Intragraft Cytokine Gene Expression Research Articles

Gene Transfer of Heme Oxygenase-1 Using an Adeno-Associated Virus Serotype 6 Vector Prolongs Cardiac Allograft Survival

Introduction. Allograft survival can be prolonged by overexpression of cytoprotective genes such as heme oxygenase-1 (HO-1). Modifications in vector design and delivery have provided new opportunities to safely and effectively administer HO-1 into the heart prior to transplantation to improve long-term graft outcome. Methods. HO-1 was delivered to the donor heart using an adeno-associated virus vector (AAV) with a pseudotype 6 capsid and vascular endothelial growth factor (VEGF) to enhance myocardial tropism and microvascular permeability. Survival of mouse cardiac allografts, fully or partially mismatched at the MHC, was determined with and without cyclosporine A. Intragraft cytokine gene expression was examined by PCR. Results. The use of AAV6 to deliver HO-1 to the donor heart, combined with immunosuppression, prolonged allograft survival by 55.3% when donor and recipient were completely mismatched at the MHC and by 94.6% if partially mismatched. The combination of gene therapy and immunosuppression was more beneficial than treatment with either AAV6-HO-1 or CsA alone. IL-17a, b, e and f were induced in the heart at rejection. Conclusions. Pretreatment of cardiac allografts with AAV6-HO-1 plus cyclosporine A prolonged graft survival. HO-1 gene therapy represents a beneficial adjunct to immunosuppressive therapy in cardiac transplantation.

Cytokine single nucleotide polymorphisms and intrarenal gene expression in chronic allograft nephropathy in children

Single nucleotide polymorphisms (SNPs) have been reported to influence cytokine production. Certain cytokine high producer genotypes have been associated with an increased risk for acute rejection and chronic allograft dysfunction (CAD) after transplantation. Our study evaluates SNP distribution for transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) in pediatric renal transplant recipients and control individuals and correlates them to corresponding intrarenal gene expression. SNPs for TGF-beta1 (codon 10 and 25), IL-10 (positions -1082, -819, -592) and TNF-alpha (position -308) were determined in 30 patients with stable graft function, in 75 patients with CAD, and in 173 control individuals by allele-specific polymerase chain reaction (PCR). Intrarenal cytokine gene expression was studied in 25 biopsies with chronic allograft nephropathy (CAN) and 27 normal kidney specimens by real-time reverse transcription (RT)-PCR. No difference in allele and genotype frequency was detected in any of the investigated groups. No correlation between genotype and intragraft cytokine gene expression was recognized in CAN patients. However, in normal kidney specimens, the low producer TGF-beta1 genotype at codon 10 was associated with significant lower TGF-beta1 mRNA expression. This association was not found for IL-10 or TNF-alpha. Our results do not support the proposition that certain specific cytokine genotypes for TGF-beta1, IL-10, and TNF-alpha are associated with CAD. Intrarenal cytokine gene expression only correlated to one TGF-beta1 SNP in normal kidney specimens. Since overall TGF-beta1 expression was higher in transplanted patients compared to controls, this suggests that SNPs may not play a significant role once the immune system is activated.

Immunotolerance of liver allotransplantation induced by intrathymic inoculation of donor soluble liver specific antigen.

To study the effects of liver specific antigen (LSA) on the immunoreaction of liver allotransplantation and its significance. Orthotopic liver transplantation was used in this study. Group I: syngeneic control (Wistar-to-Wistar); Group II: acute rejection (SD-to-Wistar). Group III: acute rejection treated by intramuscular injection of cyclosporine A (CsA) (SD-to-Wistar+CsA). Group IV: Intrathymic inoculation of SD rat LSA one week before transplantation (LSA+SD-to-Wistar). The common situation and survival time, rejection grades, NF-kappaB activity of splenocytes and intragraft cytokine gene expression were observed to analyze the acute rejection severity and immune state of animals. The common situation of Wistar-to-Wistar group was very good after the transplantation and no signs of rejection were found. Recipients of SD-to-Wistar group lost body weight progressively. All died within 9 to 13 days after transplantation with the median survival time of 10.7+/-0.51 days. It was an optimal control for acute rejection. The common situation of SD-to-Wistar+CsA group was bad during CsA medication but only with mild rejection. As for LSA+SD-to-Wistar group, 5 of 6 recipients survived for a long time and common situation was remarkably better than that of SD-to-Wistar group and SD-to-Wistar+CsA group. Its rejection grades were significantly lower than that of SD-to-Wistar group (P=0.026). Furthermore, no significant discrepancies of rejection were found between SD-to-Wistar group and LSA+SD-to-Wistar group at day7 and day12 (P=0.067). NF-kappaB activity, IFN-gamma and IL-2mRNA expression were significantly inhibited in LSA+SD-to-Wistar group compared with that of SD-to-Wistar group (P<0.05). LSA is an important transplantation antigen which involves in the immunorejection of liver transplantation directly. We reported for the first time that intrathymic inoculation of LSA can induce immnotolerance of liver allotransplantation and grafts can survive for a long time thereby, thus leading to a novel way to liver transplantation immunotolerance.

