Soheyla Saadi

Transplantation of discordant xenografts: a challenge revisited

Six years ago, Jeffrey Platt and colleagues reviewed the biological hurdles to transplanting organs between species. The ensuing years have allowed the concepts advanced at that time to be tested leading to significant progress in understanding the immunology of xenotransplantation and in developing strategies for potential clinical application. Here, William Parker and colleagues review that progress.

Regional manifestations and control of the immune system

Although immune responses are generally considered to be systemic, local events such as interaction of complement products with blood vessels and with inflammatory cells play a pivotal role in determining the nature and manifestations of immune responses. This paper will discuss how blood vessel physiology and immunity influence one another to reach homeostasis upon exposure to an infectious agent. We review new insights into the mechanisms by which the microenvironment of tissues protects against microbial invasion yet facilitates migration of leukocytes and ‘decides’ whether immunity or tolerance ensues and whether, in the face of immunity, protective responses or tissue injury ensues. These ‘decisions’ are made based on interaction of components of normal tissues such as proteoglycans and injured tissues such as cell‐associated cytokines with receptors on immune cells and blood vessels.—Saadi, S., Wrenshall, L. E., Platt, J. L. Regional manifestations and control of the immune system. FASEB J. 16, 849–856 (2002)

Modulation of eicosanoid metabolism in endothelial cells in a xenograft model. Role of cyclooxygenase-2.

Lipid inflammatory mediators are thought to play a critical role in the pathogenesis of vascular injury. Among the events which might cause the synthesis of eicosanoids in blood vessels is activation of the complement. To evaluate how complement might influence eicosanoid metabolism, we investigated endothelial cells exposed to xenoreactive antibodies and complement, as might occur in rejecting xenografts where severe vascular injury is a typical feature. While resting porcine aortic endothelial cells released only prostaglandin (PG) I2, endothelial cells stimulated with xenoreactive antibodies and complement released PGE2 and thromboxane A2 (TXA2), in addition to increased amounts of PGI2. This alteration in eicosanoid metabolism was associated with induction of cyclooxygenase (Cox)-2 and thromboxane synthase, but not Cox-1. Unlike results seen in other systems, the upregulation of Cox-2 and the subsequent release of eicosanoids by endothelial cells was not directly induced by complement but rather required production of IL-1alpha, which acted on endothelial cells as an autocrine factor. Since eicosanoids have a potent effect on inflammation, vascular tone and platelet aggregation, we postulated that the abnormalities in eicosanoid release induced by xenoreactive antibodies and complement might provide one explanation for the vascular injury, focal ischemia, and thrombosis observed in acute vascular rejection and other vasculitides mediated by complement.

Endothelial Cell Activation by Pore-Forming Structures Pivotal Role for Interleukin-1α

BACKGROUND Interaction of complement with endothelial cells (ECs) underlies the development of inflammation and coagulation in disease. Assembly of the membrane attack complex (MAC) of complement on EC membrane, like stimulation with cytokines, upregulates tissue factor and cyclooxygenase-2 but does so via the intermediary action of IL-1alpha. We asked whether the MAC activates porcine aortic and microvascular ECs in a global manner by this mechanism and whether this mechanism is used by membrane pore-forming structures. METHODS AND RESULTS Exposure of ECs to complement caused upregulation of mRNAs for E-selectin, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, Ikappa-Balpha, interleukin (IL)-1alpha, IL-1beta, IL-8, and plasminogen activator inhibitor-1 over a period of 6 hours. The expression of these genes was not a primary response to stimulation, however, because IL-1 receptor antagonist inhibited expression of these genes. Activation of ECs by complement depended on the autocrine action of IL-1alpha, because complement-mediated EC activation was inhibited by anti-IL-1alpha antibodies. Melittin and mastoparan, amphiphilic pore-forming peptides like the MAC, induced E-selectin through intermediary action of IL-1. CONCLUSIONS These findings suggest that transmembrane pore-forming proteins, as a class of molecules, activate ECs through the autocrine effects of IL-1alpha.

Human natural killer cells induce morphologic changes in porcine endothelial cell monolayers

In this study, we have investigated the early in vitro effects of natural killer (NK) cells on porcine aortic endothelial cell (PAEC) monolayers. Incubation of effector cells containing about 70% CD56+ cells on PAEC monolayer led to time-dependent changes in PAEC monolayer morphology. As little as 20 min of incubation resulted in changes in PAEC shape and in the appearance of gaps between the cells. These effects have been observed for up to 6 hr, but not before 20 min or after 6 hr. When NK-depleted effector cells were used, no morphological changes were observed in comparison with the same effectors before depletion; if CD56+ cells were added back, the effects were comparable with those on nondepleted effector cells. There was no detectable NK cell-mediated cytolytic activity during the 1-6 hr of incubation of peripheral blood lymphocytes with PAEC monolayers. These data indicate that NK cells may participate in endothelial cell changes leading to xenograft rejection.

