New information has begun to emerge on the importance of the vascular endothelium in immune-mediated events in the immediate posttransplantation period that ultimately affect graft function and survival. Circulating host leukocytes literally carpet the vascular endothelium, providing a unique interface between the host and graft.
Three experts discussed key aspects of endothelial cell biology and transplantation during the AST 4th Annual Winter Symposium. They focused on 3 key points:
- Cytotoxic T lymphocyte (CTL)-mediated injury to the vasculature;
- Induced expression (and the inducible expression) of protective genes that facilitate good long-term outcomes following allograft and xenograft transplantation; and
- Function of complement and degradation products of complement on both acute injury and on leukocyte activation and activational responses that are associated with early antigen-independent events as well as later antigen-dependent events characteristic of acute rejection.
CYTOTOXIC T LYMPHOCYTE: ENDOTHELIAL CELL INTERACTION AND ALLOGRAFT REJECTION
It has been established that acute allograft rejection in humans correlates with the presence of CTL transcripts in biopsies, and that it is possible to culture CTL from these biopsies, noted Jordan Pober, MD, PhD, of Yale University, New Haven, Conn. In addition, he said, it is evident that these cultured CTLs may be specific for donor endothelial cells but not donor leukocytes. Thus, it would appear possible in vivo in humans that there may be a CD8+ T cell that is selectively cytotoxic to target endothelial cells.
Indeed, Dr. Pober’s group has shown that if CD8+ T cells are cultured with single-donor allogenic endothelial cells and are restimulated by the same endothelial cells, they subsequently become cytotoxic specific for the stimulating endothelial cells but not a third party. It also has been demonstrated that while endothelial cells are capable of inducing the proliferation of CTLs, their ability to do so is weak compared with the lymphocytes. Supplementation with a variety of reagents does not facilitate a more potent ability of endothelial cells to generate CTLs, Dr. Pober said.
A number of studies have made it clear that CD8+ T-cell lines or in fact CD8+ T-cell clones are capable of killing endothelial cell targets but not B-cell targets and, conversely, that CD8 T cells generated by B cells are not necessarily specific for endothelial cells. The specificity of the CD8 T cells to kill endothelial cells has been demonstrated by the ability of anticlass I and anti-CD8+ antibodies to inhibit killing.
Interestingly, further characterization of endothelial-specific CTL clones shows them to have a low level of messenger RNA for interferon-gamma and a markedly high level of self-surface expression of CD40 ligand (not typically expressed as high levels on CD8+ T cells). Another feature of these endothelial cell selective alloreactive CTL lines is that they appear to inhibit the differentiation of conventional alloreactive CTLs. However, it has not been demonstrated whether this clearly correlated with any particular profile of cytokines produced by this cell type.
Thus, it is apparent that understanding the mechanisms by which endothelial-specific CTLs are generated or the specific molecule present on endothelial cells that dictates the selectivity for the endothelium is an important direction for further research.
To this end, Dr. Pober suggested that cytoprotective gene products in the endothelium might be capable of preventing CTL-mediated injury. It has been demonstrated that endothelial cells are expressed by all 12 receptor alpha chains. Furthermore, interleukin-11 (IL-11) in endothelial cells induces the correlation of STAT3 and STAT1 and IL-11 also induces the phosphorylation of MAP kinases in endothelial cells. In addition, it has been shown that pretreatment of the endothelium with IL-11 partially protects endothelial cells from CTL-mediated lysis (approximately 50% in addition). Lastly, Dr. Pober said, researchers have found that transfection of a capsase-resistant Bcl-2 protein into endothelial cells conferred resistance to CTL-mediated lysis.
The antiapoptotic gene product, Bcl-2 is typically made proapoptotic following capsase cleavage. These studies used a mutated Bcl-2, which was resistant to capsase cleavage. Once the Bcl-2 was expressed in the endothelium, it was clearly shown to be remarkably resistant to CTL-mediated lysis. Dr. Pober said that these data, therefore, provide strategies by which endothelial cells may be protected from CTL-mediated lysis: as a function of IL-11 treatment and as a function of potential gene therapy in which this capsase-resistant Bcl-2 might be used to protect the allograft from rejection.
ENDOTHELIUM AND PROTECTIVE GENES
Protective genes are a series of heterogeneous gene products expressed in endothelial cells that have been shown to have antioxidant and antiapoptotic actions and may marginate the effect of transcription factors, including NF kappa B. These genes include Bcl-2, Bcl-xL, A20, and hemoxygenase (HO-1).
Wayne Hancock, MD, PhD, from Harvard Medical School, Boston, described work showing that different protocols that mediate long-term allograft survival have different affects on the expression of protective genes following transplantation. For instance, treatment of allograft with CD40 ligand blockade in combination with donor-specific transfusion resulted in good long-term survival, minimal histologic changes within long-term surviving allografts, and the induced expression of Bcl-xL and HO-1.
In contrast, treatment of allograft with anti-CD4-promoted long-term allograft survival but with evidence of chronic disease on pathologic structure. These anti-CD4-treated allografts failed to show induction of productive genes. In addition, there was a clear difference in the number of apoptotic cells within anti-CD4 and CD40 ligand-treated allograft, with minimal apoptosis seen in the CD40 ligand-treated allograft.
These data suggest that the production of alloantibody might be a mediator of protective gene. To this end, Dr. Hancock noted, studies have shown that the transfer of alloantibody into B-cell-deficient recipients treated with anti-CD4 can indeed result in the induction of protective gene. However, there is a clear difference between animals treated with alloantibody at early times vs those treated at later times.
