PRECONDITIONING OF ORGAN DONORS WITH LOW LEVELS OF CARBON MONOXIDE REDUCES GRAFT IMMUNOGENICITY AND IMPROVES LONGTERM ALLOGRAFT FUNCTION

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

Background: Chronic rejection remains the major obstacle for successful transplantation, and to date there is no effective treatment. Events occurring prior to organ transplantation such as brain death, harvesting procedure, and ischemia-reperfusion injury lead to unspecific inflammatory damages danger signals - that increase graft immunogenicity and reduce long-term graft survival. To induce the acceptance of transplants without lifelong immunosuppression, we should mimic the bodys own way to induce tolerance, i.e. by blocking the endogenous alarm signals and inhibiting the costimulatory signals. Graft damage prior to transplantation can be avoided by optimal donor maintenance, careful organ harvesting, short preservation time (cold and warm ischemia), and donor preconditioning to make grafts more resistant to injury. Reduction of graft immunogenicity before engraftment may have a great impact on graft function and survival without the undesirable effects of immunosuppressive treatment for the organ recipient. Many studies have shown the induction of protective genes after cell injury. Overexpression of these genes has provided increased graft function and survival and has been implicated in the process of graft tolerance. One of these genes decodes the enzyme heme oxygenase-1 (HO-1), which may be the most important cytoprotective mechanisms against cellular stress. One of HO-1 downstream products, carbon monoxide (CO), has been shown to mediate its protective effects. Purpose: Here, we wanted to test the effects of exclusive carbon monoxide therapy to donor animals shortly prior to organ harvesting. Methods: Four sets of experiments were carried out in the rat (n=6/group). In all experiments, 4 hours prior to organ harvesting, donor animals were either treated with methylene chloride (MC) (1x 100 mg/kg p.o.), to deliver carbon monoxide, or remained untreated. In the first experiment, a well-established model for chronic renal allograft rejection was used (F-344 to Lewis). Recipients received no treatment, short-term (10 days) or long-term (180 days) MC treatment (100 mg/kg/day p.o.). Animals were followed for 6 months and proteinuria assessed at monthly intervals. After this period, grafts were analysed by histology and immunohistology (CD4+, CD8+, MHC class II, ED1+ cells). In the second set of experiments, using the same transplant combination, grafts from CO (MC) treated (-4 hs) and untreated donors were examined 24h after transplantation to investigate early inflammatory changes by real-time RT-PCR. In the third experiment, using an acute rejection model to investigate the role of CO induction on dendritic cells (DCs), kidneys from DA rats were transplanted into untreated LEW rats. Analysis of grafts, spleens, perigraft lymphnodes (LN) and blood of LEW recipients was performed at day 1 post transplantation. Donor-specific MHC-class II + dendritic cells were determined by haplotype-specific mAb and flowcytometry (RT1ab+, OX62+). Cell markers (CD4/CD8+ T cells, ED1+ monocytes, MHC class II+/ CD86+DC) were measured by immunohistochemical staining. T-cell alloreactivity of recipient splenocytes was measured by ELISPOT. In the fourth set of experiments we investigated the expression of 820 genes after pre-treatment with CO (MC) through DNA microarray analysis. Another set of experiments was used to assess the toxicity of the therapy using hematological and biochemical parameters and blood gas analysis. Results: Carboxyhemoglobin levels peaked by 2 h after MC administration (COHb 5.5 % 2.1%), and had returned to base-line values by 24 h. In the first experiment, using a chronic rejection model, there was no difference in the survival rate among groups 6 months after transplantation. However, proteinuria was significantly reduced following CO (MC) treatment both after a long-term administration to the recipient and after a single treatment in the donor (control: 60 20 mg/24h vs. CO (MC) recipient long-term: 11 2 mg/24h and CO (MC) donor treatment: 15 1 mg/24h, p<0.05). Morphological alterations characteristic of chronic graft deterioration were impressively improved following CO delivery in the donor (glomerulosclerosis by 180 days: 20 5 % following donor