Fig. 8.
Timing of interferon (IFN)-β administration affects cellular responses to lipopolysaccharide (LPS). The murine peritoneal macrophage cell line, RAW264.7, in the culture media was stimulated with 100 ng/ml of LPS. Il6 mRNA expression (A) and IL-6 protein secretion (B), Socs3 mRNA (C) and p21 mRNA (D) were evaluated. Quantification of mRNA has done by real-time polymerase chain reaction using TaqMan gene expression assays (Applied Biosystems, USA) with StepOne Real-Time PCR System, and IL-6 level in the culture media was measured by enzyme-linked immune sorbent assay. Expression values for each gene were calculated by normalization to Gapdh, and fold change in expression to unstimulated control was determined for each condition. Numbers for each bar indicate the duration of stimulation with IFNβ or LPS. LPS tol (+) indicates cells incubated for 12 h before stimulation with either or both IFNβ and LPS. nd indicates not done. When LPS-tolerant cells were stimulated with LPS, coincubation with IFNβ showed a marked increase in Il6 mRNA expression and secretion (A and B, right closed bar in the middle) compared with those without IFNβ (A and B, left closed bar in the middle), whereas previous administration with IFNβ before LPS stimulation did not (A and B, right gray bar). Patterns of expression of Socs3 and p21 are similar to Il6 in LPS-tolerant cells (C and D, closed bars in the middle). However, incubation with IFNβ increased the expression of Socs3 and p21 (C and D, second right gray bar), and additional LPS markedly increased their expressions (C and D, first right gray bar). Data are shown as the mean of three determinants (±SD).

Timing of interferon (IFN)-β administration affects cellular responses to lipopolysaccharide (LPS). The murine peritoneal macrophage cell line, RAW264.7, in the culture media was stimulated with 100 ng/ml of LPS. Il6 mRNA expression (A) and IL-6 protein secretion (B), Socs3 mRNA (C) and p21 mRNA (D) were evaluated. Quantification of mRNA has done by real-time polymerase chain reaction using TaqMan gene expression assays (Applied Biosystems, USA) with StepOne Real-Time PCR System, and IL-6 level in the culture media was measured by enzyme-linked immune sorbent assay. Expression values for each gene were calculated by normalization to Gapdh, and fold change in expression to unstimulated control was determined for each condition. Numbers for each bar indicate the duration of stimulation with IFNβ or LPS. LPS tol (+) indicates cells incubated for 12 h before stimulation with either or both IFNβ and LPS. nd indicates not done. When LPS-tolerant cells were stimulated with LPS, coincubation with IFNβ showed a marked increase in Il6 mRNA expression and secretion (A and B, right closed bar in the middle) compared with those without IFNβ (A and B, left closed bar in the middle), whereas previous administration with IFNβ before LPS stimulation did not (A and B, right gray bar). Patterns of expression of Socs3 and p21 are similar to Il6 in LPS-tolerant cells (C and D, closed bars in the middle). However, incubation with IFNβ increased the expression of Socs3 and p21 (C and D, second right gray bar), and additional LPS markedly increased their expressions (C and D, first right gray bar). Data are shown as the mean of three determinants (±SD).

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