The regulation and expression of secretory interleukin-1 receptor antagonist in control and endotoxin tolerant THP-1 cells

Sepsis with endotoxic shock is the number one cause of death in critical care units in the United States. During infection from many gram-negative bacteria, lipopolysaccharide (LPS) induces proinflammatory gene expression. Repeated stimulation by LPS induces a gene specific adaptive cellular process called endotoxin tolerance, a phenomenon common to sepsis whereby expression of proinflammatory genes is repressed. However, adaptation to LPS is not limited to suppression of proinflammatory mediators. This thesis will demonstrate that anti-inflammatory sIL-1 RA (sIL-1 RA) gene expression persists when the production of proinflammatory mediators is repressed in endotoxin tolerance.; To determine the mechanisms responsible for the differential expression of immunoregulatory genes in endotoxin tolerance, investigations into the regulation of proinflammatory cyclooxygenase-2 (COX-2) and anti-inflammatory sIL-1 RA were made using the THP-1 cell line as a model for sepsis. Endotoxin tolerance of COX-2 correlates with repressed transcription, yet transcription factors bind normally to the COX-2 promoter during tolerance, and truncation of the COX-2 promoter does not alleviate repression. While COX-2 is transcriptionally repressed, COX-2 mRNA degradation is not enhanced in endotoxin tolerance; however, COX-2 mRNA and protein are rapidly metabolized. Similarly, sIL-1 RA is transcriptionally repressed in endotoxin tolerance, but in contrast to COX-2, its mRNA and protein are more stable. These findings are consistent with the notion that endotoxin tolerance of COX-2 occurs by altering the transcriptional machinery controlling LPS-induced COX-2 expression, and that differential regulation of sIL-1 RA occurs by other molecular events including mRNA and protein stabilization, as well as enhanced translational efficiency. In addition, this thesis will show that the phosphatidylinositol-3′-kinase (PI3K) signaling pathway influences the expression of sIL-1 RA protein in LPS-stimulated endotoxin tolerant cells through modulating translation of nascent mRNA. Therefore, the PI3K signaling pathway may play a role in the enhanced translational efficiency of sIL-1 RA mRNA in endotoxin tolerance.