Hamster cardiac xenografts are protected against antibody mediated damage, early after transplantation to Lewis rats.

Antibodies play a crucial role in the rejection of xenografts. We tested the hypothesis that xenografts are protected against antibody-mediated attack early after transplantation in a concordant model. We investigated the role of xenoreactive antibodies as a stimulus for protection and the effects of a total blockade of the antibody response by the leflunomide analog malononitrilamide 279. Hamster cardiac xenografts were transplanted to Lewis rat recipients. Second transplants and retransplants of xenografts were performed to untreated rats that had a xenograft in place for 3 d. Untreated rats rejected hamster cardiac xenografts after 4.0 +/- 0.0 d. Significant levels of anti-donor IgM, as measured by flowcytometry, were present on day 3 after transplantation (11.2% +/- 2.8 vs. 1.2% +/- 0.0 on day 0, P < 0.001). 'Fresh' second xenografts transplanted to rats that had a first xenograft in place for 3 d and had anti-hamster antibodies, underwent hyperacute rejection. The first xenografts remained functioning. Xenografts that were removed on day 3 from untreated rats and then retransplanted remained functioning. Xenografts that were removed on d 3 from rats that had been treated with malononitrilamide 279, 15 mg/kg/d and were retransplanted underwent hyperacute rejection. IgM levels at the time of removal were 1.1% +/- 0.5 in these rats and not different from baseline (P = 0.96). We conclude that xenografts are protected against antibody-mediated damage early after transplantation. The presence of anti-donor antibodies might be an essential stimulus for the induction of protection. There seems to be a delicate balance between the injurious and protective effects of antibodies. Treatment strategies that are designed to block antibody formation completely might prevent the induction of protection.

Pre-transplant blood transfusion induces tolerance to hamster cardiac xenografts in athymic nude rats.

The effects of pre-transplant blood transfusion vary from induction of antibodies and accelerated graft rejection, to prolonged survival and even tolerance. The beneficial 'transfusion effect' in allotransplantation is believed to be merely T-cell mediated. In xenotransplantation, T-cell independent mechanisms form a major hurdle. In this study we investigated the effects of pre-transplant hamster blood transfusion on the survival of hamster cardiac xenografts in T-cell deficient athymic nude rats. Nude rats rejected xenografts after 3.8 +/- 0.5 d (n = 8), and immunocompetent Lewis rats after 4.0 +/- 0.5 d (n = 8), following a similar IgM response (P = NS). Hamster blood transfusion 3 d before transplantation in nude rats led to an IgM response and long-term xenograft survival in 17/20. Timing was of importance: blood transfusion 7 d before transplantation resulted in 45% long-term xenograft survival (n = 20). Injection of purified hamster erythrocytes, leukocytes or minced heart also led to survival of xenografts for > 100 d in nude rats, but not in all cases. Second xenografts transplanted to long-term survivors did not provoke an IigM response, and were accepted for > 100 d (n = 4). Transfer of serum from long-term survivors to untreated nude rats resulted in survival of xenografts for > 100 d (n = 4). In Lewis rats pre-transplant blood transfusion induced hyperacute rejection of xenografts after 158 +/- 128 min (n = 8, P < 0.01). We conclude that pre-transplant hamster blood transfusion can induce long-term survival of hamster cardiac xenografts in T-cell deficient athymic nude rats. This blood transfusion effect is mediated by humoral factors and can be transferred by serum. Elucidation of underlying mechanisms might provide some insight into xenotransplantation nonresponsiveness of T-cell independent immunefactors.

Hyperacute rejection in the guinea pig-to-rat model without formation of the membrane attack complex.

