ABSTRACT
Monocytes/macrophages respond to external stimuli with rapid changes in the expression of numerous inflammation-related genes to undergo polarisation towards the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype. We have previously shown that, independently of Toll-like receptor activation, extracellular RNA (eRNA) could exert pro-thrombotic and pro-inflammatory properties in the cardiovascular system to provoke cytokine mobilisation. Here, mouse bone marrow-derived-macrophages (BMDM) differentiated with mouse macrophage-colony-stimulating factor (M-CSF) were found to be skewed towards the M1 phenotype when exposed to eRNA. This resulted in up-regulated expression of inflammatory markers such as Tnf-α and Il-6, together with Il-12 and iNOS, whereas anti-inflammatory genes such as chitinase-like proteins (Ym1/2) and macrophage mannose receptor-2 (Cd206) were significantly down-regulated. Human peripheral blood monocytes were treated with eRNA and analysed by micro-array analysis of the whole human genome, revealing an up-regulation of 79 genes by at least four-fold; 27 of which are related to signal transduction and 15 genes associated with inflammatory response. In accordance with the proposed actions of eRNA as a pro-inflammatory "alarm signal", these data shed light on the role of eRNA in the context of chronic inflammatory diseases such as atherosclerosis.
Subject(s)
Cell Differentiation , Cell Lineage , Cytokines/metabolism , Inflammation Mediators/metabolism , Macrophages/metabolism , Monocytes/metabolism , RNA/metabolism , Animals , Biomarkers/blood , Cells, Cultured , Cytokines/genetics , Cytokines/immunology , Gene Expression Profiling/methods , Gene Expression Regulation , Humans , Inflammation Mediators/immunology , Macrophages/immunology , Mice, Inbred C57BL , Monocytes/immunology , Phenotype , Time FactorsABSTRACT
Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.
Subject(s)
Gene Expression Regulation/drug effects , Inflammation/genetics , Monocytes/drug effects , Thrombin/pharmacology , Transcription, Genetic/drug effects , Wound Healing/genetics , Blood Coagulation/genetics , Cells, Cultured , Factor Xa/biosynthesis , Feedback, Physiological , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Monocytes/metabolism , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Receptor, PAR-1/physiology , Thromboplastin/biosynthesis , Thromboplastin/genetics , Up-Regulation/drug effectsABSTRACT
Protease-activated receptors (PARs) are a subfamily of four G-protein-coupled receptors mediating multiple functions. PARs expression was studied in subpopulations of human lymphocytes. Our results indicate that natural killer cells expressed mRNA for PAR1, PAR2 and PAR3, CD4+ T cells expressed PAR1 and PAR2, while γδ and CD8+ T cells only expressed PAR1. PAR4 was absent at mRNA level and B cells did not express any PAR. Analyses of the cell surface PARs expression by flow cytometry were consistent with the mRNA data and also between different donors. PAR1 is the most abundant member of the PAR family present in lymphocytes.
Subject(s)
B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Killer Cells, Natural/metabolism , Receptor, PAR-1/genetics , Receptor, PAR-2/genetics , Receptors, Thrombin/genetics , B-Lymphocytes/cytology , CD4 Antigens/genetics , CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Gene Expression , Gene Expression Profiling , Humans , Killer Cells, Natural/cytology , RNA, Messenger/genetics , RNA, Messenger/immunology , Receptor, PAR-1/immunology , Receptor, PAR-2/immunology , Receptors, Antigen, T-Cell, gamma-delta/genetics , Receptors, Antigen, T-Cell, gamma-delta/immunology , Receptors, Thrombin/immunologyABSTRACT
Thrombin is a potent mitogen for many tumor cells, suggesting that this enzyme may be involved in tumor genesis and metastasis. Inhibition of thrombin expressed on the surface of tumor cells may improve outcomes in some tumor cases. For this reason, a thrombin inhibitor to be applied in antitumor therapy must have favorable pharmacokinetic attributes to exert its action as long as possible in the extravascular compartment of the extracellular space, with a short action intravascularly, avoiding bleeding and/or other undesirable side-effects. None of the thrombin inhibitors in clinical use has these properties. Here, we report for first time a direct thrombin inhibitor, named dipetarudin that could be very useful in antitumor therapy because of its pharmacokinetic behavior characterized by a rapid distribution in the extravascular space with a slow elimination from this compartment.
