Figure 4.

Reversal of the human phenotype by hamster TLR4 expression: lipid IVa and RSLA activate THP-1 monocytes that overexpress hamster TLR4. THP-1 cells were plated at a density of 2 × 10⁶ cells per well in 6-well dishes, and then transiently transfected with the reporter plasmid pELAM.luc plus either pcDNA3.1 (vector), hToll (human TLR4), or pFLAG-hamTLR4 (hamster TLR4). The next day, the cells were incubated with medium alone (0), LPS (10 ng/mL), lipid IVa (1 μg/mL), or a combination of LPS and lipid IVa (10 ng/mL plus 1 μg/mL, respectively) (a), or in a separate experiment with medium alone (0), LPS (10 ng/mL), RSLA (1 μg/mL), or a combination of LPS and RSLA (10 ng/mL and 1 μg/mL, respectively) (b) for 5 hours. Luciferase activity was measured as described in Methods and plotted as the fold induction of activity compared with vector-transfected, unstimulated controls. The values shown are mean ± SD of triplicate transfections in 1 representative experiment out of 3. Similar results were observed when cells were stimulated with 100 ng/mL LPS and a 10-fold excess of inhibitor (data not shown).