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Some problems related to discordant xenografting
ELSEVIER
Some Problems Related to Discordant Xenografting F.H. Bach
T
HE shortage of donor organs for allotransplantation and the identification of new targets for potential genetic therapies in the donor animal have both rekindled interest in xenotransplantation. Most investigators are targeting their efforts at enabling the transplantation of swine organs to humans, and using nonhuman primates as experimental recipients. I shall address problems in this paper that I believe to be relevant to such a model. It appears that utilization of the idea developed by Dr Agustin Dalmasso and myself, and published with our colleagues in 1991, is likely to make a major impact on preventing hyperacute rejection.‘.’ Expression of human inhibitors of complement, such as decay accelerating factor (DAF), in pig endothelial cells (EC) inhibits the action of complement on the porcine EC in vitro and leads to prolonged survival of the porcine organ in vivo. It is not clear that this in vivo effect can be attained consistently. Further, while difficult to interpret in terms of its relevance to a clinically acceptable protocol, the use of porcine hearts that express human DAF on the EC surface as donor organs to cynomolgus monkeys treated with heavy immunosuppression leads to survival in some cases for several weeks.3 Similar results have been obtained using soluble complement receptor 1 (sCR1) and immunosuppression.4 When hyperacute rejection is averted, the problem that we face is one that we have referred to as delayed xenograft rejection (DXR), which takes place after about 3 days.5x6If we can overcome the multiple problems associated with DXR, we will presumably have to deal with the xenograft equivalent of the T-cell-mediated allograft rejection response. The problems associated with DXR will be addressed here. We have hypothesized that DXR is related primarily to EC activation.
DELAYED
XENOGRAFT
Molecules/Activities
I find it useful to divide into two categories the problems that are associated with EC activation as it occurs in DXR. First, there is the loss from the surface of the EC of the graft of a number of molecules and/or their associated activities. These include thrombomodulin, heparan sulfate and ATPDase.‘,’ Thrombomodulin binds thrombin, which in turn catalyzes the conversion of protein C to activated protein C (aPC), which has powerful anticoagulant and anti-inflammatory functions. To the extent that thrombomodulin is lost from the surface of the EC when they are activated, there will be a concomitant loss of the anticoagulant and anti-inflammatory functions mediated by aPC. The ATPDase function is also lost with EC activation. ATPDase (e&o-ADPase) converts ATP and ADP to AMP, which is in turn converted to adenosine. The enzymatic activity of ATPDase thus removes the pro-platelet aggregatory stimulus of ADP and the pro-inflammatory stimuli mediated by ATP and ADP. In addition, adenosine acts as an anti-platelet aggregatory and anti-inflammatory stimulus. With the loss of activity, and the presumed accumulation of ADP at the site of the inflammatory lesion, the ADP stimulates platelet aggregation; ATP and ADP aggravate the inflammatory lesion, and the lack of adenosine further aggravates these responses. We have approached the problems associated with the loss of thrombomodulin and ATPDase activity by expressing these molecules on the surface of porcine EC in a manner that their activities will be maintained even in the presence of EC-activating stimuli. Thrombomodulin can be mutated to avoid a part of the loss of activity and expressed under regulation of a promoter that is not susceptible to the suppressive action of a factor that acts with EC activation to down-regulate the normal endogenous thrombomodulin. We have shown that this approach works in vitro’, and are in the process of using the same approach in vivo. For
REJECTION
A discordant xenograft such as one from pig to primate is rejected after 3 to 4 days. The immunopathology of the graft at that time shows evidence of EC activation (upregulation of the multiple pro-inflammatory genes associated with EC activation), thrombosis, a complex cytokine profile, and the infiltration into the graft of host monocytes and NK cells, both of which appear to be activated.5,6 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation
Loss of EC Surface
Proceedings,
29, 3009-3010
(1997)
From the Sandoz Center for chusetts,. This work was supported by land. Address reprint requests to Professor, Harvard University, ogy, 99 Brookline Ave, Boston,
Immunobiology,
Boston, Massa-
Sandoz Pharma, Basel, SwitzerFritz Bach, MD, Lewis Thomas Sandoz Center for ImmunobiolMA 02215.
