Fan Pan

Tacrolimus and cyclosporine differ in their capacity to overcome ongoing allograft rejection as a result of their differential abilities to inhibit interleukin-10 production.

Background. Accumulated evidence from clinical transplantation has suggested that tacrolimus-based treatment can reverse ongoing allograft rejection in patients treated with cyclosporine (CsA)-based immunosuppression, even when a high dose of antirejection rescue therapy has failed. This evidence prompted us to investigate whether these two compounds, which share an in vitro mechanism, would differ in their abilities to regulate in situ cellular and molecular events during ongoing allograft rejection. Methods. The equivalent effective doses of tacrolimus (3.2 mg/kg/day) and CsA (10 mg/kg/day), when administered orally to Lewis rats for 10 days (day 0–9), were predetermined and defined as the ability of the drug to induce a similar survival of Brown Norway rat heart allografts with an equal suppression of intragraft interleukin (IL)-2 mRNA expression. To investigate the ability of each drug to rescue ongoing allograft rejection, Lewis recipients of Brown Norway rat heart grafts were left untreated for the first 5 days after transplantation. Tacrolimus or CsA was then administered at the equivalent effective dose for 10 days (days 5–14). Heart grafts and blood samples, harvested on days 3, 5, 7, and 10, were analyzed by reverse transcriptase-polymerase chain reaction, real-time quantitative polymerase chain reaction, ELISA, and immunohistology. Results. Ongoing allograft rejection was found to be rescued by tacrolimus but not by CsA at the equivalent dose (median survival time: untreated, 6 days; tacrolimus, 18 days; and CsA, 7 days). A significant suppression of local intragraft IL-10 mRNA expression and serum protein production along with a dramatic down-regulation of functional CD8+ T and NKR-P1a+ natural killer cell local infiltration by means of decreased of cytotoxic factor release, including granzyme B and perforin 1, was found to be associated with tacrolimus but not CsA treatment. However, both drugs inhibited other immune cells (CD4+ T cell, ED2+ macrophage) and cytokines (IL-1&bgr;, IL-2, IL-4, IL-6, IL-12, interferon-&ggr;, transforming growth factor-&bgr;, and tumor necrosis factor-&agr;) at almost the same levels. The inability of CsA to overcome ongoing allograft rejection could be rescued by cotreating recipients with neutralizing anti-IL-10 antibody on day 5 and day 6 after transplantation: anti-IL-10 antibody alone did not show such an effect. Conclusions. Inhibition of IL-10 production is a critical factor in the ability of tacrolimus to reverse ongoing allograft rejection.

Microarray-based gene expression profiles of allograft rejection and immunosuppression in the rat heart transplantation model.

Background. Gene expression profiling has the potential to produce new insights into complex biologic systems. To test the value of complement DNA arrays in identifying pathways involved in organ transplant rejection, we examined the gene expression profiles of rat heart allografts from recipients treated with or without immunosuppression to prevent acute allograft rejection. Methods. Heterotopic heart transplantation was performed using ACI or Lewis donors and Lewis recipients. Recipients were treated with tacrolimus (Tac) or cyclosporine (CsA) at the equivalent effective doses, and graft hearts were harvested on days 3, 5, and 7. A commercial microarray was used to measure gene expression levels of 588 genes in day 5 grafts. Selected genes were analyzed by reverse transcriptase-polymerase chain reaction. Results. The expression levels of 118 genes were perturbed in the untreated allograft in comparison with the isograft control, of which 77 genes were categorized as candidate genes for Tac- or CsA-mediated immunosuppression or both, and 41 as genes associated with other pathways. Among the 77 candidate genes, 55 genes shared the same response to suppression by both drugs, including inducible nitric oxide synthase, interferon-&ggr;, and interferon regulatory factor 1. Drug-specific effects were observed in 22 genes: Fourteen genes were exclusively reversed by Tac and eight by CsA. Conclusions. Gene expression profiling reveals a large variety of genes affected during acute rejection, indicating that multiple metabolic pathways, including immune and nonimmune responses, are involved in the local graft rejection events. The differences and similarities of the gene expression profiles relative to the two immunosuppressants may provide more detailed therapeutic approaches for optimal immunosuppression.

FK778, a powerful new immunosuppressant, effectively reduces functional and histologic changes of chronic rejection in rat renal allografts.

