Rapid, comprehensive analysis of human cytokine mRNA and its application to the study of acute renal allograft rejection.

Cytokine mRNA analysis was performed on human renal allograft needle core biopsies by a PCR-based assay. The assay was specifically developed to be capable of simultaneous analysis of multiple interleukin transcripts (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 tissue samples. It was initially used on preparations containing known amounts of plasmid DNA encoding individual cytokine cDNA sequences, confirming that the sensitivity of this technique was both well defined and comparable for all target sequences tested. Analysis of human PBLs prior to stimulation, after polyclonal stimulation with PHA and after simultaneous treatment with PHA and MP or CyA, was also performed to show a proportional relationship between mRNA levels measured by PCR and protein release measured by ELISA (R2 = 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 ACR when compared to other causes of graft dysfunction. Transcripts from the IFN-gamma and IL-6 genes were also increased in ACR (p = 0.001 and 0.017, respectively), whereas increased IL-8 mRNA was correlated with irreversible loss of graft function (p = 0.02). TNF-alpha, IL-1 beta, 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 those 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 beta and IL-10 genes is also detected during ACR, but the presence of these transcripts is not exclusive to this process.

Duke Scholars

Author Allan Douglas Kirk Surgery, Abdominal Transplant Surgery

Author Ralph Randal Bollinger Surgery