The expression of TWEAK (TNFSF12) and TweakR/Fn14 was detected in regions rich in macrophage/foam cells in atherosclerotic plaques. The role of TWEAK in monocytes in relation to atherogenesis was investigated by analyzing the cellular events induced by TWEAK in a human macrophage-like cell line, THP-1. TWEAK induced various molecular mediators of atherogenesis, such as IL-6, MCP-1, IL-8 and MMP-9, and the induction was augmented by interferon-γ. TWEAK-induced activation of MMP-9 was mediated by activation of NF-κB. These results suggest that TWEAK is involved in atherosclerosis by inducing pro-inflammatory cytokines and extracellular matrix degrading enzymes, which reduce plaque stability. (Circ J 2004; 68: 396 - 399)

In the course of screening inhibitors of matrix metalloproteinase (MMP)-9 induction in macrophages, we isolated decursin, a coumarin compound, from the roots of Angelicae gigas. As a marker for the screening and isolation, we tested expression of MMP-9 in RAW264.7 cells and THP-1 cells after treatment with bacterial lipopolysaccharide (LPS), the TLR-4 ligand. Decursin suppressed MMP-9 expression in cells stimulated by LPS in a dose-dependent manner at concentrations below 60 μM with no sign of cytotoxicity. The suppressive effect of decursin was observed not only in cells stimulated with ligands for TLR4, TLR2, TLR3, and TLR9 but also in cells stimulated with interleukin (IL)-1β, and tumor necrosis factor (TNF)-α, indicating that the molecular target of decursin is common signaling molecules induced by these stimulants. In addition to the suppression of MMP-9 expression, decursin blocked nitric oxide production and cytokine (IL-8, MCP-1, IL-1β, and TNF-α) secretion induced by LPS. To find out the molecular mechanism responsible for the suppressive effect of decursin, we analyzed signaling molecules involved in the TLR-mediated activation of MMP-9 and cytokines. Decursin blocked phosphorylation of IκB and nuclear translocation of NF-κB in THP-1 cells activated with LPS. Furthermore, expression of a luciferase reporter gene under the promoter containing NF-κB binding sites was blocked by decursin. These data indicate that decursin is a novel inhibitor of NF-κB activation in signaling induced by TLR ligands and cytokines.

Background: Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of urokinase type plasminogen activators (uPA) and tissue type plasminogen activators (tPA), which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system.Methods: In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated byin vitroscratch-wound healing assay or Boyden chamber assay and anin vivostab wound injury model. Phagocytic activity was measured by uptake of zymosan particles.Results: The levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-γ stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP) 1/Janus kinase (JAK)/signal transducer and activator of transcription (STAT)1 axis. PAI-1 also increased microglial migrationin vivowhen injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner.Conclusion: Our results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

Summary: Glucocorticoid‐induced tumour necrosis factor receptor family related protein (GITR) is the 18th member of the tumour necrosis factor receptor superfamily (TNFRSF18) and is known to interact with its cognate ligand GITRL (TNFSF18). We investigated the potential role of GITR in the pro‐inflammatory activation of macrophages. Immunohistochemistry and in situ hybridization analyses of human atherosclerotic plaques demonstrated that GITR and its ligand are expressed mainly in lipid‐rich macrophages. We then investigated the role of GITR in human and mouse monocyte/macrophage functions. Stimulation of GITR caused nuclear factor (NF)‐κB‐dependent activation of matrix metalloproteinase‐9 (MMP‐9) and pro‐inflammatory cytokine expression in both the human and mouse monocytic/macrophage cell lines, THP‐1 and RAW264.7, respectively. These cellular responses were also observed when the THP‐1 cells were treated with phorbol‐12 myristate‐13 acetate (PMA), which is known to induce macrophage differentiation. To demonstrate that these responses are not restricted to cultured cell lines, we tested primary macrophages. Both peritoneal and bone marrow‐derived macrophages responded to GITR stimulation with induction of MMP‐9 and tumour necrosis factor‐α (TNF‐α). Furthermore, the GITR staining pattern overlapped with those of MMP‐9 and TNF‐α in atherosclerotic plaques. These data indicate that GITR‐mediated macrophage activation may promote atherogenesis via the induction of pro‐atherogenic cytokines/chemokines, and destabilize the atherosclerotic plaques via the induction of the matrix‐degrading enzyme, MMP‐9.