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, or interleukin-12) in response to LPS. Our current specific interest is in the mechanism(s) of priming of monocytes, by T cell-derived cytokines, which results in marked enhancement of subsequent LPS responses. Priming occurs following culture in either interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Two cytokines produced by monocytes are absolutely dependent upon priming: IFN-alpha and interleukin-12. Both of these cytokines are under active investigation for the anti-viral and anti-tumor properties. Priming also results in substantial enhancement of TNF production. 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 transcription 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. In addition, priming significantly affects the regulation of cytokine 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. Studies of interleukin 12 (IL-12) production have demonstrated that expression of this cytokine is controlled by the stringent regulation of the p35 subunit (previously thought to be constitutively expressed and relatively unregulated). The expression of p35 is induced only when cells are primed with IFN-gamma, followed by LPS stimulation. Neither stimulus alone is sufficient, and GM-CSF, which primes for TNF expression at equivalent levels, is a poor inducer of IL-12 p35 or the active heterodimer (p35/p40). These studies further revealed the strict temporal requirements for expression of IL-12 subunits. We have recently isolated human genomic clones for IL-12 p35. Sequencing of the 5' flanking region of this gene reveals an unconventional promoter with no definable TATA box. Current studies are underway to identify cis sequences involved in IL-12 expression induced by IFN-gamma and LPS.