Host systemic and local nitric oxide levels do not correlate with rejection of pig proislet xenografts in mice.

The rejection of pig proislet xenografts in mice is a CD4 T cell-dependent process in which macrophages play an important role. To assess the potential for activated macrophages to act as effector cells in xenograft destruction, we have examined the relationship between proislet xenograft rejection, two principal markers of macrophage activation, transcription of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO), and their temporal relationship to intragraft cytokine gene expression. Xenograft rejection in CBA/H mice correlated with early induction of intragraft host iNOS mRNA and marked intragraft production of NO (reactive nitrogen intermediates, RNI). Intragraft mRNA expression for IFN-gamma, IL-1beta and TNF, cytokines associated with macrophage activation, was also found. These findings suggested that activated macrophages could be contributing to xenograft destruction via local NO-mediated toxicity at the graft site. To test the role of NO in this model: (1) Q-fever antigen (QFA) was administered to recipient mice in order to induce high systemic RNI levels and (2) in another experiment, pig proislets were transplanted into iNOS-/- mice. Treatment with QFA correlated with prolonged xenograft survival at 7 days post-transplant. Splenocytes from QFA-treated, but not control mice at 7 and 22 days post-transplant, exhibited inhibition of secondary xenogeneic mouse antiporcine mixed lymphocyte reaction (MLR) that was reversed by culture with the NOS inhibitor N-methylarginine (NMA). Despite continued elevated NO production, xenograft protection was temporary with complete rejection by day 22. Evidence that locally produced NO was not contributing to rejection was seen when pig proislets transplanted into iNOS-/- mice were rejected with normal kinetics; in these animals intragraft NO production was not detected (despite porcine iNOS gene expression). Failure of activated macrophages to achieve indefinite xenograft survival suggests that other factors are also required. Macrophage potential to effect either destructive or protective roles after pig proislet xenotransplantation suggests that such functions may depend on the site and magnitude of macrophage activation. Together these findings clearly demonstrate that high NO levels in the periphery are not damaging to xenogeneic islet tissue, neither host nor donor NO production is essential for islet xenograft rejection and consequently elevated plasma RNI levels do not represent a direct marker for rejection.

Predominant expression of T helper 2 cytokines and altered expression of T helper 1 cytokines in long-term allograft survival induced by intrathymic immune modulation with donor class I major histocompatibility complex peptides.

We have recently demonstrated that three synthetic peptides corresponding to the alpha-helices of the alpha1 and alpha2 domains of the donor class I RT1.Aa molecule served as efficient CD4+ T-cell epitopes for indirect recognition of this molecule during cardiac allograft rejection in the PVG.R8-toPVG.1U rat strain combination. These peptides induce long-term graft survival when injected into the thymus 7 days before transplantation under the cover of transient immunosuppression with anti-rat lymphocyte serum. In this study, we analyzed intragraft cytokine gene expression to test whether immune deviation to the T helper (Th) 2 response is associated with long-term allograft survival in this model. Intragraft cytokine gene expression was analyzed using a competitive reverse transcription polymerase chain reaction method we developed for this study. Cytokine gene expression was quantified in control allografts (n=5) with acute rejection and allografts from intrathymically manipulated recipients with acute rejection (n=5), delayed rejection (n=7), or no rejection (n=8). Long-surviving allografts expressed high levels of interleukin (IL)-4, IL-10, transforming growth factor (TGF)-beta, interferon (IFN)-gamma, and undetectable levels of IL-2. Allografts that were rejected in a delayed fashion expressed mostly IL-2, IFN-gamma, and TGF-beta with low or undetectable levels of IL-4 and IL-10. Acutely rejected allografts from unmanipulated controls or peptide-manipulated recipients expressed high levels of IL-2, IFN-gamma, TGF-beta and undetectable levels of IL-4 or IL-10. All allografts also expressed T-cell receptor Cbeta gene, providing evidence for the presence of T-cell infiltrates in the grafts. These observations demonstrate that acute graft rejection in this model is associated with the expression of Th1 cytokines, IL-2, and IFN-gamma, whereas long-term survival is associated with predominant expression of Th2 cytokines, IL-4, and IL-10. The expression of IFN-gamma in long-surviving allografts in the absence of IL-2 provides evidence for altered activation of the Th1 response in this intrathymic immune modulation model.