0041-l 345/97/$17.00 PII SO041 -1345(97)00762-8
3009
BACH
3010
ATPDase, we have identified the gene encoding this molecule as CD39,1°, and are beginning work that it is hoped will yield a mutant of that gene, the activity of which is also maintained in the pro-inflammatory environment that can develop. Inhibition of the Up-Regulation Genes in EC
of the Pro-Inflammatory
A large number of genes are up-regulated with EC activation, including ones encoding adhesion molecules, prothrombotic factors, cytokines, and others. Given the complexity of this response, and the hypothesized role of these factors in xenograft rejection, we have attempted to find a target within EC that would allow us to prevent this form of EC activation. The target we have chosen is the transcrip tion factor, NF-KB. It appears, based both on our own data and that of others, that NF-KB plays a key role in the up-regulation of essentially all of the identified pro-inflammatory genes.l’-13 Thus, our goal was to block the activation of NF-KB. We have used a dominant negative mutant to achieve our goal. First, we have overexpressed IKB~, the natural inhibitor of NF-KB. I2 Overexpression of IKBCI in either the native form or as a mutant that cannot be degraded does block the transmigration of NF-KB into the nucleus and thus to a very large extent blocks the induction of the pro-inflammatory genes. We are now in the process of testing the hypothesis that blocking NF-KB in vivo will ameliorate DXR. ACCOMMODATION
AND PROTECTIVE GENES
Several years ago, I suggested that one potential therapeutic approach to achieving survival of a xenograft would be to achieve accommodation.‘4 I proposed that the term “accommodation” be used to refer to the survival of a graft in the presence of anti-graft antibodies and complement, the very factors that are thought to cause hyperacute rejection of a xenograft. I based this suggestion on the observation of several individuals studying ABO-incompatible allografts. We have recently worked with a model in which accommodation can be achieved: the transplantation of hamster hearts to rats that are treated for a few days with cobra venom factor (CVF) to inhibit complement, and with cyclosporine (CyA) for the duration of survival of the hamster heart. Under these conditions, approximately 75% of hearts survive longterm.” Relevant to the present discussion is the finding that in the accommodated (surviving) hearts, there is expression in the EC and smooth muscle cells of a number of genes. These include three genes, A20, bcl-2, and b&XL, that were first described based on their anti-apoptotic properties, as well as hemoxygenase. We refer to these genes as “protective” genes, since we believe that their expression may prevent the EC from having the type of response that would lead to rejection. The three anti-apoptoticlprotective genes all share one property in addition to their serving to prevent apoptosis:
expression of any one of these genes in EC blocks NF-KB and thus inhibits the up-regulation of the pro-inflammatory genes in the EC. These genes appear to be a natural mechanism that blocks reactions that likely account for rejection. Apoptosis of EC is seen with rejection, and we hypothesize that the up-regulation of the pro-inflammatory genes is one of the major factors in precipitating rejection. We feel that the therapeutic potential of expressing these protective genes to avoid rejection is important. THE PRESENT SITUATION
There is currently a difference of opinion in the xenotransplantation community about our progress toward clinical xenotransplantation. Some believe that by using donor organs that express human DAF plus immunosuppression as used to date, we shall be able to transplant hearts and perhaps kidneys to humans. While it seems not unreasonable to think that one could find therapeutic agents that would suppress the reactions that I have discussed as associated with DXR, others, including myself, believe that the most acceptable protocol to achieve xenotransplantation will be to engineer the donor organ with more than just the gene for DAF. One could envision that, in addition to DAF, or some other inhibitor of complement, we shall express thrombomodulin, ATPDase, and perhaps one of the protective genes discussed above. Such an approach would likely limit the need for immunosuppressive agents with their associated toxicity. REFERENCES 1. Dalmasso AP, Vercellotti GM, Platt JL, et al: Transplantation 52:530, 1991 2. Bach FH, Turman MA, Vercellotti GM, et al: Transplant Proc 23:205, 1991 3. Cozzi E, White D: Nat Med 1:964, 1995 4. Davis EA, Pruitt SK, Greene PS, et al: Transplantation 62:1018, 1996 5. Blakely ML, van der Werf WJ, Berndt MC, et al: Transplantation 58:1059, 1994 6. Bach FH, Robson SC, Winkler H, et al: Nat Med 1:869, 1995 7. Platt J, Vercellotti GM, Lindman BJ, et al: J Exp Med 171:1363, 1990 8. Robson S, Kaczmareck E, Siegel J, et al: J Exp Med 185:153, 1997 9. Wrighton CJ, Kopp CW, McShea A, et al: Transplant Proc 27:288, 1995 10. Robson S, Kaczmareck E, Siegel J, et al: J Exp Med 1997 (in press) 11. Ferran C, Millan MT, Csizmadia V, et al: Biochem Biophys Res Commun 214:212, 1995 12. Wrighton Cl, Hofer-Warbinek R, Mall T, et al: J Exp Med 183:1013, 1996 13. Read MA, Whitley MZ, Williams AJ, et al: J Exp Med 179:503, 1994 14. Back FH, Turman MA. Vercellotti GM, et al: Transplant Proc 23:205, 1991 15. Bach FH, Ferran C, Candinas D, et al: Transplant Proc 29:56, 1997
