A SYSTEM FOR ASSESSMENT OF MONOKINE GENE-EXPRESSION USING HUMAN WHOLE-BLOOD

Monocyte derived cytokines (monokines) are important mediators in infl ammatory diseases and cancer. Control of monokine expression is also a major therapeutic target in autoimmune inflammation. Whole blood cult ures permit examination of monokine expression under conditions which emulate the en-vivo environment whilst avoiding many of the artefacts associated with monocyte separation and culture. Here we describe a sy stem for measuring interleukin-1 beta, interleukin-1 alpha, interleuki n-6 and tumour necrosis factor-alpha mRNA in stimulated human whole bl ood ex-vivo which can be applied to specimens from treated patients. O ligodeoxyribonucleotide probes are designed to allow standardisation o f hybridisation and washing procedures. Washing and reprobing of membr anes in appropriate sequence permits measurement of each monokine mRNA and mRNA for glyceraldehyde-3-phosphate dehydrogenase in only 7 mi of lipopolysaccharide-stimulated human blood. The method has been used s uccessfully in studies of dexamethasone and methotrexate action on lip opolysaccharide stimulated IL-beta gene expression.