FIGURE 3.

FheCL1 does not affect MyD88-dependent signaling through TLR4. A, BALB/c mice were given a single intraperitoneal injection of 50 μl of FheCL1 (1 mg/ml) or PBS and 2 h later peritoneal macrophages were removed. A, these macrophages were then stimulated with varying concentrations of LPS (0–5 μg/ml) for 12 h, and secreted cytokines were measured by ELISA. Data shown are the means + S.E. of triplicates and are representative of two individual experiments. B, macrophages removed from either PBS- or CL1-treated mice were analyzed for the surface expression of CD14 or TLR4/MD-2 by flow cytometry. C, TLR4 mRNA levels in peritoneal macrophages isolated from either PBS- or FheCL1-treated BALB/c mice incubated in the presence or absence of LPS was assessed by RT-PCR. D, peritoneal macrophages isolated from both PBS- and FheCL1-treated mice were stimulated with LPS (1 μg/ml) for 6 h, lysed, and examined for levels of mRNA expression of IL-6, TNFα, IL-12, and iNOS by RT-PCR. Data shown are representative of three independent experiments. E, densitometric analysis of amplification products for IL-6, TNFα, IL-12, and iNOS. Values for mRNA are expressed in relative absorbance units and are standardized per unit of β-actin per sample. The data are obtained from the mean of three independent experiments; FITC, fluorescein isothiocyanate; PE, phycoerythrin.