Some Problems Related to Discordant Xenografting F.H. Bach
T
HE shortage of donor organs for allotransplantation and the identification of new targets for potential genetic therapies in the donor animal have both rekindled interest in xenotransplantation. Most investigators are targeting their efforts at enabling the transplantation of swine organs to humans, and using nonhuman primates as experimental recipients. I shall address problems in this paper that I believe to be relevant to such a model. It appears that utilization of the idea developed by Dr Agustin Dalmasso and myself, and published with our colleagues in 1991, is likely to make a major impact on preventing hyperacute rejection.‘.’ Expression of human inhibitors of complement, such as decay accelerating factor (DAF), in pig endothelial cells (EC) inhibits the action of complement on the porcine EC in vitro and leads to prolonged survival of the porcine organ in vivo. It is not clear that this in vivo effect can be attained consistently. Further, while difficult to interpret in terms of its relevance to a clinically acceptable protocol, the use of porcine hearts that express human DAF on the EC surface as donor organs to cynomolgus monkeys treated with heavy immunosuppression leads to survival in some cases for several weeks.3 Similar results have been obtained using soluble complement receptor 1 (sCR1) and immunosuppression.4 When hyperacute rejection is averted, the problem that we face is one that we have referred to as delayed xenograft rejection (DXR), which takes place after about 3 days.5x6If we can overcome the multiple problems associated with DXR, we will presumably have to deal with the xenograft equivalent of the T-cell-mediated allograft rejection response. The problems associated with DXR will be addressed here. We have hypothesized that DXR is related primarily to EC activation.
DELAYED
XENOGRAFT
Molecules/Activities
I find it useful to divide into two categories the problems that are associated with EC activation as it occurs in DXR. First, there is the loss from the surface of the EC of the graft of a number of molecules and/or their associated activities. These include thrombomodulin, heparan sulfate and ATPDase.‘,’ Thrombomodulin binds thrombin, which in turn catalyzes the conversion of protein C to activated protein C (aPC), which has powerful anticoagulant and anti-inflammatory functions. To the extent that thrombomodulin is lost from the surface of the EC when they are activated, there will be a concomitant loss of the anticoagulant and anti-inflammatory functions mediated by aPC. The ATPDase function is also lost with EC activation. ATPDase (e&o-ADPase) converts ATP and ADP to AMP, which is in turn converted to adenosine. The enzymatic activity of ATPDase thus removes the pro-platelet aggregatory stimulus of ADP and the pro-inflammatory stimuli mediated by ATP and ADP. In addition, adenosine acts as an anti-platelet aggregatory and anti-inflammatory stimulus. With the loss of activity, and the presumed accumulation of ADP at the site of the inflammatory lesion, the ADP stimulates platelet aggregation; ATP and ADP aggravate the inflammatory lesion, and the lack of adenosine further aggravates these responses. We have approached the problems associated with the loss of thrombomodulin and ATPDase activity by expressing these molecules on the surface of porcine EC in a manner that their activities will be maintained even in the presence of EC-activating stimuli. Thrombomodulin can be mutated to avoid a part of the loss of activity and expressed under regulation of a promoter that is not susceptible to the suppressive action of a factor that acts with EC activation to down-regulate the normal endogenous thrombomodulin. We have shown that this approach works in vitro’, and are in the process of using the same approach in vivo. For
REJECTION
A discordant xenograft such as one from pig to primate is rejected after 3 to 4 days. The immunopathology of the graft at that time shows evidence of EC activation (upregulation of the multiple pro-inflammatory genes associated with EC activation), thrombosis, a complex cytokine profile, and the infiltration into the graft of host monocytes and NK cells, both of which appear to be activated.5,6 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation
Loss of EC Surface
Proceedings,
29, 3009-3010
(1997)
From the Sandoz Center for chusetts,. This work was supported by land. Address reprint requests to Professor, Harvard University, ogy, 99 Brookline Ave, Boston,
Immunobiology,
Boston, Massa-
Sandoz Pharma, Basel, SwitzerFritz Bach, MD, Lewis Thomas Sandoz Center for ImmunobiolMA 02215.
