FIG. 5.

Positive and negative regulation of TLR mRNA in MC by stimulus or challenge with PgLPS or EcLPS. (A and B) MC were stimulated at 37°C for 24 h with 1,000 ng of PgLPS (Pg), EcLPS (Ec), or no LPS (---), challenged with PgLPS (PgPg) or EcLPS (EcEc) for an additional 1 h, pelleted, washed, and processed for total RNA and for analysis of (A) TLR2 mRNA and (B) TLR4 mRNA by quantitative real-time PCR as described in Materials and Methods. The assay was performed three separate times, and the means and standard errors of three separate analyses are shown. (C and D) Fluorescence-activated all sorter analysis of human MC stimulated as in panels (A and B), except that the challenge occurred for 24 h to enable the proteins to be transcribed. A detailed account of the method is given in Materials and Methods. Histograms show mean fluorescence intensity, while numbers in parentheses indicate the geometric means of MC stimulated with the indicated regimen of stimulus and challenge. Results are representative of five separate experiments. FLT-Height, fluorescence intensity of TLRs.