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Objective: C‐C motif chemokine ligand 2 (CCL2) is a potent chemokine that plays an important role in host defense and inflammatory diseases in the central nervous system. Toll‐like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double‐stranded RNAs. In the present study, we examined whether a TLR3 agonist induces CCL2 expression in brain microvascular endothelial cells, and investigated the mechanism underlying upregulation of CCL2 expression through TLR3 signaling. Methods: A human brain microvascular endothelial cell line, human cerebral microvascular endothelial cell (hCMEC)/D3 was treated with a TLR3 agonist, polyinosinic‐polycytidylic acid (poly IC). The mRNA and protein expression of CCL2 were evaluated using real‐time reverse transcription polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. The role of TLR3 and nuclear factor‐κB (NF‐κB) were examined with RNA interference. We also examined the effect of pretreatment with an NF‐κB inhibitor, SN50, an interferon regulatory factor 3 inhibitor MR67307 and human type I interferon neutralizing antibody mixture on poly IC‐induced CCL2 expression. Results: Expression of CCL2 was induced by poly IC. Poly IC‐induced CCL2 expression was decreased by knockdown of TLR3 or NF‐κB p65 and by pretreatment with SN50. Neither MR67307 nor human type I interferon neutralizing antibody mixture affected the CCL2 expression induced by poly IC. Conclusions: CCL2 expression was induced by poly IC through TLR3 signaling, and NF‐κB is involved in this reaction. CCL2 produced by human brain endothelial cells might induce the infiltration of monocytes followed by immune and inflammatory reactions in the brain, and could be involved in the pathogenesis of viral encephalitis. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Brain microvascular endothelial cells (BMEC) are the major component of the blood–brain barrier, and play a critical role in protecting the brain from pathogens. Although Toll‐like receptor 3 (TLR3) is known as an important pattern‐recognition receptor against viral double‐stranded RNA, the downstream reactions of TLR3 in human BMEC are not fully characterized. The purpose of the present study was to investigate the role of TLR3 signaling in the expression of retinoic acid‐inducible gene‐I (RIG‐I), melanoma differentiation‐associated gene 5 (MDA5) and C‐X‐C motif chemokine ligand 10 (CXCL10) in human BMEC. Methods: The hCMEC/D3 cells, a human BMEC cell line, were cultured and stimulated with polyinosinic‐polycytidylic acid, a synthetic TLR3 agonist. Quantitative real‐time reverse transcription polymerase chain reaction, western blotting and enzyme‐linked immunosorbent assay were used to examine the expression of interferon‐β, RIG‐I and CXCL10. RNA interference against TLR3, interferon‐β, RIG‐I or MDA5 was also carried out. Results: Expression of RIG‐I, MDA5 and CXCL10 were induced by polyinosinic‐polycytidylic acid, and this was inhibited by knockdown of either TLR3 or interferon‐β. Knockdown of RIG‐I or MDA5 did not affect the CXCL10 expression in the early phase (4 h), but decreased the CXCL10 induction in the late phase (8 or 24 h). Conclusions: RIG‐I, MDA5 and CXCL10 are induced through TLR3 activation in hCMEC/D3 cells, and RIG‐I and MDA5 might function to enhance and/or prolong the reactions mediated by CXCL10. These molecules might play a distinct role in innate immune reactions against double‐stranded RNA viruses in human BMEC. [ABSTRACT FROM AUTHOR]

