Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation

The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p-PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p-PBMC with human platelets to assess the capacity of p-PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 microg/ml), were performed with platelets in platelet-rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p-PBMC or human PBMC (h-PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p-PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p-PBMC or h-PBMC. Aggregation of human platelets in PRP, induced by p-PBMC, was inhibited when DAPA (100 microm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p-PBMC to aggregate human platelets. No deposition of C3c fragments on p-PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p-PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.     

Ajoene inhibits the activation of human endothelial cells induced by porcine cells: implications for xenotransplantation

Abstract: Ajoene, is an organosulfur compound derived from garlic that strongly inhibit platelet aggregation, proliferation of human lymphocytes induced by phytohemagglutinin, and in general, blocks membrane-mediated signaling of cell activation. As a thrombotic microangiopathy frequently complicates procedures designed to induce pig-to-baboon chimerism by infusion of large amounts of pig progenitor cells in baboons, it was thought that ajoene might be useful to prevent such complication. For such purpose, we studied the effects of ajoene on the activation of human umbilical vein endothelial cells (HUVEC) induced by pig peripheral blood mononuclear cells (p-PBMC). Co-cultures of p-PBMC with HUVEC results in activation of the HUVEC as shown by over-expression of E-selectin and vascular cells adhesion molecule-1 (VCAM-1). Ajoene (25 μ m ) strongly inhibits HUVEC activation induced by tumor necrosis factor-α (TNF-α) or p-PBMC as shown by a down regulation of VCAM-1 and of E-selectin expression. After 5 or 8 h of pre-treatment with Ajoene, HUVEC incubated with TNF and p-PBMC showed an E-selectin or VCAM-1 expression, respectively, at levels similar to the positive control indicating that the inhibitory effect is transient. Ajoene at concentration of 25 μ m or lower did not affect HUVEC viability. Based on the finding that Ajoene has a strong, although transient, inhibitory effect on the activation of the endothelium induced by pig cells and its known anti-platelet activity, it is suggested that this garlic compound could be useful to prevent the development of microangiopathy and thrombotic disorders seen in primates infused with pig cells.     

The pathobiology of pig‐to‐primate xenotransplantation: a historical review

The immunologic barriers to successful xenotransplantation are related to the presence of natural anti‐pig antibodies in humans and non‐human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose‐α1,3‐galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti‐pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3‐galactosyltransferase gene‐knockout [GTKO] pigs) and express one or more human complement‐regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade‐based immunosuppressive regimen, prevents early antibody‐mediated and cellular rejection. However, low levels of anti‐non‐Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation‐anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation‐regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non‐human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.     

Platelet-mediated activation of endothelial cells: implications for the pathogenesis of transplant rejection

BACKGROUND: Platelets exert their normal functions at sites of endothelial disruption by plugging discontinuities in blood vessels and secreting products that promote thrombosis, inflammation, and the healing of wounds. Whether platelets might induce these changes in xenograft blood vessels, leading to development of acute vascular rejection, has been uncertain. METHODS: To examine the role of human platelets in modulation of xenograft endothelium, pig endothelial cells were treated with human platelets. RESULTS: Treatment of quiescent porcine endothelial cells with human platelets modulated the endothelial cells. Whereas resting human platelets caused little change in normal porcine endothelial cells, platelets activated with small amounts of thrombin induced striking changes in the endothelial cells, including the induction of tissue factor activity, the expression of E-selectin, and the secretion of endothelin-1. These changes were induced, at least in part, by interleukin-1 (IL-1) associated with the platelet surface and were modified by the secretion of transforming growth factor-beta (TGF-beta). CONCLUSION: These findings may explain how the activation of platelets at an early point in the rejection of vascularized organ xenografts or in chronic diseases might contribute to thrombotic, ischemic, and inflammatory changes characteristic of an organ xenograft undergoing rejection.     

