RAPID, COMPREHENSIVE ANALYSIS OF HUMAN CYTOKINE MESSENGER-RNA AND ITSAPPLICATION TO THE STUDY OF ACUTE RENAL-ALLOGRAFT REJECTION

Cytokine mRNA analysis was performed on human renal allograft needle c ore biopsies by a PCR-based assay. The assay was specifically develope d to be capable of simultaneous analysis of multiple interleukin trans cripts (IL-1-IL-12), as well as those of other relevant cytokines, by one person in less than 1 day from cultured cells or directly from tis sue samples. It was initially used on preparations containing known am ounts of plasmid DNA encoding individual cytokine cDNA sequences, conf irming that the sensitivity of this technique was both well defined an d comparable for all target sequences tested. Analysis of human PBLs p rior to stimulation, after polyclonal stimulation with PHA and after s imultaneous treatment with PHA and MP or CyA, was also performed to sh ow a proportional relationship between mRNA levels measured by PCR and protein release measured by ELISA (R(2) = 0.86). This correlation was not adversely altered by pharmacologic immunosuppression by MP or CyA . Thus, this method of PCR primer design and usage was appropriate for the clinical study of cytokine mRNA levels during allograft rejection . Direct study of cytokine mRNA in allograft biopsy tissue showed that IL-2 was specifically and significantly (p = 0.006) elevated during A CR when compared to other causes of graft dysfunction. Transcripts fro m the IFN-gamma and IL-6 genes were also increased in ACR (p = 0.001 a nd 0.017, respectively), whereas increased IL-8 mRNA was correlated wi th irreversible loss of graft function (p = 0.02). TNF-alpha, IL-1 bet a, and IL-10 gene transcripts were also detected during ACR, but were not quantitatively increased compared to other forms of graft injury ( p > 0.2). We conclude that acute cellular rejection is associated with intragraft mRNA from the IL-2 gene. Other transcripts, including thos e from the IFN-gamma, IL-6, and IL-8 genes, are detected in increased amounts during this process. Messenger RNA from the TNF-alpha, IL-1 be ta and IL-10 genes is also detected during ACR, but the presence of th ese transcripts is not exclusive to this process.