Objective Brain microvascular endothelial cells are one of the cell types that form the blood-brain barrier, and play an important role in the defense system of the brain. Toll-like receptor 3 ( TLR3) is a pattern-recognition receptor against double-stranded RNA, and TLR3 signaling is important in antiviral innate immune reactions. However, TLR3 signaling in brain microvascular endothelial cells is not well understood. We aimed to investigate the role of TLR3 signaling in chemokine CCL5 production in human brain microvascular endothelial cells. Methods The hCMEC/D3 human brain microvascular endothelial cells were cultured, and treated with an authentic TLR3 agonist polyinosinic : polycytidylic acid. The expression of CCL5 was examined using quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Involvement of TLR3, interferon ( IFN)-β, or IFN-λ1, IFN-regulatory factor 3 or nuclear factor-κB p65 in this reaction was examined using RNA interference. Translocation of p65 into the nucleus was examined using immunofluorescence staining. Results Treatment of cells with polyinosinic : polycytidylic acid induced the expression of CCL5, IFN-β and IFN-λ1, and also the translocation of p65 into the nucleus. Knockdown of TLR3, IFN-regulatory factor 3 or p65 inhibited the induction of these molecules, while knockdown of neither IFN-β nor IFN-λ1 affected the expression of CCL5. Conclusions TLR3 activation by polyinosinic : polycytidylic acid induces the expression of CCL5 in cultured hCMEC/D3 cells, and IFN-regulatory factor 3 and p65 are involved in this reaction. CCL5 induced by TLR3 signaling in brain microvascular endothelial cells might contribute to antiviral protective reactions and/or detrimental responses associated with viral infection in the brain. [ABSTRACT FROM AUTHOR]

Aim It has been reported that the innate immune system plays a pivotal role in the pathogenesis of immunoglobulin A nephropathy (IgAN). To explore non-invasive monitoring of disease activity in children with IgAN, we examined whether expressions of mRNA for innate immunity-associated functional molecules: CC ligand chemokine 5 ( CCL5), fractalkine/ CX3 CL1, interferon-γ-induced protein 10 ( IP-10), monocyte chemoattractant protein 1 ( MCP-1), retinoic acid-inducible gene- I ( RIG- I), and toll-like receptor 3 ( TLR3) in urinary sediment from patients with IgAN correlate with histologic parameters. Methods Twenty consecutive children with IgAN and four children with thin basement membrane disease (serving as a non-inflammatory control) were enrolled in this pilot study. Urinary mRNA expressions of target genes were examined real-time quantitative polymerase chain reaction. Results The expressions of CCL5, fractalkine and RIG- I were significantly increased in IgAN (all P < 0.05). Although no significant correlation was observed between mRNA expressions of these three molecules and clinical parameters, such as levels of urinary protein excretion, degrees of occult blood in urine and serum albumin, the expression of fractalkine was significantly correlated with the histological activity index ( P = 0.022) and the chronicity index ( P = 0.005). Furthermore, intense glomerular immune activity of fractalkine was observed in biopsy specimens in patients with moderately to severe proliferative IgAN. Conclusion Regional expression of fractalkine may be involved in the pathogenesis of childhood IgAN. Although our present findings remain preliminary, measurement of mRNA expression of fractalkine in urinary sediment could be used as a non-invasive method for predicting histologic severity in IgAN in children. Further studies of this issue are needed. [ABSTRACT FROM AUTHOR]

Aim Mizoribine ( MZR) is a selective inhibitor of the inosine monophosphate dehydrogenase - a key enzyme in the de novo pathway of guanine nucleotides - that was developed in Japan. Besides its immunosuppressive effects, MZR has recently been reported to suppress the progression of histologic chronicity via suppression of macrophage infiltration of the interstitium in selected patients with lupus nephritis. Methods We examine the direct effect of MZR in human mesangial cells on the expression of functional molecules including monocyte chemoattractants in cultured human mesangial cells ( MCs) treated with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, that makes 'pseudoviral' infection, and analyzed the expression of target molecules by reverse transcriptase-polymerase chain reaction and Western blotting. Thereafter, the effect of MZR on the expressions was examined. Results Pretreatment of cells with MZR partially, but significantly, attenuates the expression of monocyte chemoattractant protein ( MCP)-1 mRNA and protein, whereas the poly IC-induced expressions for the other functional molecules, such as CCL5, fractalkine and IL-8 were not influenced by MZR treatment. On the other hand, pretreatment of cells with tacrolimus did not suppress the expression of MCP-1 mRNA. Conclusion Mizoribine itself selectively attenuated the expression of MCP-1 both mRNA and protein levels in MCs treated with poly IC; that is, a possible model of 'pseudoviral' infection, which may be involved in the pathogenesis of lupus nephritis. [ABSTRACT FROM AUTHOR]