Expression of tissue factor mRNA in cardiac xenografts: clues to the pathogenesis of acute vascular rejection.

BACKGROUND Acute vascular rejection destroys vascularized xenografts over a period of hours to days and is now considered the major hurdle to the clinical application of xenotransplantation. The hallmark of acute vascular rejection is diffuse intravascular coagulation; however, the pathogenesis of coagulation is a matter of controversy. One line of evidence points to activated endothelial cells and another to activated inflammatory cells as a source of tissue factor and thus as a primary cause of this lesion. The distinction between the two mechanisms inducing coagulation in the xenograft provides an opportunity for specific intervention. METHODS To explore these mechanisms, we studied the expression of tissue factor mRNA by in situ reverse transcriptase-polymerase chain reaction in relation to the histopathologic manifestations of acute vascular rejection in guinea pig hearts transplanted into rats treated by cobra venom factor to avoid the hyperacute rejection. RESULTS Three hours after transplantation and before the deposition of fibrin, tissue factor mRNA was expressed in the endothelial cells lining small and medium blood vessels and in smooth muscle cells of guinea pig cardiac xenografts. Sixteen hours after transplantation, while rat tissue factor mRNA was expressed only in occasional infiltrating cells, cardiac xenografts showed prominent deposits of fibrin in small vessels. Maximum expression of tissue factor on rat infiltrating cells was observed 48 hr after transplantation. CONCLUSIONS These results suggest that in acute vascular rejection, coagulation is initiated on the donor vascular system, while the procoagulant characteristics of infiltrating cells may reflect a response to tissue injury rather than a cause.

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-&bgr;). 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.

Pathways to Acute Humoral Rejection

Acute humoral rejection, also known as acute vascular rejection, is a devastating condition of organ transplants and a major barrier to clinical application of organ xenotransplantation. Although initiation of acute humoral or vascular rejection is generally linked to the action of antibodies and complement on the graft, other factors such as ischemia, platelets, T cells, natural killer cells, and macrophages have also been implicated. Central to any understanding of the pathogenesis of acute humoral rejection, and to developing means of preventing it, is to know whether these factors injure the graft independently or through one or few pathways. We addressed this question by examining early events in a severe model of vascular rejection in which guinea pig hearts transplanted heterotopically into rats treated with cobra venom factor (CVF) develop disease over 72 hours. The early steps in acute vascular rejection were associated with expression of a set of inflammatory genes, which appeared to be controlled by availability of interleukin (IL)-1. Interruption of IL-1 signaling by IL-1 receptor antagonist (IL-1ra) averted expression of these genes and early tissue changes, including coagulation and influx of inflammatory cells. These findings suggest IL-1 plays an important role in initiation of acute humoral rejection.

The role of complement in transplantation.

The complement system contributes critically to the barrier to transplantation of cells and organs. In the case of tissues and organs transplanted between individuals of the same species, that is in allotransplantation, the barrier posed by complement is seemingly eclipsed by the barrier posed by cellular immune responses. In the case of cells and organs transplanted between individuals of disparate species, that is xenotransplantation, the complement system has been thought to pose a nearly insurmountable barrier. With our understanding on how the complement system contributes to rejection, it is now clear that the complement system is more important in allotransplantation and more forgiving in xenotransplantation than was previously thought.

IMMUNOLOGY OF XENOTRANSPLANTATION

The transplantation of tissues and organs between individuals of different species, that is, xenotransplantation, engenders a variety of immune responses. Xenogeneic immune responses mediated by naturally-occurring antibodies and complement lead to hyperacute and acute vascular rejection of vascularized organ grafts and may also cause vascular rejection of cell and tissue grafts. Under some circumstances, however, a vascularized organ graft may evade humoral rejection despite the presence of anti-donor antibodies in the circulation of the recipient; this condition is called accommodation. Xenogeneic immune responses mediated by T lymphocytes and natural killer cells may cause acute cellular rejection. The extent to which cellular rejection of xenografts resembles cellular rejection of allografts remains to be determined. New insights into the molecular mechanisms underlying the immune responses to xenotransplantation has shed light on the pathogenesis of immunological disease and has allowed the development of specific immunomodulatory strategies that may facilitate clinical application of xenotransplantation.