The interleukin-1 beta (IL-1beta) gene, which encodes for a protein that plays a critical role in modulating inflammation, is down-regulated in lipopolysaccharide (LPS)-tolerant neutrophils (PMN) and macrophages (MAC). This suppression of the IL-1beta gene in LPS-tolerant PMN and MAC can be overcome by treating the cells with cycloheximide (CHX), suggesting that a labile repressor protein controls LPS tolerance. This project will investigate the regulation of LPS-tolerance at the level of the IL-1beta gene, and at earlier points in signal transduction. Models of in vivo- induced tolerance of blood PMN and MAC obtained from patients with the systemic inflammatory response syndrome (SIRS), and a model of in vitro- induced LPS tolerance in THP-1 MAC, will be used.
Aim 1. Studies of LPS tolerance at the level of the IL-1beta gene will: (i) determine whether reduced transcription or enhanced degradation of IL-1beta mRNA occurs in LPS-tolerant leukocytes, using nuclear run-on and mRNA degradation assays, (ii) define the specificity of LPS tolerance by investigating TNF alpha and prostaglandin G/H synthase-2 genes, (iii) identify the cis-acting elements of the IL-1beta gene that are responsible for LPS tolerance, using THP-1 cells transfected with plasmids containing regulatory elements of the IL-1beta gene and a reporter, (iv) investigate the proteins that interact with the cis-elements responsible for LPS tolerance, using electrophoretic mobility-shift, DNA or RNA footprinting, and immunologic assays.
Aim 2. Experiments of signal transduction events that lie proximal to the IL-1beta gene, and that might indirectly be responsible for LPS tolerance will assess protein kinases C (PKC), protein tyrosine kinases (PTK), and extracellular signal related kinases (ERK). Enzymatic and immunoassays will be used.
Aim 3. Extracellularly added anti-inflammatory agents, including dexamethasone and IL-4, will be used in an attempt to produce the phenotypic changes found in LPS-tolerant PMN and MAC obtained from humans with SIRS. Studies will focus on the IL-1 and Type 2 IL-1 receptor genes. This research may provide insight into the molecular basis of tolerance of leukocytes to LPS-induction of cytokine genes.