Pre-transplant donor-specific transfusions induce allograft rejection and IL-2 gene expression in the WKY → F344 functional tolerance model of rat lung transplantation

Our previous studies have shown that a spontaneous functional tolerance develops in a rat model of lung transplantation (WKY→F344). The tolerance observed in this model may be due to the minor histocompatible differences in this combination, however, the possibility of a tolerogenic effect related specifically to the lung allograft must be considered. To further examine this model, the effect of pre-transplant donor-specific spleen cell transfusions (DSTs) was examined on the functional tolerance state seen in this model. F344 rats received WKY spleen cells on days −45 and −30 before lung transplantations. Control F344 rats received lung transplants without DSTs. Recipients in both groups were killed on day 7, 14, 21 and 49 post-transplant, and allograft rejection (AR) was graded histologically (stage 0–IV). Intragraft cytokine gene transcripts were examined on day 7 and 14 post-transplant using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to investigate the underlying immunological events occurring in each group. In addition, allogeneic (WKY) and third party (BN) skin grafts were placed on lung recipients at day 35 post-transplant to evaluate the development of systemic tolerance. It was seen that control animals showed moderate to severe lymphocytic infiltrations (stage II–III AR) in the first 3 weeks followed by spontaneous recovery with stage I–II AR on day 49. In marked contrast, DST-treated animals showed more aggressive AR with severe lymphocytic infiltration and haemorrhagic infarction (stage III–IV AR) by day 14–21, without any evidence of recovery on day 49. WKY skin grafts showed prolonged survival in control animals, but were promptly rejected in DST-treated animals. Intragraft cytokine gene expression in control animals was characterized by no or weak expression of IL-2 and high IL-10, while DST-treated animals showed high levels of IL-2 transcripts. IL-2:IL-10 and IL-2:IL-4 ratios were significantly increased in DST-treated animals compared with controls on day 7 post-transplant.

Intragraft cytokine gene expression: implications for clinical transplantation.

As our knowledge of the cytokine network in experimental transplant models grows, we need to understand how and to what extent cytokines mediate the various donor-directed immune events in clinical situations. This overview of clinical cytokine measurements shows that specific intragraft cytokine messenger RNA (mRNA) expression profiles can be associated with acute rejection, that they may reflect the efficacy of immunosuppression, and that they can identify patients at risk for the development of early chronic rejection. The literature also shows that acute rejection and immunological quiescence in humans are not restricted to the cytokine patterns defined in the type 1/type 2 paradigm. This apparent lack of association may be caused by the immunosuppression used in the clinic but may also be the result of the infinite diversity of donor and recipient factors, in which polymorphisms in cytokines and cytokine receptor genes may play a central role.

Cytokines and Autoimmune β Cell Destruction in NOD Mouse Fetal Pancreas Isografts in

The role of cytokines in a model of cyclophosphamide (CP)-accelerated β cell destruction in fetal pancreas isografts transplanted into NOD mice was studied. One group of prediabetic NOD mice was injected with CP at a dose of 300 mg/kg ip and 7 days later isografts of organ cultured fetal pancreas (FP) were transplanted under the kidney capsule of these and untreated control mice. The mice were killed at several time points posttransplantation and the histological appearance of the host pancreas used to evaluate the disease progress in the grafts since previous studies had shown good corellation between isograft and native pancreas pathology. Intragraft cytokine gene expression was monitored by reverse-transcriptase polymerase chain reaction (RT-PCR) at the same time points and the expression levels between the experimental groups compared to normal kidney tissue. In comparison to isografts from non-CP injected mice, isografts from CP-treated mice showed increased expression of IFN-γ, TNF-α, TNF-β, IL-5, and eotaxin but no increase in IL-10 expression. The enhanced transcription of these cytokines correlated with massive infiltration of immune cells and ongoing β cell destruction in the host pancreas of the CP-treated recipients.

