Tumor necrosis factor (TNF) is widely considered to be an important element in mediating inflammation and septic shock. The ability to accurately measure TNF production has been limited by the sensitivity of existing immunochemical methods. These methods prelude determining TNF levels in the small specimens obtained by bronchoalveolar lavage and cannot measure TNF production as a precise point in time since they are inherently steady- state. The limitations of these immunochemical methods can be overcome by utilizing the recently developed polymerase chain reaction (PCR). PCR allows the determination of mRNA levels (and thus, indirectly TNF production) in relatively small numbers of cells. We are currently utilizing PCR to measure TNF mRNA levels in macrophages isolated by lavage from human volunteers who have been administered endotoxin. Initial results have shown that we can determine TNF mRNA levels in less than one million alveolar macrophages. We are trying to improve the sensitivity of the assay to detect mRNA levels in one hundred thousand cells, a number readily obtained by lavage. We have radioactive and non-radioactive dot blots of PCR products. Additionally, a cDNA transcription vector has been constructed which enables us to produce, in vitro, TNF mRNA in measurable quantities which can then be used as an unambiguous standard. Finally, we are beginning to look at the use of PCR to determine the mRNA levels for other lymphokines which mediate macrophage activity in inflammation.
