INTRAGRAFT EXPRESSION OF GRANULYSIN AND PERFORIN IN ACUTE HUMAN RENAL ALLOGRAFT REJECTION

Abstract

174 Introduction: Granulysin (GL) is a novel cytolytic molecule that may be synergistic with the other cytolytic granule constituents, granzymes and perforin, in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. We have recently presented data that GL is present in mononuclear cells (MNC), polymorphs and eosinophils by tissue immunohistochemistry in renal allograft specimens. In the current study we have expanded our observations by staining larger numbers of biopsy specimens with acute rejection and graft dysfunction. Methods: Renal biopsy specimens were obtained from children and adult renal transplant recipients during episodes of graft dysfunction (GD) and acute rejection (AR) episodes. Murine anti-human GL antibody was used to examine GL expression by immunohistochemistry. Competitive RT-PCR was performed on similar samples. Results: 28 renal allograft adult and pediatric tissue samples were examined for GL expression by IH and by RT-PCR. Tissue was obtained as core biopsies, wedge biopsies or as transplant nephrectomies. 18 patients had AR, 6 had GD and there were 4 donor biopsies. Nephrectomy specimens had vast numbers of strongly positive MNC in contrast to the core biopsy AR specimens, where the staining was patchy. GL staining of MNC was largely granular in appearance, localizing to the interstitial and peri-tubular MNC infiltrate and staining infiltrating intra-tubular and endothelial MNC. Occasional PMN and rare eosinophil staining was detected in all biopsies including the 4 donor biopsies. GL positive casts were found in abundance in the urinary space in allograft biopsy specimens with AR. Perforin staining was more patchy than GL staining in AR biopsies and largely involved the tubular epithelial cells rather than infiltrating MNCs. Donor biopsies showed very rare PMN staining for perforin. GL/GAPDH message assessed by RT-PCR was not significantly different between samples with AR versus GD obtained by core biopsy. However, message was greatest in transplant nephrectomy specimens (AR=0.001, GD=0.0004, Nephrectomy=0.003). CONCLUSIONS: There appears to be a positive correlation between MNC GL expression and AR. In addition, GL tissue expression may serve as an indicator of both the tempo of AR and its response to steroids. Increased immunosuppression to treat AR is likely suppressing GL expression. This may explain why we see patchy staining in our core biopsy AR specimens as these patients are still receiving immunosuppression. GL staining of urinary casts suggests that GL may be found in the urine of AR patients. We are currently investigating this possibility. The presence of rare PMN and eosinophil staining in all tissue specimens (AR, GD and donor) is a novel finding as GL expression has only been found in CTL and NK cells till now. Further studies need to be done to ascertain other GL expressing cell types.