Human CD154 induces activation of porcine endothelial cells and up-regulation of MHC class II expression

BACKGROUND: CD40 is expressed on a number of antigen-presenting cells and also on vascular endothelium. It has been shown that engagement of CD40 on vascular endothelium by CD154 on platelets and CD154-bearing cell lines leads to the induction of adhesion molecule expression. Having cloned porcine CD40, and shown that it is capable of binding human CD154, we investigate whether human CD154 can activate porcine endothelial cells (EC) through CD40 ligation. METHODS: Human Jurkat clone D1.1 (CD154+), or clone E6.1 (CD154-), were co-cultured with EC from pig aorta and human aorta and umbilical vein for various times in the presence or absence of blocking antibody to CD154. RESULTS: Human and pig EC were shown to express CD40 by flow cytometry by using soluble human CD154 (CD154Ckappa). Co-culture of pig EC with CD154-expressing Jurkat D1.1 cells led to the induction of E-selectin by 6 hr (peak 24 hr) and vascular cell adhesion molecule-1 (VCAM-1) by 6 hr (peak 48 hr). Similar results were also observed with human EC. Porcine EC were induced to up-regulate major histocompatibility complex class II at 24 hr by co-culture with Jurkat D1.1 cells through a CD40-dependent mechanism. In contrast, no up-regulation was observed on human EC. CONCLUSIONS: A number of cells can express CD154, including T cells, natural killer cells, and platelets, and these could signal graft EC through the CD40 pathway. These results demonstrate a possible role for the CD40 pathway in the activation of vascular endothelium in the rejection of porcine xenografts.     

The fate of human platelets perfused through the pig liver: implications for xenotransplantation

Burlak C, Paris LL, Chihara RK, Sidner RA, Reyes LM, Downey SM, Tector AJ. The fate of human platelets perfused through the pig liver: implications for xenotransplantation.
Xenotransplantation 2010; 17: 350–361. © 2010 John Wiley & Sons A/S. Abstract: Background: Pig liver xenotransplantation could offset the shortage of livers available for orthotopic liver transplantation. Studies in pig and baboon liver xenografts revealed the main obstacle to be a lethal thrombocytopenia that occurred within minutes to hours of transplantation. Methods: We have created a model of xenotransplantation‐induced thrombocytopenia using ex vivo pig liver perfusion with human platelets. Thrombocytopenia was examined using fluorescently labeled platelets during the ex vivo perfusion and coculture with primary liver sinusoidal endothelial cells (LSEC). Results: Ex vivo liver perfusion revealed that 93% of human platelets were removed from circulation after 15 min. Endothelial cells and platelets were not activated based on tissue factor release into the perfusate. Biopsies from the ex vivo perfusion at 15 and 30 min and in vitro analysis indicated that human platelets are phagocytosed by pig LSEC and degraded in phagosomes. Sixty to 120 min after the addition of platelets to the ex vivo perfusion system, we observed platelet fragments and degraded platelets in hepatocytes. Platelet phagocytosis was not mediated by opsonization as Fc blocking had no effect on platelet phagocytosis. In vitro uptake of human platelets by primary LSEC cultures peaked at 15 min followed by a greater than 55% decrease in platelet fluorescence after 3 h. Primary pig LSEC phagosomes containing human platelets were colocalized with lysosomes positive for lysosome‐associated membrane protein‐1 (LAMP1), indicating the formation of mature phagosomes within pig LSEC. Conclusions: Our observation of pig LSEC phagocytosis of human platelets describes a novel mechanism of large‐particle uptake in the liver. The creation of a model system to study xenotransplantation‐induced thrombocytopenia makes possible the investigation into mechanisms that mediate platelet loss.     

The role of natural IgM in the hyperacute rejection of discordant heart xenografts.