The guinea pig (GP)-to-rat transplantation model has been widely used to study hyperacute rejection (HAR) of xenografts. In this model heart graft survival beyond 8 days has never been reported. In contrast, survival times of kidney and heart grafts up to 62 days have been reported in the discordant pig-to-primate model. It is not clear why it is so much more difficult to obtain long-term graft survival in the GP-to-rat model as compared to the pig-to-primate model. We hypothesized that mechanisms other than activation of complement may be involved in the rejection of guinea pig grafts by rat recipients. Therefore, we have studied in detail the rejection of GP aortic grafts by rat recipients, either PVG/c+ (complement competent, group 1), or PVG/c- (complement C6 deficient, group 2). PVG/c- rats are not able to form the membrane attack complex (MAC) of complement. Forty-four GP-to-rat aortic transplantations were performed successfully. Recipient rats were sacrificed at various intervals after transplantation (4, 24 and 48 h, and 7 and 28 days, three to six animals per time point per group). Twenty-four hours after transplantation the number of cells in the media was significantly decreased from 11.1 +/- 0.9 cells/mm2 to 3.1 +/- 2.8 cells/mm2 in group 1, whereas the number of medial cells in group 2 remained the same. The number of medial cells was significantly decreased in both groups at 48 h post-transplantation (group 1: 1.8 +/- 2.2 cells/mm2; group 2: 5.5 +/- 3.0 cells/mm2). At that time no infiltrating cells were apparent in the grafts of either two groups. Seven days after transplantation, the number of medial cells remained low in group 1 (1.8 +/- 2.9 cells/mm2) but was increased in group 2 (10.7 +/- 2.6 cells/mm2) as a consequence of infiltrating immune cells. These infiltrating cells consisted mainly of macrophages, but also T cells and NK cells. At 28 days after transplantation the grafts in both groups were completely reorganized and no distinction could be made between media and adventitia. These results show that rejection of GP grafts by rat recipients can occur in the absence of both MAC of complement and immune competent cells. This MAC and immune cells independent type of rejection has not been described before and may explain the difficulty in obtaining long-term graft survival in the GP-to-rat xenotransplantation model.

IgG, but not IgM, mediates hyperacute rejection in hepatic xenografting.

We reported previously that no classical features of hyperacute rejection (HAR) could be found in liver grafts in the guinea-pig (GP)-to-rat model and that recipients died shortly after transplantation of non-immunologic causes. Thus, the GP-to-rat model is not suitable for studying the mechanisms of discordant liver xenograft rejection. In the hamster to rat model, long-term survival of a liver graft is possible, but extremely low levels of xenoreactive natural antibodies are present. To mimic a discordant situation with pre-formed IgM and IgG antibodies, we sensitized rats 1 or 5 weeks before grafting. Specific anti-hamster IgM antibodies were found in recipients sensitized at week -1 but not week -5. Anti-hamster IgG was present in all recipients, albeit considerably higher in animals sensitized 5 weeks before grafting. In these two models, we examined the mechanism of HAR of liver grafts and compared this with heart xenografts. Control heart and liver grafts were rejected 4 and 7 days after transplantation respectively. Liver grafts in recipients sensitized at week -5 showed venous congestion and bleeding after reperfusion, indicating HAR, however this was not observed after sensitization at week -1. This surprising finding was confirmed by histology. Massive extravasation, edema, and acute liver cell degradation were noticed in grafts subjected to HAR. Liver grafts of recipients sensitized at week -1 showed only minimal changes. Heart grafts were rejected hyperacutely in both sensitization models. IgG antibodies could be detected on liver grafts in the group sensitized at week -5 but not in the group sensitized at week -1. Minimal IgM depositions were found on liver grafts of animals sensitized 1 week before grafting. Rejected heart grafts from similar sensitization groups showed identical antibody depositions; only IgM depositions were massive. Complement depositions were found in all groups. These results indicate that IgG, but not IgM, mediates HAR in hepatic xenografting. Such a predominance of IgG over IgM does not exist for heart grafts.

Chronic rejection of concordant aortic xenografts in the hamster-to-rat model.

