New Insights into Endotoxin Tolerance

Endotoxin tolerance is an immunosuppressive state that renders immune cells unable to generate an inflammatory response to subsequent challenges following an initial exposure to endotoxin. This phenomenon prevents the excessive production of inflammatory mediators that when unregulated can be very detrimental to the host. The central mediator of inflammation is tumor necrosis factor-alpha (TNF-alpha) and is typically used to monitor the inflammatory response and induction of tolerance. This led to the hypothesis that the degree of endotoxin tolerance was dependent on the production of TNF-&agr; following lipopolysaccharide (LPS) stimulation. Human monocytic THP-1 cells were stimulated with LPS in the presence of anti-TNF-alpha and anti-TNF-alpha receptor antibodies to determine if the induction of tolerance would be impaired without TNF-alpha signaling. Anti-TNF-alpha and anti-TNF-alpha receptor treatment reduced the production of TNF-&agr; and IL-1beta during the primary LPS stimulation; however, the tolerant-like state was observed during subsequent LPS challenges.;Over the past several decades, researchers have investigated and identified biochemical mechanisms involved in regulating the production of inflammatory mediators. However, most of these investigations have overlooked the contribution of LPS-induced cell death to the diminished response to subsequent challenges. Absolute cell loss and cell viability was determined following LPS stimulation and challenge in THP-1 cells. LPS-induced a dose-dependent loss and reduced viability of THP-1 cells. The cell loss and reduced cell viability of LPS-stimulated THP-1 contributed to the attenuated production of TNF-alpha but viable THP-1 cells still demonstrated impaired TNF-alpha secretion indicating the involvement of biochemical regulatory mechanisms.;Finally, the ability to simultaneously observe mRNA and protein expression in an individual cell would be valuable technique when analyzing a heterogeneous population of cells. Flow cytometry provides an opportunity to investigate individual cell expression, however methods capable of demonstrating mRNA and protein expression are not available. A self-assembling oligonucleotide fluorescent Universal Reporter (UR) probe was designed and a method to label mRNA and protein in suspended murine splenocytes was optimized for analysis by flow cytometry. The UR probe specifically recognized CD45 mRNA expression in CD45+ splenocytes. In addition, TNF-alpha mRNA and intracellular protein expression was observed in PMA/Ionomycin-stimulated murine CD45+CD3e+CD4+ splenocytes.