The mechanism of xenograft hyperacute rejection in discordant species combinations remains controversial. The purpose of this work was to study the role of natural antibodies in the hyperacute rejection of guinea pig hearts transplanted into rats, a highly discordant combination. This study was conducted in vitro, ex vivo, and in vivo. The endothelial cells of the graft being the first targets damaged in the process of hyperacute rejection, the binding of rat natural antibodies to guinea pig endothelial cells was studied by immunofluorescence. The study was carried out in vitro on guinea pig endothelial cells in culture, and ex vivo on isolated guinea pig hearts perfused with either rat serum or immunoglobulins or immunoglobulin fragments bearing the antigen-binding site. In vitro and ex vivo, rat natural IgM were found to bind specifically to guinea pig endothelial cells, since IgM fragments bearing the antigen-binding site (Fab mu and Fab' mu) could be detected on these cells. IgM fragments were able to inhibit the fixation of native IgM molecules. In contrast, rat IgG only bound to endothelial cells through Fc portions. Thus rat natural IgM might play a role in hyperacute rejection by binding to the graft endothelial cells and triggering the complement cascade activation. In order to test the role of natural IgM in vivo, isolated guinea pig hearts were first perfused with rat Fab' mu, which inhibit the binding of IgM and are unable to activate the complement cascade. These hearts were then transplanted into Lewis rats. The rejection time of Fab' mu-perfused guinea pig hearts was prolonged compared with hearts perfused with buffer or IgG F(ab')2. Therefore, in the guinea pig to rat combination, preventing the binding of the recipient's natural IgM to the graft endothelium delays the hyperacute rejection. In addition, natural IgM are likely to play a greater role than natural IgG.     

Progress in xenotransplantation following the introduction of gene-knockout technology

The production of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pigs has overcome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies that has inhibited progress in pig-to-primate organ xenotransplantation for many years. Survival of GT-KO pig organs in nonhuman primates is currently limited by the development of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potential causative factors include vascular endothelial activation from preformed anti-nonGal antibodies or cells of the innate immune system that recognize nonGal pig antigens directly, and coagulation dysregulation associated with molecular incompatibilities between pig and primate. Carefully isolated pancreatic islets from wild-type (genetically unmodified) adult pigs express minimal Gal epitopes, allowing survival sometimes for weeks or months after transplantation into nonhuman primates receiving immunosuppression directed only at T-cell function. However, there is a considerable immediate loss of islets, probably related to activation of coagulation and complement cascades. Further genetic manipulation of organ-source pigs is therefore required to overcome these problems. GT-KO pigs expressing a human complement-regulatory protein, e.g. decay-accelerating factor, and/or an 'anti-coagulant' gene, e.g. human tissue factor pathway inhibitor, might prevent the change in vascular endothelium from an anti-coagulant to a procoagulant phenotype, and protect the islets from early loss.     

Antibodies to human adhesion molecules and von Willebrand factor: In vitro cross-species reactivity in the xenotransplantation setting

Abstract: Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD31, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD 106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.     

Platelet adherence to isolated rat hepatic sinusoidal endothelial cells after cold preservation

BACKGROUND: Platelet adhesion to hepatic sinusoidal endothelial cells (SEC) is a major mechanism of cold preservation injury. This study was performed to determine whether cold preservation leads to adhesion between SEC and platelets in the absence of other cell types, whether adherent platelets become activated upon adhesion, and whether there is increased expression of the platelet receptor von Willebrand factor (vWF) on cold preserved SEC. Because we previously showed that cold causes actin disassembly and matrix metalloprotease (MMP) secretion by SEC, we also questioned whether these events are related to increased adhesion of platelets to SEC after preservation. METHODS: Isolated SEC were cold preserved for 8 hr and rewarmed briefly. Biotinylated platelets were added to the plates and adhesion was assessed. Activation of platelets was determined by staining with antibody to P-selectin. Expression of vWF was assessed with a specific antibody. RESULTS: Cold preservation induced increased adhesion of platelets to SEC in the absence of other cell types. Adherent platelets were activated. Preservation increased the expression of vWF on SEC. Pretreatment with phalloidin or treatment with MMP inhibitors partly prevented platelet adhesion and activation, as well as vWF expression. Treatment of SEC at 37 degrees C with recombinant human MMPs for 24 hr also rendered the SEC more adherent for unactivated platelets. CONCLUSION: Cold preservation of SEC results in increased expression of the platelet receptor vWF, increased platelet adhesion, and platelet activation. This is at least partly mediated by actin disassembly and MMP secretion.     