Efficacy of inhaled cyclosporine in lung transplant recipients with refractory rejection: Correlation of intragraft cytokine gene expression with pulmonary function and histologic characteristics

Refractory rejection is a major cause of morbidity and death among lung transplant recipients. Traditional rescue therapies have proved only modestly successful. We recently demonstrated the safety of inhaled cyclosporine for patients with end-stage chronic rejection; this trial was extended to patients with refractory acute rejection. The present study was to determine whether effective inhaled cyclosporine therapy was correlated with suppression of cytokine gene expression. Twelve lung transplant recipients were studied. Maintenance therapy, cyclosporine or FK 506, azathioprine, and prednisone, was continued, and inhaled cyclosporine at a dose of 300 mg/day was added. Pulmonary function testing and histologic characteristics from transbronchial biopsy specimens were used to assess efficacy of therapy. Bronchoalveolar lavage (BAL) and peripheral blood cells were analyzed for the presence of messenger RNA by using 32P-labeled primers of cytokines interleukin-2 (IL-2), IL-6, IL-10, and interferon-gamma (gamma) via reverse transcriptase-polymerase chain reaction. Nine of 12 patients (five with acute rejection, four with chronic rejection) exhibited histologic resolution of rejection within 3 months of inhaled cyclosporine therapy. Pulmonary function (forced expiratory volume in 1 second) improved from pretherapy levels in the patients with acute rejection (p < 0.05). All of the nine histologic responders exhibited 4- to 150-fold decreases (p < 0.05) in IL-6 and interferon-gamma messenger RNA levels in the BAL, whereas the three patients who failed exhibited persistent or increased cytokine profiles. IL-2 and IL-10 in BAL and peripheral blood lymphocyte cytokines were not informative. These results indicate that inhaled cyclosporine is effective therapy for refractory pulmonary rejection and that its mechanism of action is associated with suppression of proinflammatory cytokines IL-6 and interferon-gamma within the allograft.

IN VIVO DEPLETION OF CD8+ T CELLS RESULTS IN Th2 CYTOKINE PRODUCTION AND ALTERNATE MECHANISMS OF ALLOGRAFT REJECTION

A current hypothesis states that Th1 cytokines promote allograft rejection and that Th2 cytokines promote graft acceptance. We present data that question the tolerogenic activity of Th2 cytokines, and we suggest that Th2 cytokines may evoke allograft rejection by recruitment of alternate effector mechanisms. Unmodified rejection of mouse heterotopic cardiac allografts is associated with the accumulation of large numbers of donor-reactive CD8+ CTL within the allograft, which is indicative of a Th1-driven cellular response. However, when recipients are depleted of CD8+ CTL, rejection still occurs and is associated with an aggressive cellular infiltrate rich in eosinophils, large mononuclear cells, and fibroblast-like cells. Eosinophils, which are responsive to the Th2 cytokines IL-4 and IL-5, are not present in unmodified rejecting allografts. Differential production of Th1 versus Th2 cytokines was further suggested by altered levels of IgG2a (promoted by IFN gamma) and IgG1 (promoted by IL-4) alloantibody in the sera of these mice; IgG2a dominated the alloantibody response in unmodified allograft recipients, whereas IgG1 levels increased in recipients depleted of CD8+ CTL. Altered intragraft cytokine gene expression was verified by RT-PCR; Th1 (IL-2, IFN gamma), but not Th2 (IL-4, IL-5, IL-10), cytokine mRNAs were readily detectable in the allografts of unmodified recipients. In contrast, both Th1 and Th2 cytokine genes were expressed in the allografts of mice depleted of CD8+ CTL. These data suggest that donor-reactive CD8+ CTL inhibit intragraft production of Th2 cytokines, thereby promoting a Th1 dominated-rejection response. Elimination of CD8+ cells allows Th2 cytokine production, which may have deleterious, rather than protective, effects.

The effects of inhaled cyclosporine A (CYA) in lung transplant recipients: correlation between histology and intragraft cytokine gene expression

The effects of inhaled cyclosporine A (CYA) in lung transplant recipients: correlation between histology and intragraft cytokine gene expression