ABSTRACT
Unmethylated CpG-DNA motifs from pathogens are detected by the pattern recognition receptor toll-like receptor 9 (TLR9), eliciting an inflammatory immune response. These DNA sequences have been identified as potent immune modifiers and are used as adjuvants in vaccine research. Since we previously found TLR9 expression and function in human endothelial cells, we have here investigated whether endothelial cells play a role in the recognition of respective ligands and whether their response might contribute to vaccination success. We determined the effect of CpG-DNA on the inflammatory response of human endothelial cells of aortic or skin microvascular origin (HAoEC, HDMEC and HMEC-1) and compared the effects to those of two identically treated human macrophage cell lines. Using the same CpG-DNA D19(chimera) sequence in both cell types, we find the known up-regulation of pro-inflammatory cytokines in macrophages but consistent and significant inhibition of the pro-inflammatory response (IL-6, IL-8, and IFN-beta1) in endothelial cells. This inhibition is accompanied by enhanced proliferation and an increase in IL-10 gene expression. This anti-inflammatory response persists even in the presence of pro-inflammatory cytokines and low LPS concentrations, and is overruled only in the presence of relatively high concentrations of LPS. By testing different sequences, we find the strongest response with phosphorothioate bonds. Our results demonstrate an important regulatory function of endothelial cells in inflammatory responses, and the apparent Th2-like endothelial response in the human system may contribute significantly to the adjuvant activity of CpG-DNA.
Subject(s)
Adjuvants, Immunologic/pharmacology , CpG Islands , Endothelial Cells/drug effects , Macrophages/drug effects , Oligodeoxyribonucleotides/pharmacology , Th2 Cells/drug effects , Adjuvants, Immunologic/genetics , Cell Line, Tumor , Cell Survival/drug effects , CpG Islands/immunology , Endothelial Cells/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunity, Innate/drug effects , Interferon-beta/antagonists & inhibitors , Interleukin-10/genetics , Interleukin-6/antagonists & inhibitors , Interleukin-8/antagonists & inhibitors , Ligands , Macrophages/immunology , Oligodeoxyribonucleotides/genetics , Real-Time Polymerase Chain Reaction , Th2 Cells/immunology , Toll-Like Receptor 9/immunologyABSTRACT
Human heat shock protein 60 (Hsp60) exhibits immunoregulatory properties, primarily by inducing pro-inflammatory responses in innate immune cells. Extensive analyses identified specific receptor structures for the interaction of Hsp60 with these cells. The existence of distinct receptor structures responsible for Hsp60 binding and for Hsp60-induced release of pro-inflammatory mediators has been demonstrated, implying that the interaction of Hsp60 with innate immune cells is a multifaceted process. Distinct Hsp60 epitopes responsible for binding to innate immune cells and for the activation of these cells have been identified. Depending on the cell-type, the amino acid (aa) region 481-500 or the regions aa241-260, aa391-410 and aa461-480 are involved in Hsp60-binding to innate immune cells. An entirely different Hsp60-region, aa354-365 was found to bind lipopolysaccharide, thereby mediating the pro-inflammatory effects of Hsp60. Because of its immunoregulatory properties, Hsp60 has been proposed to act as intercellular danger signal, controlling innate and adaptive immune reactions.