Relative effects of GAL+ and GALlow/- porcine hematopoietic cells on primate platelet aggregation and endothelial cell activation: implications for the induction of mixed hematopoietic chimerism in the pig-to-primate model

Abstract: Background: The induction of porcine hematopoietic cell chimerism in preconditioned baboons has been hampered by the development of thrombotic microangiopathy. As pigs that lack expression of Galα1,3 Gal (Gal) may become available in the near future, we have explored the effects of porcine hematopoietic cells that express low or no Gal (Gal^low/?) on baboon platelet aggregation and on human umbilical vein endothelial cell (HUVEC) activation. Methods: Porcine mobilized peripheral blood progenitor cells (PBPC; Gal⁺) and bone marrow mononuclear cells (BM; Gal⁺ or Gal^low/?) were investigated for their potential to (i) induce aggregation of baboon platelets, and (ii) to activate endothelial cells as measured by increased expression of vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and E‐selectin on HUVEC. α‐Galactosidase‐treated PBPC were also investigated for their effect on platelet aggregation. Results: Gal⁺ PBPC and Gal⁺ BM cells (10⁷) induced aggregation of baboon platelets by 42 and 31%, respectively, whereas Gal^low/? BM cells did not induce any platelet aggregation. α‐Galactosidase‐treated PBPC induced less platelet aggregation than untreated PBPC. Gal⁺ PBPC and Gal⁺ BM cells (10⁷) increased expression of VCAM‐1, ICAM‐1 and E‐selectin on HUVEC, whereas Gal^low/? BM cells did not. Conclusions: In contrast to Gal⁺ PBPC or BM, Gal^low/? BM cells do not induce aggregation of baboon platelets or activate HUVEC. The induction of tolerance through mixed hematopoietic cell chimerism may be facilitated when α‐galactosyltransferase‐knockout pigs become available.     

Adult porcine islets produce MCP-1 and recruit human monocytes in vitro

Abstract:  Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4⁺ T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1β and TNF-α. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of α- and β-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1β nor TNF-α were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1β and TNF-α were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.     

Identification and characterization of monoclonal antibodies that partially block human natural antibody binding to pig endothelial cell xenoantigens

Abstract: The shortage of human donors for clinical transplantation has led to a serious consideration of the use of non-human species as organ donors. The major barrier to the clinical use of xenografts from species such as the pig in human transplantation has been the aggressive nature of the immune-mediated rejection of the graft. We have recently identified the molecular weights of several endothelial cell surface proteins that may be targets of human antibody-mediated responses to pig aortic endothelial cells (PAEC). In this series of experiments, we produced a panel of rat monoclonal antibodies (Mabs) to PAEC in an effort to identify Mabs that detect pig xenoantigens. Mabs were selected based on flow cytometric binding to PAEC, pig platelets, and various pig cell lines, including a pig kidney cell line (LLC-PK1) reported to react with human natural antibodies (HNA). Eleven of the eighty-three antibodies produced were cytotoxic for PAEC. Six of the cytotoxic clones recognized a 44 kDa protein and two of the clones recognized a 115 kDa protein expressed on the surface of PAEC. Since PAEC target antigens recognized by human natural antibodies include both 115 and 44 kDa antigens, these Mab clones were selected for further study. Several distinct patterns of tissue reactivity were demonstrated within this group of antibodies by immunohistochemical analysis; however all monoclonal antibodies were highly reactive with endothelial cells in all tissues examined. Two monoclonal antibodies recognizing antigens that are highly expressed on pig endothelial cells (92–98%) and pig platelets (74–92%), but moderately expressed on pig splenocytes (33–38%), were capable of reproducibly blocking 48–53% of human IgM binding to pig endothelial cells when analyzed with flow cytometry. This data suggests that these Mabs may recognize epitopes of potential significance in the human-to-pig xenograft reaction.     

