EXPRESSION OF GRANULYSIN IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) CORRELATES WITH ACUTE RENAL ALLOGRAFT REJECTION IN CHILDREN

Abstract

171 Introduction: Granulysin (GL) is expressed by CTL and NK cells and causes target cell death by granule mediated cytolysis and apoptosis. We have previously shown that is expressed in PBMC, mononuclear cells and eosinophils by tissue immunohistochemistry on renal transplant biopsy specimens. Methods: We looked for GL expression in PBMC of 20 pediatric renal transplant recipients with acute rejection (AR) and 7 graft dysfunction without acute rejection (GD). Expression analysis for GL was done using competitive reverse transcriptase polymerase chain reaction (RT-PCR) with message for glyceraldehyde phosphate dehydrogenase (GAPDH) serving as transcription control. We also collected urine during AR and in the peri-transplant period from 10 children and performed ELISA on centifuged sediment using murine anti-human monoclonal antibodies. Results: The GL/GAPDH was 0.099 in the AR vs 0.010 in the GD cohort (p< 0.005). There was a significant reduction in the GL/GAPDH value in the AR group, after treating AR with high dose steroids, suggesting that steroids may down-regulate GL expression in PBMC. To examine this further, we looked at GL expression in the peri-transplant period in 10 pediatric renal transplant recipients with living related transplant donation on our standard immunosuppression (Dacluzimab, Prednisone, CsA and Azathioprine). Blood was drawn on the day before transplant (D-1), day of transplant (D0) and days 1 and 7 post-transplant (D1 and D7). The mean GL/GAPDH values were 0.017 (D-1), 0.002 (D0), 0.008 (D1) and 0.015 (D7) suggesting that GL message is reduced with onset of high dose immunosuppression. Furthermore, in vitro FACS staining of PHA-induced PBMC GL expression was reduced after treatment with dexamethasone but not after treatment with CsA. GL was barely detectable in the urine prior to transplantation but increased significantly on D0 and D1 post transplant and with acute rejection episodes. Quantitative ELISA is being set up to investigate this as a non invasive marker of acute rejection. Conclusions: We have found that GL mRNA is significantly elevated in the PBMC of children with acute renal allograft rejection as compared to other causes of graft dysfunction. In addition, we have presented evidence, both in vitro and in vivo, that immunosuppression with steroids leads to a decreased GL expression. We continue to investigate the role of the novel molecule GL in the transplanted human kidney and its possible use as a marker for rejection.