Background. FK778 is a new derivative of the active leflunomide metabolite A77 1726. It effectively prevented acute allograft rejection in several experimental transplant models, and it is currently in phase II trials in human transplant recipients. In this study, we examined the effects of FK778 in a well-established model of chronic renal allograft rejection in the rat. Methods. Kidneys of Lewis (LEW) and F344 rats were orthotopically transplanted into bilaterally nephrectomized LEW recipients as the isograft and allograft control, respectively. Allograft recipients were orally administered FK778 at doses of 3 mg/kg per day, 10 mg/kg per day, and 20 mg/kg per day for 10 days. Blood and 24-hr urine samples were collected once a week after grafting for plasma creatinine, allo-specific antibodies, and proteinuria determination. Kidney grafts were harvested on the 90th day after transplantation and subjected to histologic, immunohistologic, and reverse transcriptase-polymerase chain reaction analysis. Histologic sections were semiquantitatively scored using criteria adapted from the Banff’ classification for transplant pathologic conditions. Results. Recipients treated with FK778 for 10 days exhibited a dose-dependent decrease in proteinuria and plasma creatinine for the entire 90-day period after transplantation when compared with the allograft control. FK778, at doses of 10 mg/kg per day and 20 mg/kg per day, remarkably reduced chronic histologic changes, including tubular atrophy, glomerulosclerosis, fibrointimal hyperplasia, and transplant glomerulopathy. In addition, FK778 treatment was associated with decreased intragraft mononuclear cell infiltration, serum allo-specific immunoglobulin (Ig)M and IgG antibody production, and intragraft transforming growth factor &bgr; messenger RNA expression in those recipients surviving 90 days after transplantation when compared with the allograft control. Conclusion. FK778 effectively reduces functional and histologic chronic kidney allograft rejection in the rat.

Deletion of DOCK2, a regulator of the actin cytoskeleton in lymphocytes, suppresses cardiac allograft rejection

Allograft rejection is induced by graft tissue infiltration of alloreactive T cells that are activated mainly in secondary lymphoid organs of the host. DOCK2 plays a critical role in lymphocyte homing and immunological synapse formation by regulating the actin cytoskeleton, yet its role in the in vivo immune response remains unknown. We show here that DOCK2 deficiency enables long-term survival of cardiac allografts across a complete mismatch of the major histocompatibility complex molecules. In DOCK2-deficient mice, alloreactivity and allocytotoxicity were suppressed significantly even after in vivo priming with alloantigens, which resulted in reduced intragraft expression of effector molecules, such as interferon-γ, granzyme B, and perforin. This is mediated, at least in part, by preventing potentially alloreactive T cells from recruiting into secondary lymphoid organs. In addition, we found that DOCK2 is critical for CD28-mediated Rac activation and is required for the full activation of alloreactive T cells. Although DOCK2-deficient, alloreactive T cells were activated in vitro in the presence of exogenous interleukin-2, these T cells, when transferred adoptively, failed to infiltrate into the allografts that were transplanted into RAG1-deficient mice. Thus, DOCK2 deficiency attenuates allograft rejection by simultaneously suppressing multiple and key processes. We propose that DOCK2 could be a novel molecular target for controlling transplant rejection.

A blocking anti-CD28-specific antibody induces long-term heart allograft survival by suppression of the PKC theta-JNK signal pathway.

This study investigated the effects of a blocking anti-CD28 antibody (Anti-CD28-PV1-IgG3) in vitro and in vivo. Anti-CD28-PV1-IgG3, a hamster-mouse chimeric antibody against murine CD28, which does not provide CD28-positive signaling during TCR-driven T cell activation, enabled long-term survival of heart allografts across a complete mismatch of the MHC in rats. Among the T cell signaling proteins tested in the spleens from recipients, we found that recipients treated with anti-CD28-PV1-IgG3 exhibited suppression of alloantigen-initiated proximal TCR signaling events, including Lck, Zap70, Vav, and PI3K expression, and their PKC&thgr;- and JNK-regulated expression/activation. This leads to attenuation of intragraft T cell infiltration and expression of T cell effector molecules. These results indicate that targeting the CD28 receptor with a blocking antibody leads to long-term allograft survival by reducing activation of alloantigen-mediated key signaling events in T cells that might be crucial for full T cell activation.

Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

Background Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. Methods Splenocytes from C57BL/6 J (B6) AID−/− mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID−/− mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. Results The AID−/− splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID−/− recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID−/− recipients with further reduction of intimal hyperplasia and fibrosis scores. Conclusions The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.