in mechanical vascular injury (alloeffector mechanisms do not pertain). In general, the alloimmune vascular injury caused by transplantation is quite brisk and severe. This is an important difference when one considers the work by Zernecke et al,14 in which following carotid wire injury, there was no significant difference in neointimal formation when CD73-null marrow was transplanted into wild-type recipients. Based on knowledge of the effects of CD73 and downstream adenosine and its signaling mechanisms in immune regulation, one could speculate that transplantation of CD73-null marrow might increase allograft vasculopathy. The reasoning behind this hypothesis is that CD73-dependent adenosine generation induces a form of leukocyte–endothelial cell crosstalk that results in reduced leukocyte adhesion to the endothelium and decreased transmigration into tissues in the setting of certain types of inflammatory responses.10 In the present transplant experiments, there was an opportunity to discern whether there was a local vascular effect of CD73 based on its expression on circulating leukocytes or whether the effect was attributable to CD73 present on cells resident in the graft at the time of transplantation. Our data clearly demonstrate that CD73 in either or both locations can play a role in restoring vascular homeostasis to cardiac allografts.
Another recent study has shown that both recipient- and donor-derived cells contribute to the regeneration of damaged cells in cardiac allografts.29 The interaction between endothelial cells and lymphocytes attenuates CD73 activity,30 whereas CD73-dependent adenosine generation induces a novel form of leukocyte–endothelial cell crosstalk that results in reduced leukocyte adhesion to the endothelium and decreased transmigration into tissues in the setting of hypoxia-associated inflammatory responses.10 Therefore, it is possible that the intragraft level of CD73 expression effects on the outcome of cardiac allografts. Because recipient-derived cells infiltrate into allografts over time and injury to the donor-derived cells in allografts is a progressive process, our model results in a total CD73 expression in cardiac allografts that fluctuates with time. It is interesting to note that allotransplants of CD73−/− donors or recipients were found to have lower levels of CD73 expression throughout the posttransplantation period, resulting in increased cardiac graft damage. In our cardiac isograft transplantation experiment, the homologous combination of CD73−/− donors and recipients resulted in an accelerated inflammatory response when compared with a heterologous combination of CD73−/− donors and CD73+/+ recipients and vice versa (T. Hasegawa, D.J. Pinsky, unpublished data, 2007). Our in vitro coculture studies involving CD73−/− T lymphocytes and CD73+/+ endothelial cells supplemented with APCP (an inhibitor of CD73) significantly enhanced the transendothelial migration of T lymphocytes, as well as TNF-α and VCAM-1 expression. Thus, CD73 expressed on both local and circulating cells could contribute to preserving vascular homeostasis after cardiac transplantation.
In summary, these experiments demonstrate that both local and circulating CD73 contribute to allograft-protection in cardiac transplantation, leading to improved allograft survival and protection against CAV development. Mechanisms underlying this protection likely include (1) the maintenance of graft barrier function resulting from a concurrent upregulation of A2BAR in the I/R phase; (2) suppression of the inflammatory response, possibly attributable to an upregulation of A2BAR; and (3) suppression by CD73 of the transit of effector leukocytes across graft endothelium. These studies point to CD73 as residing at the nexus of inflammatory and vascular reactions that can protect a vulnerable graft and its vasculature from immune attack.
We gratefully acknowledge Dr Linda F. Thompson for critical reading of the manuscript, as well as insightful comments.
Sources of Funding
This work was supported by the A. Alfred Taubman Medical Research Institute, the Ruth Professorship, the Scleroderma Research Foundation, the Cardiovascular Medical Research and Education Fund, and NIH grants R01HL055397, R01HL085149, R01HL086676, P01HL089407, and T32HL007853.
Original received May 27, 2008; revision received October 27, 2008; accepted October 30, 2008.
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