Human platelets activate porcine endothelial cells through a CD154-dependent pathway.

BACKGROUND: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40. METHODS: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E. RESULTS: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner. CONCLUSION: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.     

Ex vivo perfusion of HLA‐E/CD46 transgenic pig limbs with human blood: evaluation of NK cell recruitment

In vitro, interactions between human NK cells and porcine endothelial cells (pEC) are characterized by NK cell recruitment and cytotoxicity. NK cells play a role in allo‐ and xenotransplantation due to the incompatibility of the MHC class I molecules expressed on transplanted cells and tissues on one hand, and the MHC class I specific inhibitory NK receptors of the recipient on the other hand. In this context we published previously that: (i) the expression of human HLA‐E on pEC partially protects from polyclonal huNK cytotoxicity and completely protects from killing by NKG2Abright NK clones in vitro; but does not affect the adhesion of huNK cells to pEC or the heteroconjugate formation between huNK and porcine cells (1, 2); (ii) lymphoblasts and pEC derived from HLA‐E/human beta2microglobulin transgenic pigs are effectively protected against huNK cell‐mediated cytotoxicity and inhibit the secretion of IFN‐gamma by co‐cultured huNK cells in vitro, depending on CD94/NKG2A expression on the NK cells (3); (iii) HLA‐G expression on pEC protects partially against direct huNK cytotoxicity but not against ADCC (4); (iv) HLA‐G expression inhibits rolling adhesion of activated huNK cells on pEC (5, 6); (v) HLA‐Cw3 expression on pEC protects against NK cell‐mediated cytotoxicity (7), HLA‐Cw4 reduced NK cell adhesion and cytotoxicity; HLA‐Cw4 and HLA‐Cw3 co‐expression is not sufficient to completely overcome NK cytotoxicity via recognition of the KIR CD158a (KIR2DL1) and CD158b (KIR2DL2/3) receptors (8). The aim of this study was to evaluate NK cell recruitment and infiltration of porcine tissues using an ex vivo perfusion system of HLA‐E transgenic pig legs with human blood. Over the past year, a pilot study has been set up in order to establish an experimental protocol that would allow for the study and the evaluation of the rejection mechanisms occurring during xenotransplantation and especially following reperfusion of pig organs with human blood (9). Amputated pig legs were perfused with heparinised human blood over a period of 12h. Blood samples were collected at several time points and blood cell populations were analyzed by flow cytometry (FACS) analysis. Two wild type and 2 HLA‐E/CD46 transgenic pig legs were perfused ex vivo. A strong diminution of NK cells was observed after 7 h of perfusion in both the wild type and the transgenic legs. However, this decrease occurred earlier in the wild type leg than in the transgenic. In addition, the apparition of a SLA‐I+ cells population of pig origin of higher granularity than the lymphocyte population over time was observed. We hypothesized that these cells could be pig endothelial cells detached from the vessel wall due to xenorejection, or alternatively originating from the pig bone marrow. These results confirmed previous in vitro studies demonstrating that pEC damage was mediated by human NK cells. Moreover, the expression of HLA‐E/CD46 provided a partial protection with regard to NK cell recruitment and tissue infiltration. From these preliminary experiments the following conclusions were drawn: Pig leg perfusion with human blood was feasible for up to 12 h with stable perfusion parameters thus extending the observation time compared to previously described pig kidney, lung and beating heart perfusion systems. pH and potassium were maintained at normal levels and muscle stimulation resulted in contraction throughout the entire perfusion. Thus the pig leg reperfusion system appears to be a stable and optimized system to study rejection mechanisms over several hours in the pig‐to‐human xenotransplantation setting. This system allows for the study of the dynamics of the different cell populations in the blood. Indeed, a strong diminution of NK, NKT and T cells was observed. In addition, this system enables to study the apparition of cells of pig origin in the circulating blood. In conclusion, this system represents a powerful tool to study the basic molecular mechanisms taking place in a setting of xenotransplantation, and in particular to evaluate the protection from early cell‐mediated rejection mechanisms in tissues originating from transgenic pigs. References: Forte P, Lilienfeld BG, Baumann BC, Seebach JD. Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol 2005; 175: 5463–5470. Lilienfeld BG, Crew MD, Forte Pet al.Transgenic expression of HLA‐E single chain trimer protects porcine endothelial cells against human natural killer cell‐mediated cytotoxicity. Xenotransplantation 2007; 14: 126–134. Weiss EH, Lilienfeld BG, Muller Set al. HLA‐E/human beta2‐microglobulin transgenic pigs: protection against xenogeneic human anti‐pig natural killer cell cytotoxicity. Transplantation 2009; 87: 35–43. Seebach JD, Pazmany L, Waneck GLet al. HLA‐G expression on porcine endothelial cells protects partially against direct human NK cytotoxicity but not against ADCC. Transplant Proc 1999; 31: 1864–1865. Forte P, Matter‐ Reissmann UB, Strasser Met al.Porcine aortic endothelial cells transfected with HLA‐G are partially protected from xenogeneic human NK cytotoxicity. Hum.Immunol 2000; 61:1066–1073. Forte P, Pazmany L, Matter‐ Reissmann UBet al. HLA‐G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol. 2001; 167: 6002–6008. Seebach JD, Comrack C, Germana Set al. HLA‐Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells. J Immunol 1997; 159: 3655–3661. Forte P, Baumann BC, Schneider MKet al. HLA‐Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells. Xenotransplantation 2009; 16: 19–26. Constantinescu MA, Knall E, Xu Xet al. Preservation of amputated extremities by extracorporeal blood perfusion; a feasibility study in a porcine model. J Surg.Res 2010.     

