Rac mediates TNF-induced cytokine production via modulation of NF-kappaB.

TNF is a key factor in a variety of inflammatory diseases. Here we report that TNF induced pro-inflammatory cytokine synthesis of IL-6 and IL-8 is mediated by the Rho GTPase Rac. TNF induces p42/p44, p54 and p38 MAPK kinase; these kinases have been implicated in control of cytokine synthesis. However, over-expression of a dominant negative form of Rac strongly inhibited TNF-induced p42/44 MAPK kinase activation, but had little effect upon JNK and no effect upon p38 MAPK activity. Another key signalling pathway controlling cytokine expression is NF-kappaB. When analyzing TNF-induced NF-kappaB activity via luciferase-reporter assays or via EMSA, we were able to show that the dominant negative version of Rac could completely abrogate TNF-induced NF-kappaB activity. In addition, we also observed that inhibition of the ERK pathway led to a reduction in TNF-induced NF-kappaB transcriptional activity; this was accompanied by an ablation of TNF-induced p65 phosphorylation at serine 276. This would suggest that TNF-induced activation of Rac, lies upstream of NF-kappaB activation, and that the inhibition of this pathway results in inhibition of cytokine production.

Adenoviral targeting of signal transduction pathways in synovial cell cultures.

Methods for high efficiency gene transfer into primary cells of various lineages and disease states are desirable, as they remove the uncertainties associated with using transformed cell lines. Adenoviruses have evolved to deliver their genes into cells with high efficiency and in recent years have been exploited as a gene transduction system. Prior to the discovery of adenoviruses, efficient expression of transgenes was only possible by cloning stably transfected cells; this was limited to cell lines and was not an option for primary cells. Here we describe a method of transgene expression, which enables previously untransfectable cells, such as primary myeloid cells or diseased synovium, to express protein at extremely high levels with nearly 100% of cells expressing the transgene. This allows us to examine the effect of target genes on signaling pathways in primary cells without the need for cell sorting or the simultaneous transfection of reporter genes. This is very important in studies of tissues such as rheumatoid synovium where sorting of cells will damage the biological value of the system.