Bacterial lipopolysaccharide (LPS) is a potent stimulatory agent for monocytes and macrophages. The functions induced by LPS are critical to effective host defense, but LPS can also induce pathological complications when responses continue unchecked. The long-term goal of this project is to elucidate molecular events that are involved in LPS stimulation of human monocytes. The focus of these studies is the regulation of the production of potent, bioactive cytokines (such as tumor necrosis factor, interferon, interleukin-1) in response to LPS. Our current specific interest is in the mechanism(s) of priming of monocytes which results in marked enhancement of subsequent LPS responses. Priming occurs following culture in either interferon-gamma (IFN--) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Priming is an absolute requirement for LPS-induced IFN-a production, and results in substantial enhancement of TNF production as well. Analysis of specific RNA for these cytokines indicated that their enhanced expression in primed monocytes occurs at the RNA level. There is some evidence that the effects on TNF mRNA expression are transcriptional; nuclear run-on analysis suggested that LPS induces trancription and that this is enhanced by priming, but not to a degree that explains the vast increase in mRNA accumulation. Priming also has dramatic effects on NF- kB transcription factor activation; NF-kB has been implicated in mediating LPS-induced transcriptional responses. The current emphasis in the laboratory is on the regulation of specific mRNA stability. The cytokines under study (TNF, IFN) are unique with respect to possessing 3'-flanking sequences thought to be associated with mRNA instability. Exhaustive studies of mRNA half-life of TNF demonstrated that priming results in a substantial increase in TNF mRNA stability. Further studies are designed to elucidate potential RNA-protein interactions involved in this phenomenon. A parallel series of studies has examined the expression of an important new cytokine that interfaces monocyte-T cell responses, IL-12. These studies have shown that IFN-gamma selectively primes for the expression of LPS-induced IL-12 p35 and p40 chains.
