Immunosuppression in Xenotransplantation with Wf10 (2002)

Xenotransplantation may eradicate the serious shortage of organs for clinical transplantation. Immunosuppression, however, seems to be necessary, but although immunosuppressive therapy has improved, it is still not perfect. A very useful xenotransplantation model is transplantation of hamster heart to rat, a so-called concordant model, leading to rejection after 3 days with formation of xenoantibodies. The production of xenoantibodies from B cells may be stimulated by T cells and the antibodies together with antigens will then activate the complement system resulting in rejection of the transplanted organ. In concordant xenotransplantation, macrophages and activated lymphocytes play an important role in the rejection process. These combined forces are both altered during treatment with the anti-inflammatory drug Wf10 (McGrath et al. 1998). The aim of the present study was to investigate the effect of Wf10 on rejection time, pathology and the composition of peripheral lymphocytes. Animals. Heart transplantation with Syrian hamster hearts was performed. Lewis rats weighing 200–300 g (MOL:SPRD) served as recipients and outbred Syrian hamsters (MOL:Breeding centre, Skensved, Denmark) as donors. The animals were 3 to 4 months of age and were maintained under standard laboratory conditions, receiving human care. The animals were divided into five groups: 1) 10 animals that received no treatment or transplant (controls), 2) 5 animal that received Wf10 in a high dosis, 3) 10 animals that received a transplant, 4) 6 animals that received Wf10 in a low dosis and a transplant, and 5) 11 animals that received Wf10 in a high dosis and a transplant. Immunosuppression. Wf10, 0.5 ml, was administered intraperitoneally daily from day 2 until the end of the experiment. Two dilutions of the solution were tested. Low dosis: 1+4 dilution with 0.9% NaCl and high dosis: 1+1 dilution with 0.9% NaCl. Transplantation procedure. The transplant was placed in the abdomen and connected to the abdominal aorta and vena cava. The function of the transplant was followed daily by abdominal palpation. The experiment finished when the heartbeat stopped as described by Steinbrüchel (1996). Flow cytometry. Samples of heparinized, peripheral blood were obtained. Surface staining was based on other studies (Kemp et al. 2001). In brief, 50-υl aliquots were labelled for 20 min. at room temperature with directly fluorochrome-conjugated monoclonal antibodies to CD3 (clone G4.18; Pharmingen, San Jose, CA, USA), CD4 (clone ox-35; Pharmingen) or CD8 (clone ox-8; Pharmingen) or with non-specific isotype control antibodies, and analysed on a FACScan flow cytometer (Becton Dickinson). Samples were live-gated on lymphocytes by forward and side scatter, and data for 10,000 events were collected. Statistical analysis. Different groups were compared by one way ANOVA on ranks. Values of P<0.05 were considered significant. Results are presented as means and 95% confidence intervals. To investigate the influence of Wf10 on survival of transplanted animals we administered Wf10 in low and high dose (table 1). A statistically significant difference was found when comparing the groups with treated animals to the group with untreated animals. The survival time was increased when Wf10 was administered in a low dose. Furthermore, Wf10 increased survival time further when administered in a high dose. To investigate the influence of Wf10 on lymphocyte sub-populations in the blood we measured the frequency of CD3+, CD4+ and CD8+ cells of 5 groups of animals (fig. 1). The control group had significantly higher frequencies of both CD4+ and CD8+ T cells compared to the rest of the groups. Furthermore, the frequencies of CD4+ and CD8+ T cells were significantly higher in the transplanted animals without Wf10 treatment when compared to animals that had received Wf10 treatment. In contrast, no difference was seen when comparing the groups that had received Wf10 treatment, neither when non-transplanted animals were compared to transplanted animals nor when the dose of Wf10 was changed. Expression of CD3 (black bars), CD4 (grey bars) or CD8 (white bars) on lymphocytes in 42 rats. 1) the control group, 2) rats treated with high levels of Wf10, 3) animals with a transplant, 4) animals with a transplant treated with Wf10 in a low dose and 5) animals with a transplant treated with a high dose of Wf10. Means and 95% confidence intervals are shown. Wf10 is a tetrachlorodecaoxygen anion complex. It is produced in two forms, a chlorite matrix approved for clinical use in a systemic form (Wf10) and a more dilute topical form (oxoferin). Neither Wf10 nor its metabolites can be measured in vivo, a fact that limits information but its function is thought to be due to chlorite molecules acting with haemoproteins. When given systemically, Wf10 affects the immune system in several ways, two seem to be prominent, macrophages are activated, but antigen presentation by macrophages is diminished (McGrath et al. 1998). In the present study the survival time of hamster hearts transplanted into normal untreated rats was approximately 3.5 days, which is a fairly constant survival time with rather small variations. This is consistent with earlier studies, yet with a tendency to a slightly longer survival in splenectomized animals (Kemp 1996). In the present study survival was prolonged in experiments where the rats were treated with Wf10. The larger doses resulted in the longest survival time. Although the prolongations were statically significant, they were very small when compared to cyclosporine and leflunomide. The general pattern of the transplanted hearts was outspoken rejection, although delayed in the treated animals, with disintegration of the myocardium, and with heavy deposition of IgG and IgM in the walls of the small vessels (not shown) corresponding to the existence of xenoantigens in these structures. All the described alterations were in accordance with the terminal phase of rejection. Wf10 alone without transplantation did not induce these changes (Hansen et al. 2001). CD4+ and CD8+ T cells greatly affect survival of xenografts (Wecker 1994). Therefore, we investigated the effect of Wf10 on CD4+ and CD8+ T cells both in transplanted and in non-transplanted animals. Wf10 significantly reduced the number of both CD4+ and CD8+ T cells in the peripheral blood of the rats and no differences were found when comparing transplanted and non-transplanted animals treated with Wf10. Transplantation without Wf10 treatment also induced a decrease of peripheral T cells, but significantly lower when compared to Wf10-treated animals. Our hypothesis is that Wf10 induces a general down-regulation of T cells, whereas the down-regulation seen after transplantation reflects a temporary reallocation of T cells away from the periphery into the transplant. Wf10 prolongs survival of xenografts in this model but only for a few days. In this model we obtained graft survival of more than 3 months with cyclosporine and leflunomide (Kemp 1996), of another magnitude than the few days seen in the present study. In a heavily immunosuppressed rat, we found graft survival of half a year. Thus, the question of whether Wf10 has any relevance in the treatment of xenotransplantation is still open. However, Wf10 alone does not seem to be an immunosuppressive candidate for xenotransplantation, but it may decrease the effective dose of drugs such as cyclosporine and leflunomide in order to improve renal tolerance. Gitte Grauvert and Diana S. Dræby are thanked for excellent technical assistance. Dr. Th. Isensee and the firm Oxochemie for supplying the Wf10.