Several groups have demonstrated that it is possible to obtain long-term graft survival of concordant xenografts. One of the important questions that remains is whether xenografts are susceptible to chronic rejection. To answer this question we used the aorta transplantation model. One centimetre of hamster aorta was interposed in the abdominal aorta of Lewis rat recipients. The recipients were either untreated (group 1), or treated with 10 mg/kg cyclosporine (CsA), given intramuscularly three times a week (group 2). Rats were sacrificed at day 7, 14, 21, 28, 56 and 84 and the thickness of the intima, the media and the adventitia was measured. Furthermore, the cellularity of the media and the adventitia was assessed by counting the number of nuclei per 0.05 mm2 and immunohistochemistry of the aortic grafts was performed. Graft arteriosclerosis developed in aortic xenografts of both group 1 and group 2. In group 1, intimal lesions were already present from day 21 onwards in all rats, whereas in group 2 they were present only in 33% (2/6) of the rats. At day 84 all the grafts in group 1 were totally occluded, while those in group 2 were still open. The thickness of the media was slightly increased in both groups during the whole observation period, mainly due to edema. Although a few infiltrating macrophages could be seen, the number of nuclei per 0.05 mm2 of the media remained constant during the first 21 days, but declined sharply from day 21 onwards, as a consequence of disappearing myocytes. Thickness of the adventitia in both groups increased after transplantation due to infiltrating macrophages and T cells, reaching a peak at day 14. After day 14 the adventitial thickness in group 1 decreased rapidly to reach values comparable to group 2 from day 28 onwards. In conclusion, graft arteriosclerosis, as a sign of chronic rejection, occurs in concordant aortic xenografts. The lesions in the xenografts develop extremely rapidly, and compared to data from the literature, faster than in aortic allografts. The process of chronic rejection in aortic xenografts can be reduced by CsA.

Ketanserin reduces graft arteriosclerosis after allogeneic aorta transplantation in rats.

The serotonin-2 receptor antagonist ketanserin has been suggested to diminish arteriosclerotic development by its effect on platelet function and on vascular smooth muscle cells. We investigated the ability of ketanserin in reducing immune-mediated arteriosclerosis using the BN-WAG and WAG-BN rat aortic transplantation models. Ketanserin (10 mg/kg/day) administered in drinking water significantly reduced posttransplant arteriosclerotic thickening of the intima in the BN-WAG rat model to 102 +/- 23 mu m as compared with 171 +/- 60 mu m in untreated BN-WAG allografts 8 weeks posttransplantation (p < 0.05). In the opposite WAG-BN combination, at 4 weeks posttransplantation, no significant reduction in intimal thickening was attained (112 +/- 42 vs. 152 +/- 49 mu m). Platelet aggregation to increasing amounts of collagen did not show a correlation between the effect of ketanserin on platelet function and reduction in intimal thickening. Ketanserin had no effect on systolic blood pressure or mononuclear cell infiltration. We conclude that ketanserin reduces graft arteriosclerosis by a mechanism other than by inhibition of platelet function, decrease in blood pressure, or immunosuppression. Because of this antiarteriosclerotic effect, ketanserin therapy might be beneficial to the long-term survival of vascular allografts.

Suppression of acute rejection prevents graft arteriosclerosis after allogeneic aorta transplantation in the rat.

Rat aortic allografts develop arteriosclerotic alterations in the vascular wall that are histologically similar to those observed in chronic rejecting vascular allografts. We used cyclosporine and rapamycin (RAPA) in two different rat strain aorta transplantation models to investigate the effect of immunosuppression on posttransplant graft arteriosclerosis. High dose CsA (25 mg/kg, 3 times/week) treatment significantly inhibited intimal proliferation in the "strong" WAG-BN model (P < 0.01) as well as in the "weak" BN-WAG combination (P < 0.001), compared with untreated allogeneic controls. In the latter combination, even fewer intimal lesions were present than in WAG autotransplants, suggesting that CsA may also inhibit the arteriosclerotic response to mechanical injury. RAPA (3 mg/kg, 3 times/week) was as effective as CsA in reducing intimal lesions (P < 0.01 and P < 0.001 in the BN-WAG and WAG-BN models, respectively). Low dose CsA (5 mg/kg, 3 times/week) was only partially effective in preventing intimal lesions. Histology of the nontreated allografts showed ongoing acute rejection in the adventitial layer. The degree of cellular infiltration correlated with the severity of arteriosclerotic lesions. High dose CsA and RAPA treatment prevented adventitial infiltration in both models, while low dose CsA was only moderately effective. In conclusion, in the present study, the degree of arteriosclerotic involvement after allogeneic aorta transplantation was related to the severity of cellular adventitial infiltration. The myointimal thickening was inhibited by effective immunosuppressive treatment. These observations may imply that chronic rejection develops after ineffective immunosuppression.