0041-l 345/97/$17.00 PII SO041 -1345(97)00762-8
3009
BACH
3010
ATPDase, we have identified the gene encoding this molecule as CD39,1°, and are beginning work that it is hoped will yield a mutant of that gene, the activity of which is also maintained in the pro-inflammatory environment that can develop. Inhibition of the Up-Regulation Genes in EC
of the Pro-Inflammatory
A large number of genes are up-regulated with EC activation, including ones encoding adhesion molecules, prothrombotic factors, cytokines, and others. Given the complexity of this response, and the hypothesized role of these factors in xenograft rejection, we have attempted to find a target within EC that would allow us to prevent this form of EC activation. The target we have chosen is the transcrip tion factor, NF-KB. It appears, based both on our own data and that of others, that NF-KB plays a key role in the up-regulation of essentially all of the identified pro-inflammatory genes.l’-13 Thus, our goal was to block the activation of NF-KB. We have used a dominant negative mutant to achieve our goal. First, we have overexpressed IKB~, the natural inhibitor of NF-KB. I2 Overexpression of IKBCI in either the native form or as a mutant that cannot be degraded does block the transmigration of NF-KB into the nucleus and thus to a very large extent blocks the induction of the pro-inflammatory genes. We are now in the process of testing the hypothesis that blocking NF-KB in vivo will ameliorate DXR. ACCOMMODATION
AND PROTECTIVE GENES
Several years ago, I suggested that one potential therapeutic approach to achieving survival of a xenograft would be to achieve accommodation.‘4 I proposed that the term “accommodation” be used to refer to the survival of a graft in the presence of anti-graft antibodies and complement, the very factors that are thought to cause hyperacute rejection of a xenograft. I based this suggestion on the observation of several individuals studying ABO-incompatible allografts. We have recently worked with a model in which accommodation can be achieved: the transplantation of hamster hearts to rats that are treated for a few days with cobra venom factor (CVF) to inhibit complement, and with cyclosporine (CyA) for the duration of survival of the hamster heart. Under these conditions, approximately 75% of hearts survive longterm.” Relevant to the present discussion is the finding that in the accommodated (surviving) hearts, there is expression in the EC and smooth muscle cells of a number of genes. These include three genes, A20, bcl-2, and b&XL, that were first described based on their anti-apoptotic properties, as well as hemoxygenase. We refer to these genes as “protective” genes, since we believe that their expression may prevent the EC from having the type of response that would lead to rejection. The three anti-apoptoticlprotective genes all share one property in addition to their serving to prevent apoptosis:
expression of any one of these genes in EC blocks NF-KB and thus inhibits the up-regulation of the pro-inflammatory genes in the EC. These genes appear to be a natural mechanism that blocks reactions that likely account for rejection. Apoptosis of EC is seen with rejection, and we hypothesize that the up-regulation of the pro-inflammatory genes is one of the major factors in precipitating rejection. We feel that the therapeutic potential of expressing these protective genes to avoid rejection is important. THE PRESENT SITUATION
There is currently a difference of opinion in the xenotransplantation community about our progress toward clinical xenotransplantation. Some believe that by using donor organs that express human DAF plus immunosuppression as used to date, we shall be able to transplant hearts and perhaps kidneys to humans. While it seems not unreasonable to think that one could find therapeutic agents that would suppress the reactions that I have discussed as associated with DXR, others, including myself, believe that the most acceptable protocol to achieve xenotransplantation will be to engineer the donor organ with more than just the gene for DAF. One could envision that, in addition to DAF, or some other inhibitor of complement, we shall express thrombomodulin, ATPDase, and perhaps one of the protective genes discussed above. Such an approach would likely limit the need for immunosuppressive agents with their associated toxicity. REFERENCES 1. Dalmasso AP, Vercellotti GM, Platt JL, et al: Transplantation 52:530, 1991 2. Bach FH, Turman MA, Vercellotti GM, et al: Transplant Proc 23:205, 1991 3. Cozzi E, White D: Nat Med 1:964, 1995 4. Davis EA, Pruitt SK, Greene PS, et al: Transplantation 62:1018, 1996 5. Blakely ML, van der Werf WJ, Berndt MC, et al: Transplantation 58:1059, 1994 6. Bach FH, Robson SC, Winkler H, et al: Nat Med 1:869, 1995 7. Platt J, Vercellotti GM, Lindman BJ, et al: J Exp Med 171:1363, 1990 8. Robson S, Kaczmareck E, Siegel J, et al: J Exp Med 185:153, 1997 9. Wrighton CJ, Kopp CW, McShea A, et al: Transplant Proc 27:288, 1995 10. Robson S, Kaczmareck E, Siegel J, et al: J Exp Med 1997 (in press) 11. Ferran C, Millan MT, Csizmadia V, et al: Biochem Biophys Res Commun 214:212, 1995 12. Wrighton Cl, Hofer-Warbinek R, Mall T, et al: J Exp Med 183:1013, 1996 13. Read MA, Whitley MZ, Williams AJ, et al: J Exp Med 179:503, 1994 14. Back FH, Turman MA. Vercellotti GM, et al: Transplant Proc 23:205, 1991 15. Bach FH, Ferran C, Candinas D, et al: Transplant Proc 29:56, 1997