CD14 Mediates Binding of High Doses of LPS but Is Dispensable for TNF-α Production
Figure 4
Binding of high doses of sLPS to the surface of RAW264 cells is mediated by CD14 and SR. (a, b) Binding of sLPS-biotin to the surface of RAW264 cells. Cells were preincubated with 0.05% NaN3 (30 min, 37°C) and either left untreated (ns) or exposed to 5 or 10 μg/mL of the anti-CD14 antibody or 10 μg/mL isotype-matched control IgG (cIgG) or 50 μg/mL dextran sulfate (DS), or 50 μg/mL chondroitin sulfate (CS), or the mixture of 5 μg/mL anti-CD14 antibody and 50 μg/mL dextran sulfate. After 30 min (37°C), cells were stimulated with 1 μg/mL sLPS-biotin (1 h, 37°C) in the constant presence of 0.05% NaN3. The amount of sLPS-biotin bound to the cell surface was assessed by dot-blot analysis of cell homogenates using streptavidin-peroxidase (a). (c, d) Internalization of sLPS-biotin. Cells were preincubated with 10 μg/mL anti-CD14 antibody or 10 μg/mL isotype-matched control IgG or 50 μg/mL dextran sulfate or 50 μg/mL chondroitin sulfate or a mixture of the anti-CD14 and dextran sulfate for 30 min at 37°C. Subsequently, the samples were supplemented with 1 μg/mL of sLPS-biotin for 1 h and washed and incubated for another 1 h at 37°C prior to homogenization and dot-blot analysis (c). (b, d) Quantification of cell surface-bound (b) and internalized (d) sLPS-biotin based on densitometric analysis of dot-blots. Data are mean ± SEM from three experiments. *Significantly different from cells exposed to control IgG.