Since viral infections activate type I interferon ( IFN) pathways and cause subsequent release of IFN-dependent proinflammatory chemokines and cytokines, the innate immune system plays an important role in the pathogenesis of lupus nephritis ( LN). It has been reported that human myxovirus resistance protein 1 ( Mx1), a type I IFN-dependent transcript, acts against a wide range of RNA viruses. Although the expression of Mx1 in biopsy specimens obtained from patients with dermatomyositis and cutaneous lupus has been described, the expression of Mx1 in human mesangial cells ( MCs) has remained largely unknown. We treated normal human MCs in culture with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of Mx1 by reverse transcription-polymerase chain reaction and western blotting. To elucidate the poly IC-signalling pathway, we subjected the cells to RNA interference against IFN-β. We also conducted an immunofluorescence study to examine mesangial Mx1 expression in biopsy specimens from patients with LN. Poly IC-induced Mx1 expression in MCs are shown both time- and dose-dependently, and RNA interference against IFN-β inhibited poly IC-induced Mx1 expression. Intense glomerular Mx1 expression was observed in biopsy specimens from patients with LN, whereas negative staining occurred in specimens from patients with IgA nephropathy or purpura nephritis. These preliminary observations support, at least in part, the theory of innate immune system activation in the pathogenesis of LN. [ABSTRACT FROM AUTHOR]

Background Propolis is a bee product with various biological properties, including an antiviral activity when taken orally. However, its mechanisms at the cellular and molecular level are not well understood. Results We investigated the effect of propolis on antiviral signaling in A549 cells transfected with double-stranded RNA ( dsRNA), a model for viral infection. Pretreatment of the cells with propolis inhibited poly I:C (synthetic dsRNA)-induced interferon ( IFN)-β expression. Propolis had no effect on the dsRNA-induced expression of RIG-I-like receptors ( RLRs), which are known as intracellular viral RNA sensors. As to the effect on antiviral executor genes, propolis enhanced myxovirus resistance 1 ( MX1) expression, whereas interferon-inducible gene 6-16 ( G1P3) and 2'-5'-oligoadenylate synthetase ( OAS) were unaffected. All of these genes belong to the IFN-inducible genes, suggesting that the effect of propolis on antiviral signaling is not necessarily mediated by the autocrine regulation by IFN-β. Propolis pretreatment inhibited dsRNA-induced interleukin-8 ( IL8) and CCL5 expression, and consequently lowered polymorphonuclear leukocyte ( PMN) chemotactic activity in the cell-conditioned medium. Conclusion Taken together, these results suggest that propolis may suppress excess inflammatory responses without affecting the innate immunity during viral infection. [ABSTRACT FROM AUTHOR]

DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up-regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1α (HIF-1α) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1α but not with ARNT1. The binding of HIF-1α to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved. [ABSTRACT FROM AUTHOR]

ISG20 is an ribonuclease specific for single-stranded RNA and considered to play a role in innate immunity against virus infections. We herein show that both poly IC, an authentic double-stranded RNA, and IFN-γ induced ISG20 expression in cultured HUVEC. Poly IC-induced ISG20 expression was inhibited by LY294002, an inhibitor of PI3K, or by RNA interference against IFN regulatory factor three. ISG20 expression was not induced by IFN-β, loxoribine or CpG oligonucleotide. These results suggest that ISG20 induction by poly IC may not be dependent on the IRF-3-mediated type I IFN induction pathway in HUVEC. ISG20 may be involved in innate immunity against viral infection in vascular endothelial cells. [ABSTRACT FROM AUTHOR]

Treatment of cells with double-stranded RNA (dsRNA) in vitro mimics viral infection and regulates expression of various genes. We addressed the mechanisms of leucocyte traffic across the vascular endothelium induced by dsRNA. The present study focused on the expression of galectin-9, which is one of key molecules in the regulation of the interaction between vascular wall and white blood cells. Human umbilical vein endothelial cells (HUVEC) in culture were treated with polyinosinic-polycytidylic acid (poly IC), and expression of mRNA and protein of galectin-9 was analysed by reverse transcription polymerase-chain reaction (RT-PCR) and Western blotting. Poly IC enhanced the expression of galectin-9 mRNA and protein in concentration- and time-dependent manners. This effect of poly IC was almost completely suppressed by the pretreatment with 2-aminopurine, an inhibitor of dsRNA-dependent kinase. Poly IC treatment of HUVEC also enhanced the adherence of EoL-1 cells to the cells, which was inhibited by co-treatment with lactose. We conclude that poly IC upregulates galectin-9 expression in the vascular endothelium and this may explain part of the mechanism for leucocyte traffic through the vascular wall. [ABSTRACT FROM AUTHOR]