treatment; 30 8% following longterm CO (MC) administration, and 8010% in controls, p<0.0001 and p<0.001, respectively); arteriosclerosis (p<0,01), tubular atrophy (p<0.0001), and fibrosis (p<0.0001) were also reduced in both groups. While ED1+ monocytes-macrophages, CD4+ T cells and MHC II expression were significantly (p<0.01) reduced by 6 months in animals receiving CO (MC) long-term; cellular infiltrates and MHC II expression were comparable in grafts receiving CO (MC) as donor treatment, over a short-term recipient treatment or in untreated controls. In the second experiment, 24h after transplantation, grafts from CO (MC) treated donors showed a reduced expression of CD3 mRNA (p<0.05, n=4), reflecting a reduced infiltration of T-cells; Th1-Th2 shift of cytokines (increased expression of IL-10 and IL-4, and decreased IFN-γ and IL-2) and increased expression of anti-apoptotic gene Bcl-2. In the third experiment, using an acute rejection model, donor treatment for induction of CO significantly reduced the number of donorspecific DCs in the graft, recipient spleen, blood, and regional lymphnodes (p=0.07, p=0.018, p=0.033, p=0.07) and intragraft CD4+ T-cells. The reduced amounts of donor-derived DC in the CO (MC) treated group were associated with reduced frequencies of alloreactive T cells, as measured by ELISPOT at day 1 (IFN-γ+ cells/CD4+ T cells: p<0.01) and reduced graft infiltration of CD4+ T-cells (p<0.05). In the fourth experiment, CO (MC) treatment to the donor prior to harvesting resulted in a different pattern of gene expression compared to recipients of untreated grafts, with increased expression of anti-inflammatory genes and reduction of pro-inflammatory genes. After donor pre-treatment with CO the expression of adhesion molecules (integrins 5,6,7, and 9, and PECAM), NF-KB, kinases (MAPKs 3,7,9,14), TGF1-B, and TNF-a was reduced, while the expression of heat-shock proteins (HSP-70, HSP86) and IL-18 was increased. CO (MC) therapy was well tolerated and toxicological assays showed only minor changes in electrolyte balance, and hepatoxicity in the long-term (for 6 months) treated group. Conclusion: Carbon monoxide delivery in the donor was associated with reduced graft immunogenicity and marked improvement of long-term graft function. Because the HO-1/CO system uses a multitude of protective pathways, its modulation seems to be a promising approach to improve graft survival. CO donor pre-treatment is a simple strategy to reduce ischemia-reperfusion injury and graft immunogenicity in order to improve long-term graft function. The reduced graft immunogenicity associated with donor pre-treatment may also decrease the demand of immunosuppressive drugs after transplantation, reducing the risks of immunosuppression. The brief and low-dose carbon monoxide therapy in human cadaver donors could reduce the concerns about CO toxicity, and could still have a major impact on transplantation outcome. This could be particularly beneficial for transplantation of marginal grafts, which are intrinsically highly immunogenic. Studies assessing CO preconditioning in more clinical relevant models, like large mammals, brain-dead, marginal and pre-sensitised donors, should be performed before its use in clinical settings is considered. Questions relating to the optimal dose of CO, way of administration, time interval before harvesting, interaction of CO with other drugs and methods to monitor the efficacy of CO therapy remain open. Even if CO therapy will not be safe enough to be used in the clinic, the modulation of an intermediate or down-stream mediator of CO should be encouraged, since it uses a multitude of protection pathways CO is articularly attractive to improve graft survival. In a relatively short time, CO exposure has changed from an absolute contra-indication for organ transplantation to a promising therapeutic tool against ischemia/reperfusion graft rejection. This is to our knowledge the first study to show the beneficial effects of exclusive donor preconditioning with CO in a chronic rejection model in the rat.

ASSUNTO(S)

chronic rejection imunologia do transplante kidney transplantation methylene chloride cirurgia de transplantes carbon monoxide cirurgia experimental donor pre-treatment chronic rejection microcirurgia experimental immunogenicity preconditioning ischemia/reperfusion injury

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