Supplementary Material for: Lipopolysaccharide and TNF-α Produce Very Similar Changes in Gene Expression in Human Endothelial Cells

Intracellular signaling pathways regulated by Toll-like receptor 4 (TLR4) and tumor necrosis factor-α (TNF-α) both activate NFĸB. This suggests that lipopolysaccharide (LPS) and TNF-α should alter transcription of a common set of genes. We tested this hypothesis by treating first passage human umbilical endothelial cells (HUVEC) for 6 h with LPS (50 ng/ml + 1 µg/ml CD14) or TNF-α (10 ng/ml) and analyzing changes in gene expression by microarray analysis (Affymetrix GeneChips). LPS and TNF-α increased expression of 191 common genes and decreased expression of 102 genes. Regulated transcripts encoded for a large number of chemokines, adhesion molecules, procoagulant factors, and molecules that affect cell integrity. Based on the microarray analysis and subsequent confirmation of specific genes by Northern analysis, all 203 genes altered by LPS were altered by TNF-α. An additional 17 genes were induced only by TNF-α and the expression of 46 was reduced. There were, however, some differences in the kinetics of changes. We also showed that endogenous CD14 was present on these early passage cells and exogenous CD14 was not necessary for most of the LPS response. An autocrine effect from LPS induced expression of TNF-α also was ruled out by blocking TNF-α with monoclonal antibodies. In conclusion, LPS induces a robust alteration in gene expression in HUVEC that is very similar to that induced by TNF-a. This LPS effect on endothelium could play an important role in the innate immune response.