LPS pretreatment of mouse peritoneal macrophages differentially modulates TNFα and iNOS expression

Abstract

Many studies have established that pretreatment of mouse macrophages with LPS will alter subsequent responsiveness of these cells to stimulation either with LPS or other stimuli. Incubation of C3Heb/FeJ mouse macrophages with low sub-stimulatory (e.g. 0.10 ng/ml) LPS for 6 h, followed by activation with 100 ng/ml of LPS results in up-regulation of LPS-dependent TNFα production and suppression of the ability of these cells to secrete nitric oxide (NO). To assess whether these two responses might be co-ordinately regulated, immunocytochemical analyses of LPS-pretreated macrophages were carried out using anti-TNFα and anti-iNOS antibodies, both with and without a period of LPS pretreatment. As anticipated, the detection of individual iNOS-expressing macrophages in LPS-stimulated cultures accurately reflected threshold doses of LPS required for detection of NO in culture supernatants in terms of frequency of percentage of the total population positive for expression of iNOS protein. Pretreatment with sub-stimulatory doses of LPS significantly reduced the frequency of these iNOS-expressing macrophages responsive to subsequent LPS stimulation, supporting the concept of iNOS down-regulation at the pretranslational level. In contrast, the detection of TNFα-expressing macrophages in LPS-stimulated cultures did not correlate directly with the detection of TNFα in culture supernatants. Further, pretreatment with sub-stimulatory doses of LPS did not always correlate with the frequency of TNFα-expressing cells in response to LPS treatment. These results support the concept that different regulatory mechanisms may be operative in differential regulation of macrophage TNFα and NO responsiveness by pretreatment of macrophages with sub-stimulatory doses of LPS.

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