A system for assessment of monokine gene expression using human whole blood.
Monocyte derived cytokines (monokines) are important mediators in inflammatory diseases and cancer. Control of monokine expression is also a major therapeutic target in autoimmune inflammation. Whole blood cultures permit examination of monokine expression under conditions which emulate the in-vivo environment whilst avoiding many of the artefacts associated with monocyte separation and culture. Here we describe a system for measuring interleukin-1 beta, interleukin-1 alpha, interleukin-6 and tumour necrosis factor-alpha mRNA in stimulated human whole blood ex-vivo, which can be applied to specimens from treated patients. Oligodeoxyribonucleotide probes are designed to allow standardisation of hybridisation and washing procedures. Washing and reprobing of membranes in appropriate sequence permits measurement of each monokine mRNA and mRNA for glyceraldehyde-3-phosphate dehydrogenase in only 7 ml of lipopolysaccharide-stimulated human blood. The method has been used successfully in studies of dexamethasone and methotrexate action on lipopolysaccharide stimulated IL-beta gene expression.
Joyce
D A
DA
Sir Charles Gairdner Hospital, Nedlands, Western Australia.
Steer
J H
JH
Beilharz
M W
MW
Stranger
R
R
D015152:Blotting, Northern / Q000379:methods; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000096:biosynthesis / Q000097:blood / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D015846:Monokines / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000097:blood; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D015846:Monokines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; D007371:Interferon-gamma; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008727:Methotrexate