SEMIQUANTITATIVE ANALYSIS OF CYTOKINE GENE-EXPRESSION IN BLOOD AND CEREBROSPINAL-FLUID CELLS BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION

An easy, reproducible and semi-quantitative, non-radioactive method fo r the analysis of mRNA expression for various cytokines, (i.e., Interl eukin (IL)-1 beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, lymp hotoxin (LT), transforming growth factor (TGF)-beta, interferon (IFN)- gamma and endothelin-1 (ET-1)) in cells from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) has been established. B y means of polymerase chain reaction primers that cover a splice junct ion, amplification of contaminating DNA was omitted. Densitometric sca nning of ethidium bromide-stained agarose gels proved to be very sensi tive for semiquantitative analysis of PCR products. Serial tenfold dil utions of cDNA revealed a log-linear regression from 10(6) to 10(2) ce lls under optimal cycle conditions. The intra- and inter-assay variabi lity of the method was below 10%. With this assay, the cytokine expres sion pattern of as few as 10(4) mononuclear cells from blood or CSF wa s determined. This method made it possible to detect differences in th e cytokine gene expression pattern of mononuclear cells from patients with different neurological diseases. CSF cells from 43 patients with various neurological diseases were analyzed. TNF-alpha, LT, and IL-1 m RNA were prominent in the CSF cells of most patients with bacterial me ningitis. TNF-alpha, LT, IFN-gamma and IL-6 mRNAs were detected in pat ients with active multiple sclerosis, whereas TNF-alpha, IL-6, and end othelin-1 mRNA expression was found frequently in patients with HIV en cephalitis. Pro-inflammatory cytokines were rarely detected in CSF cel ls from patients with non-inflammatory diseases of the central nervous system. In blood mononuclear cells from patients with multiple sclero sis, TNF-alpha mRNA expression was associated with disease activity. T he sensitivity, specificity, velocity and reliability of this assay co nsiderably facilitates the analysis of cytokine production in mononucl ear cells even in conditions where only a limited number of cells is a vailable for analysis.