Summary Peroxisome proliferator-activated receptor-γ (PPAR-γ)is a member of nuclear hormone receptor superfamily, and is knownto play a role in various biological processes including inflammatoryresponses and adipocyte differentiation. CX3CL1/fractalkineis a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes.Endothelial cells produce fractalkine when stimulated with cytokinessuch as interleukin-1 (IL-1), tumour necrosis factor-α andinterferon-γ (IFN-γ). We herein report that 15-deoxy-n12,14 -prostaglandinJ2 (15d-PGJ2 ), a PPAR-γ agonist,inhibits the expression of fractalkine induced by IFN-γ orIL-1β in human endothelial cells. Agonist for PPAR-α (WY14643)or PPAR-γ (ciglitazone) did not inhibit the cytokine-inducedfractalkine expression, and the effect of 15d-PGJ2 maybe independent of PPAR. 15-Deoxy- D12,14 prostaglandinJ2 also inhibited the adhesion of blood mononuclear cellsto endothelial monolayers treated with IFN-γ or IL-1β.The data suggest that 15d-PGJ2 regulates inflammatoryreactions, at least in part, through the inhibition of fractalkineexpression and leucocyte traffic through the endothelium. [ABSTRACT FROM AUTHOR]

α-Synuclein was originally identified as the presynaptic nerve terminal protein. Recently, we reported that α-synuclein is also expressed in cultured human astrocytes and that its levels are increased by stimulation with interleukin-1β, suggesting that it may be involved in inflammatory processes. We therefore investigated the effect of inflammatory stimuli on α-synuclein expression in human macrophages. α-Synuclein mRNA and protein were detected in cultured human macrophages and levels of α-synuclein protein were increased by stimulation with lipopolysaccharide and interleukin-1β in a time- and concentration-dependent manner. Immunofluorescent staining showed that α-synuclein protein was expressed within the cytoplasm and nucleus. Furthermore, α-synuclein immunoreactivity was present in alveolar macrophages from human lung tissues. These findings suggest that the function of α-synuclein is not exclusive to the nervous system and that α-synuclein may play a role in inflammatory processes and immune responses. [ABSTRACT FROM AUTHOR]

Summary Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL-6 expression by the ubiquitin-protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG-132, a protease inhibitor, and the levels of IL-6 mRNA and protein were measured by reverse transcription-PCR and ELISA. MG-132 increased the expression of IL-6 mRNA and protein; and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK1/2). MG-132 treatment was also found to enhance the level of phosphorylated MEK1/2. Treatment of the cells with actinomycin D inhibited IL-6 expression in response to MG-132, suggesting the transcriptional upregulation of IL-6 under proteasomal inhibition. We conclude that a protease inhibitor MG-132 upregulates IL-6 expression in vascular endothelial cells, at least in part, through the activation of MEK1/2. [ABSTRACT FROM AUTHOR]

The article presents a research study which investigated the role of Th1/Th2 proinflammatory cytokines in the development and progression of IgA nephropathy (IgAN) in children. It studied seven children with freshly diagnosed IgAN and mRNA expression was studied on urine samples. The study suggested that Th1 polarity might be involved in the pathogenesis of IgAN. It theoretically speculated that measuring the expressions of T-bet/GATA3 genes might provide clue pathogenic mechanisms of IgAN.

Retinoic acid-inducible gene-I (RIG-I) is considered to play a role in innate immunity against virus infections. We showed by immunohistochemical study that RIG-I expression is upregulated in vivo in hepatic Kupffer cells and in splenic reticular cells of mice infected with Listeria monocytogenes. Both heat-killed L. monocytogenes and live L. monocytogenes induced the expression of RIG-I in cultured RAW264.7 murine macrophage-like cells in vitro. RIG-I may also be involved in innate immunity against Listeria infection.