Production of cloned pigs expressing human thrombomodulin in endothelial cells

For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.     

Porcine endothelium supports transendothelial migration of human leukocyte subpopulations: anti-porcine vascular cell adhesion molecule antibodies as species-specific blockers of transendothelial monocyte and natural killer cell migration

BACKGROUND: In cases where hyperacute rejection has been prevented, pig to primate organ transplantation results in a delayed rejection mediated by graft-infiltrating leukocytes. The migration of human leukocytes across porcine endothelium is poorly characterized, but may offer targets for species-specific antirejection therapy. METHODS: Transwell tissue culture inserts with endothelial cells growing on polycarbonate filters were used to characterize the migration of peripheral blood monocuclear cells and purified leukocyte subpopulations across pig and human endothelial cells and cell lines. Endothelial cell morphology was evaluated by scanning and transmission electron microscopy, and the contribution of different adhesion receptor pairs to transendothelial migration was evaluated by antibody blocking experiments. RESULTS: There were no evident quantitative or qualitative differences in the capacity of human and porcine endothelium to support transendothelial migration of human leukocytes [T, B, and natural killer (NK) cells, monocytes, and neutrophils]. Monocytes and large granular CD3+ lymphocytes migrated most efficiently across the endothelium. Antiporcine vascular cell adhesion molecule-1 antibodies blocked transendothelial migration of human monocytes and NK cells across tumor necrosis factor-alpha stimulated pig endothelium by at least 60%. Anti-CD18 antibodies had no effect on the migration of human NK cells across pig endothelium, whereas they partly blocked migration of NK cells across human endothelium and migration of monocytes across porcine endothelium. Interleukin-2 stimulated, but not unstimulated, T and NK cells were cytotoxic to porcine endothelium. CONCLUSIONS: Porcine endothelium supports transendothelial migration of human leukocyte subpopulations as efficiently as human endothelium. Incompatibilities in some adhesion receptor pairs may be compensated for by other adhesion receptor pairs, as exemplified by human NK cells whose migration across human, but not pig, endothelium was blocked by anti-CD18 antibodies. Antiporcine vascular cell adhesion molecule-1 antibodies may be used as species-specific blockers of transendothelial NK cell and monocyte migration, and as such may prove to be useful inhibitors of cellular organ xenograft rejection.     

Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells

Ezzelarab C, Ayares D, Cooper DKC, Ezzelarab MB. Human T‐cell proliferation in response to thrombin‐activated GTKO pig endothelial cells. Xenotransplantation 2012; 19: 311–316. © 2012 John Wiley & Sons A/S. Abstract: Background: Thrombin formation is a key feature in the activation of coagulation in pig xenograft recipients. As thrombin is known to activate endothelial and immune cells, we explored whether thrombin activation of pig endothelial cells (EC) was associated with an increased human T‐cell response. Methods: α1,3‐galactosyltransferase gene‐knockout (GTKO) pig aortic EC (pAEC) were activated by porcine interferon‐gamma (pIFNγ), human (h)IFN‐γ, or thrombin. Swine leukocyte antigen (SLA) class I and class II expression were measured. Human peripheral blood mononuclear cells (PBMC) and CD4⁺ T‐cell proliferation in response to activated pAEC, the effect of thrombin on pig CD80/CD86 mRNA, and the effect of thrombin inhibition by hirudin were evaluated. Results: After pAEC activation, SLA I expression did not change, and only pIFNγ upregulated SLA II expression. PBMC proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. CD4⁺ T‐cell proliferation to pIFNγ‐ and thrombin‐activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non‐activated pAEC. Thrombin inhibition by hirudin reduced thrombin‐induced upregulation of pAEC CD86 mRNA, and significantly reduced human PBMC proliferation to pAEC in comparison with thrombin alone (P < 0.05). Conclusions: Thrombin upregulates CD86 mRNA on pAEC, which is associated with increased human T‐cell proliferation against pAEC. Hirudin reduces CD86 mRNA in thrombin‐activated pAEC and is associated with downregulation of the human T‐cell proliferative response. The transplantation of organs from GTKO pigs transgenic for human thrombomodulin, and/or endothelial protein C receptor, in addition to therapeutic regulation of thrombin activation may reduce the cellular response to a pig xenograft and thus reduce the need for intensive immunosuppressive therapy.     

Monocyte-Induced Endothelial Calcium Signaling Mediates Early Xenogeneic Endothelial Activation

Hallmarks of delayed xenograft rejection include monocyte infiltration, endothelial cell activation and disruption of the endothelial barrier. The monocyte is an important initiator of this type of rejection because monocytes accumulate within hours after xenografting and prior monocyte depletion suppresses the development of this type of rejection. However, the mechanisms that mediate monocyte-induced xenograft injury are unclear at present. Here we report that human monocytes activate xenogeneic endothelial cells through calcium signals. Monocyte contact with porcine but not human endothelium leads to an endothelial calcium transient mediated via a G-protein-coupled receptor (GPCR) that results in up-regulation of porcine VCAM-1 and E-selectin. Although human monocyte adhesion was greater to porcine than to human endothelium, especially when studied under laminar flow, blockade of the xeno-specific endothelial calcium signals did not reduce adhesion of human monocytes to porcine endothelium. Human monocyte contact to porcine endothelium also resulted in reorganization of the F-actin cytoskeleton with a concomitant increase in endothelial monolayer permeability. In contrast to the effect on adhesion, these changes appear to be regulated through endothelial calcium signals. Taken together, these data suggest that human monocytes are capable of activating xenogeneic endothelial cells through calcium transients, as well as other distinct pathways.