Magdi H. Yacoub

Direct recognition of SLA- and HLA-like class II antigens on porcine endothelium by human T cells results in T cell activation and release of interleukin-2.

To investigate whether human T cells can directly recognize pig xenoantigens, highly purified human CD4+ and CD8+ T cells were incubated with pig aortic endothelial cells (PAEC). The response was measured by [3H]thymidine uptake and release of bioactive interleukin-2. A detailed examination of MHC expression by cultured PAEC and tissue sections of porcine aorta and heart showed porcine endothelial cells (EC) to be constitutively positive for SLA class II and antigens that crossreact with HLA class II molecules. Low level expression of B7 receptors was detected by binding of both human and mouse CTLA-4-Ig to untreated PAEC, which was enhanced significantly by treatment with recombinant porcine interferon-gamma. Human T cells, purified by positive selection and residual DR+ cells removed by lymphocytolysis, were shown to be functionally free of monocytes. Untreated PAEC elicited strong proliferation by human CD4+ T cells: CD8+ T cells also proliferated, but more weakly. This response was inhibited by CTLA-4-Ig. Blocking studies were performed with mAbs that bind to PAEC and not human EC (MSA3, TH16B), an mAb that binds to human and porcine EC (DA6.231), and L243, which binds to human and not porcine EC. The proliferative response of CD4+ T cells to PAEC was inhibited significantly by mAbs against swine and human determinants. In contrast, the response of CD4+ T cells to human EC was inhibited only by mAbs against human determinants. Experiments that directly compared the CD4+ and CD8+ T cell responses to PAEC and the human EC line EAhy.926, both with and without prior treatment with species-specific interferon gamma, demonstrated greater proliferation and 5-10 times more interleukin-2 in response to pig EC than to human EC.

Expression of human ecto-5'-nucleotidase in pig endothelium increases adenosine production and protects from NK cell-mediated lysis

Ecto‐5′‐nucleotidase (E5′N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. We evaluated whether expression of human E5′N on pig endothelial cells (EC) attenuates human NK cell‐mediated cytotoxicity. A pig EC line was stably transfected with human E5′N and human NK cell adhesion and cytotoxicity toward pig EC cultures was measured by flow cytometry and intracellular enzyme release. E5′N activity in pig EC lysates increased from 0.68 ± 0.07 to 1013 ± 293 nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37 ± 0.05 to >300 nmol/min/mg protein in non‐transfected and transfected cells, respectively. The rate of adenosine production in transfected cells increased also with ATP as the extracellular substrate. Cytotoxicity of human NK cells was reduced from 10.7 ± 0.4% and 11.1 ± 1.1% with non‐transfected pig EC to 5.2 ± 0.2% and 5.0 ± 0.2% in transfected cells with 50 μM and 250 μM AMP, respectively. Reduction of cytotoxicity in E5′N‐transfected EC was abolished by the E5′N inhibitor and was mimicked in non‐transfected EC by the addition of adenosine, demonstrating the key role of adenosine produced by E5′N in inhibiting NK cell cytotoxicity. We suggest that overexpression of E5′N in EC of transgenic pigs is a possible strategy to ameliorate rejection after xenotransplantation.

Heart Transplanation: The End of the Beginning

Heart transplantation has passed through several stages of evolution in a relatively short period of time. About 60 years ago it was referred to as a ‘fantasy for the future’. Almost exactly 40 years ago, the clinical introduction of the procedure was greeted with great enthusiasm by both patients and the public and to some extent by scientists. This was followed shortly afterward by manifest disappointment to the extent that it was regarded as ‘experimental’, ‘unethical’ and ‘not worthy of further development’ due to many factors, including the perception that the dramatic early benefit to the patient is temporary and can only be measured in days, weeks or months. Fortunately, sustained efforts in few institutions served to prove that heart transplantation is compatible with long-term survival beyond 20 years in a significant minority of patients as documented by Deuse et al. (Stanford University) (1) in this issue of the journal.

ADENINE INCORPORATION IN HUMAN AND RAT ENDOTHELIUM

Adenine (ADE) reutilisation is an important pathway of adenylate pool regeneration. Data on the rate of this process in different types of cells, its regulation and the importance of species differences is limited. In this study we evaluated adenine incorporation rate and the effect of metabolic factors on this process in human and rat endothelium and compared it to adenine phosphoribosyltransferase (APRT) activity. Microvascular endothelial cells from human (HE) and rat (RE) hearts and a transformed human microvascular endothelial cell line (HMEC-1) were investigated. The rate of adenine incorporation into the adenine nucleotide pool under control conditions was 3.1+/-0.3, 82.8+/-11.1 and 115.1+/-11.2 pmol/min per mg protein for HE, RE and HMEC-1, respectively. In the presence of 2.5 mM ribose or elevated inorganic phosphate concentration in the medium (4.8 mM), few changes were observed in all types of cells. In the presence of both ribose and high inorganic phosphate, the rate of adenine incorporation for RE and HMEC-1 was not significantly different from control, while in HE the rate of adenine incorporation into adenine nucleotides was increased by 75%. Activities of APRT in RE and HMEC-1 were 237.7+/-23.2 and 262.0+/-30.6 pmol/min per mg protein respectively while the activity in HE was markedly lower 48.7+/-3.0 pmol/min per mg protein. In conclusion, nucleotide synthesis from adenine seems to be a slow process in human cardiac microvascular endothelium but it is fast and efficient in rat heart microvascular endothelial cells. Low APRT activity in normal human endothelial cells seems to be the most likely mechanism for this. However, adenine incorporation rate and APRT activity could be greatly enhanced in human endothelium, as demonstrated in transformed cells.

Nucleotide metabolic mismatches in mammalian hearts: implications for transplantation

Introduction Human donor organ shortages have led surgeons and scientists to explore the use of animals as alternative organ sources. Acute thrombovascular rejection (AVR) is the main hurdle in xenotransplantation. Disparities in nucleotide metabolism in the vessels of different species may contribute significantly to the microvascular component of AVR. Methods We evaluated the extent of nucleotide metabolism mismatch in selected organs and endothelial cells of different mammals with particular focus on the changes in activity of ecto-5’-nucleotidase (E5’N) elicited by exposure of porcine hearts or endothelial cells to human blood (ex vivo) or human plasma (in vitro). Results E5’N activity in the rat heart was significantly higher than in other species. We noted a significant difference (p<0.001) in E5’N activity between human and pig endothelial cell lines. Initial pig aortic endothelial E5’N activity decreased in vitro after a three-hour exposure to human and porcine plasma while remaining constant in controls. Ex vivo perfusion with fresh human blood for four hours resulted in a significant decrease of E5’N activity in both wild type and transgenic pig hearts overexpressing human decay accelerating factor (p<0.001). Conclusions This study provides evidence that mismatches in basal mammalian metabolic pathways and humoral immunity interact in a xenogeneic environment. Understanding the role of nucleotide metabolism and signalling in xenotransplantation may identify new targets for genetic modifications and may lead to the development of new therapies extending graft survival.

Exposure to human blood inactivates swine endothelial ecto-5′- nucleotidase

Ecto-5′-nucleotidase (E5′N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5′N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5′N activity to 62 and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5′N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5′N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.