Figure 3

Induction of LPS tolerance in dendritic cells. A. Induction of LPS tolerance in dendritic cell cultures at the level of TNF mRNA at day 7 dendritic cells were cultured for additional 2 days with LPS at 5 ng/ml and then washed and stimulated at 500 ng LPS/ml. Cells were lysed at 2 hours and RNA was extracted, transcribed into cDNA and amplified by RT-PCR and data were normalized relative to levels of the house keeping gene alpha-Enolase. Given is the average of 7 experiments. * = p < 0.05 compared to 0/LPS. B. Induction of LPS tolerance in dendritic cell cultures at the level of TNF protein. Day 7 dendritic cells were cultured for additional 2 days with LPS at 5 ng/ml and then washed and stimulated at 500 ng LPS/ml for 6 hours. Supernatants were then harvested and were tested for TNF protein by ELISA. Given is the average of 3 experiments. * = p < 0.05 compared to 0/LPS. C. Induction of LPS tolerance in CD83⁺ dendritic cells at the level of TNF mRNA. Day 7 CD83⁺ dendritic cells were isolated by MACS to > 90% purity and these cells were then cultured for additional 2 days with LPS at 5 ng/ml, washed and stimulated at 500 ng LPS/ml. Cells were lysed and RNA was extracted, transcribed into cDNA and amplified by RT-PCR and data were normalized relative to levels of the house keeping gene alpha-Enolase. Given is the average of 3 experiments. * = p < 0.05 compared to 0/LPS.