ABSTRACT
AIMS: In this work we aimed to investigate the expression of TLR9 protein in the murine hepatocyte cell line TIB-73, compared to macrophage-like J774 cells, by Western blot analysis, and the role played by ERK 1/2 MAP kinase in the intracellular signals triggered by stimulation with CpG and non-CpG phosphodiester-ODN, and their more stable phosphorothioate-modified analogues. RESULTS: TIB-73 hepatocytes express TLR9 protein. CpG and non-CpG ODN stimulation activated ERK 1/2 MAPK signal pathway in both hepatocytes and J774 murine macrophages. As expected, their phosphorothioate-CpG and non-CpG ODN analogues induced higher levels of ERK1/2 phosphorylation in TIB-73 cells, even higher than that induced in J774 cells under the same conditions. Phosphorylation of ERK 1/2 induced by synthetic ODN is dose-response dependent, being maximal at 100 microg/ml. Pretreatment of hepatocytes with an inhibitor of MEK-1 abrogated phosphorylation of ERK1/2 kinase. CONCLUSIONS: TIB-73 hepatocytes constitutively express TLR9 and respond to synthetic ODN stimulation through a high ERK1/2 phosphorylation independent of CpG motifs. Slight differences were found on ERK1/2 activation when using phosphorothioate versus phosphodiester oligonucleotides.
Subject(s)
Oligodeoxyribonucleotides/metabolism , Oligodeoxyribonucleotides/pharmacology , Animals , Cell Line , Hepatocytes/metabolism , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Oligonucleotides/metabolism , Phosphorylation , Signal Transduction/drug effects , Toll-Like Receptor 9/metabolismABSTRACT
Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.
Subject(s)
Hepatocytes/microbiology , Oligodeoxyribonucleotides/pharmacology , Reactive Oxygen Species/pharmacology , Salmonella typhimurium/drug effects , Salmonella typhimurium/growth & development , Animals , Cell Line , CpG Islands , Cytokines , Female , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Peroxides/metabolism , Receptors, Cell Surface/metabolism , Superoxide Dismutase/metabolism , Toll-Like ReceptorsABSTRACT
The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.