This research also indicates that treatment with alloantibody resulted in negligible protective gene expression at day 14 but marked protective gene expression (expression of IL-4, A20, and HO-1 by day 28, which persisted to day 100). The expression of Th2 cytokines, IL-4, IL-10, and IL-13 appeared to induce the expression of antiapoptotic Bcl- xL in endothelial cells, suggesting that these cytokines may be functional in the induction of these molecules in vivo.
To address the specific inducibility of HO-1, it has been shown that the predominant molecules capable of inducing HO-1 in the endothelium are cobalt protoporphyrin (COPP) and IL-6. Treatment of allografts in vivo with anti-CD4 and COPP has been found to result in protection against graft vascular arteriosclerosis and correlated nicely with the expression of HO-1. Thus, it appears that the induction of protective genes is linked to the suppression of acute and chronic allograft rejection. It also appears that specific manipulation of HO-1 is an attractive tool to limit the development of arteriosclerosis. And, Dr. Hancock noted, the induction of protective genes following xenograft rejection is a feature of accommodation.
ALLOGRAFT INJURY: ANTIBODY AND COMPLEMENT MEDIATION
It has been well described in previous literature that complement binds to the endothelium at both early and late phases following transplantation. However, the focus of new studies presented by William M. Baldwin III, MD, PhD, of the Johns Hopkins University School of Medicine, Baltimore, appears to be more the ability of complement, particularly C3, to result in specific recruitment and activation of leukocytes.
For instance, the C3 deposition following ischemia that occurs in approximately 20% of allograft recipients might be potent for the chemoattraction and binding of neutrophils, and perhaps monocyte/macrophages into allografts in the course of ischemic reperfusion injury, Dr. Baldwin said. In addition, antibodies created in the first few weeks following transplantation might be functional to bind complement and thus promote the recruitment and activation of leukocytes.
Approximately 40% of patients develop anti-HLA antibodies following transplantation, some of which are complement-fixing. Following binding of an alloantibody to the endothelium, if that antibody binds complement then it is possible that this complex might facilitate the recruitment of leukocytes and thus be pro-inflammatory. Furthermore, split products, such as CDA, C3A and C5A, might also be potent for the recruitment of macrophages and thus promote acute rejection.
Indeed, it has been demonstrated that the infusion of a complement-fixing IgG2b antibody into B-cell knockout recipients has detrimental affects on survival. It also has been clearly shown that the infusion of the complement-binding alloantibody to the endothelium is linked with complement activation and recruitment of macrophages and rejection. Binding of the membrane attack complex (MAC) to the endothelium, which is known to result in the activation of the endothelium, as well as the expression of adhesion molecules and chemoattractants, such as MCP1 and IL-8, also play a role in rejection.
Using a rodent model in which C6 deficiency inhibits the formation of the MAC, Dr. Baldwin’s group demonstrated that there are remarkable differences in pathologic structure during rejection. In particular, they showed that C6-sufficient animals in which MAC was present resulted in the activation of the endothelium and the expression of von Willebrand factor; whereas in C6-deficient animals in which there was not insufficient MAC production there was less endothelial activation and less von Willebrand factor release. There also appears to be a correlation between the deposition of MAC and arteriosclerosis in that C6-deficient animals seem to have minimal arteriosclerosis in large vessels but still develop it in smaller vessels.
These findings suggest that there may indeed be heterogeneity in vessels for the development of arteriosclerosis. Thus, understanding the mechanisms by which complement activation product binds to and result in leukocyte-dependent arteriosclerosis might have important implications for chronic transplantation rejection.
SUMMARY: IMPLICATIONS FOR CLINICAL PRACTICE
These studies have a number of important clinical implications. First and foremost, if it is true that CTLs are critical mediators of acute and potentially chronic allograft rejection, then understanding the molecular basis for endothelial-specific targets of CTL-mediated vasculitis will allow us to develop specific therapies. Furthermore, understanding the mechanisms by which the endothelium might become resistant to CTL (CTL-4 and others)-mediated injury will result in potential therapies for multiple disorders.
In particular, the possibility of applying gene transfer techniques to selectively overexpress certain molecules, such as mutate Bcl-2 protein that might protect the endothelium, could have important clinical impact. In addition, the ability to treat patients with molecules that might selectively induce protective genes, such as HO-1, will be of benefit in the clinic.
Lastly, the documentation of C3 deposition in allografts, something not commonly performed in allograft biopsies, might allow transplant clinicians to identify patients at risk for complement-dependent injury. And treatment of patients with agents that might dysregulate complement-dependent deposition and/or activation might have potential therapeutic benefits.
- Pober J. Cytotoxic T lymphocyte: endothelial cell interaction and allograft rejection. In: Program and abstracts of the American Society of Transplantation Fourth Annual Winter Symposium; Jan. 13-17, 2000; Fajardo, Puerto Rico.
- Hancock W. Endothelium and protective gene in allotransplantation and xenotransplantation. In: Program and abstracts of the American Society of Transplantation Fourth Annual Winter Symposium; Jan. 13-17, 2000; Fajardo, Puerto Rico.
- Baldwin W. Endothelium and protective gene in allotransplantation and xenotransplantation. In: Program and abstracts of the American Society of Transplantation Fourth Annual Winter Symposium; Jan. 13-17, 2000; Fajardo, Puerto Rico.
- Pober JS. Immunobiology of human vascular endothelium. Immunol Res. 1999;19:225-232.
- Baldwin WM III, Samaniego-Picota M, Kasper EK, et al. Complement deposition in early cardiac transplant biopsies is associated with ischemic injury and subsequent rejection episodes. Transplantation. 1999;68:894-900.