Mechanisms of Lipoplysaccharide-induced Lipopolysaccharide Tolerance in the Expression of TNF-alpha and IL-8 in Peripheral Blood Monocytes

Abstract

Background : Monocytes/macrophages play a central role in determining the host response during Gram-negative infection through secretion of a variety of mediators after stimulation of LPS. Even though cytokine production has been shown to play an important role in host defense during sepsis, cytokine release may also lead to tissue injury. Thus, regulation of macrophage response to LPS is critical for host survival during Gram-negative sepsis. In animals exposed to nonlethal doses of endotoxin, a characteristic hyporesponsiveness to subsequent administration of endotoxin has been observed. This phenomenon was known as 'LPS tolerance'. However, little information is available regarding the underlying mechanism of LPS tolerance. Method : Peripheral blood monocyte(PBMC) was isolated from peripheral blood of normal volunteers by adhesion purification method. To evaluate the conditions to obtain LPS tolerance, preculture was carried out with LPS at 10ng/ml for 24 hours. For stimulation, culture plates were washed two times and were stimulated with LPS at for 4, 6 and 26 hours. To assess the underlying mechanisms of LPS tolerance, autologous serum, PMA, anti-CD14 Ab, Indomethacin or were added to preculture solution respectively. Cytokine concentrations in culture supernatants were measured using ELISA for TNF- and IL-8 and mRNA of TNF- and IL-8 were determined by Northern blot analysis. Results : The exposure of PBMC to low dose of LPS suppressed the cytokine production and mRNA expression of TNF-, but not IL-8. Anti-CD14 Ab partially recovered production of TNF- which was suppressed by preculture with low dose LPS. The preculture with PMA induces LPS tolerance, as preculture with low dose LPS. Conclusion : LPS tolerance to TNF- is regulated pretranslationally and is influenced by protein kinase C pathway and CD14.