CD14 Mediates Binding of High Doses of LPS but Is Dispensable for TNF-α Production
Figure 5
Disparate requirements for CD14 participation in the binding of high doses of LPS and production of TNF-α in J774 cells.(a, b) Binding of sLPS-biotin to the surface J774 cells. Cells were preincubated with 0.05% NaN3 (30 min, 37°C) and either left untreated (ns) or supplemented with 5, 10, or 15 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 a mixture of 5 μg/mL anti-CD14 antibody and 50 μg/mL dextran sulfate for 30 min (37°C). Subsequently, 1 μg/mL of sLPS-biotin was added to the cultures for 1 h in the presence of 0.05% NaN3. (a) Dot-blot analysis of sLPS-biotin in cell homogenates. (b) Densitometric analysis of dot-blots exemplified in (a). Data are mean ± SEM from three experiments. (c) LigandTracer real-time analysis of the binding and internalization of sLPS-AF488. Cells were preincubated for 15 min at 37°C with a mixture of 10 μg/mL anti-CD14 antibody and 50 μg/mL dextran sulfate (DS) or with 50 μg/mL chondroitin sulfate (CS) and transferred into the LigandTracer. After 30 min of the baseline setting (37°C), cells were exposed to 3 μg/mL of sLPS-AF488 for 1 h (association phase), washed to remove the unbound LPS, and monitored for another 1 h to measure retention of sLPS-AF488 in cells. Traces with open symbols and closed symbols reflect sLPS-AF488 binding and internalization in CS-treated and anti-CD14/DS-treated cells, respectively. Concanavalin A-FITC (100 μg/mL; ConA) was added to the anti-CD14/DS-treated culture for 40 min to ensure that the cells were still adherent. (d, e) Production of TNF-α (d) and RANTES (e) in cells pretreated with 10 μg/mL of the anti-CD14 antibody or isotype-matched control rat IgG2b and stimulated with 10–1000 ng/mL of sLPS. Data are mean ± SEM from three experiments. *Significantly different from cells exposed to control IgG.
