Matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro

Interleukin (IL)-1β plays an important role in promoting osteoarthritis (OA) lesions by inducing chondrocytes to secrete matrix metalloproteinases (MMPs), which degrade the extracellular matrix and facilitate chondrocyte apoptosis. Matrine was shown to exert anti-inflammatory effects. However, the role of matrine in OA is still unclear. Therefore, in this study, we investigated the effects of matrine on the expression of MMPs in IL-1β-treated human chondrocytes and the underlying mechanism. The cell viability of chondrocytes was detected by MTT assay. The cell apoptosis of chondrocytes was measured by flow cytometric analysis. The protein production of MMPs was determined by ELISA. The protein expression of phosphorylation of mitogen-activated protein kinases (MAPKs) and the inhibitor of kappaB alpha (IκBα) was determined by Western blot. Matrine significantly inhibited the IL-1β-induced apoptosis in chondrocytes. It also significantly inhibited the IL-1β-induced release of MMP-3 and MMP-13, and increased the production of TIMP-1. Furthermore, matrine inhibits the phosphorylation of p-38, extracellular regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and IκBα degradation induced by IL-1β in chondrocytes. Taken together, our results show that matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro. Therefore,-matrine may be beneficial in the treatment of OA.     

Hemin inhibits NO production by IL-1β-stimulated human astrocytes through induction of heme oxygenase-1 and reduction of p38 MAPK activation

Background  

Heme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO) by interlukin (IL)-1β-activated human astrocytes.     

Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes

It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species and possesses diverse biological activities including anti-inflammatory properties. However, the role of diosgenin in inflammatory responses in OA chondrocytes is still unclear. Therefore, in this study, we investigated the anti-inflammatory properties of diosgenin in human OA chondrocytes. We found that diosgenin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) induced by interleukin-1-beta (IL-1β). Diosgenin significantly inhibited the IL-1β-stimulated expression of metalloproteinase-3 (MMP-3), MMP-13, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human OA chondrocytes. In addition, diosgenin suppressed the degradation of IκB-α in IL-1β-induced human OA chondrocytes. Taken together, this study showed that diosgenin can effectively inhibit the IL-1β-induced expression of inflammatory mediators, suggesting that diosgenin may be a potential agent in the treatment of OA.     

Coincidence of autoantibody production with the activation of natural killer T cells in α-galactosylceramide-mediated hepatic injury

Natural killer T (NKT) cells are known to be specifically activated by α-galactosylceramide (α-GalCer) via their interaction with CD1d. At that time, NKT cells mediate autoreactivity and eventually induce hepatic injury. As these immune responses resemble acute autoimmune hepatitis, it was examined whether autoantibody production and the activation of autoantibody-producing B-1 cells were accompanied by this phenomenon. Autoantibodies against Hep-2 cells and double-stranded DNA were detected in sera as early as day 3 (showing a peak at day 14) when mice were treated with α-GalCer. On day 3, B220(low) cells appeared in the liver. These B220(low) cells were CD5(-) (i.e. B-1b cells) and CD69(+) (an activation marker). Primarily, such B220(low) cells were present in the peritoneal cavity, but the proportion of B220(low) cells increased with the administration of α-GalCer even at this site. In parallel with the appearance of B220(low) cells in the liver, hepatic lymphocytes acquired the potential to produce autoantibodies in in vitro cell culture in the presence of lipopolysaccharide. These results suggested that hepatic injury induced by α-GalCer administration resembled acute autoimmune hepatitis and that the major effector lymphocytes were NKT cells with autoreactivity and autoantibody-producing B-1 cells.     

Fibronectin regulates the activation of THP-1 cells by TGF-β1

OBJECTIVE AND DESIGN: To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. MATERIAL OR SUBJECTS: THP-1 monocytic cell line. TREATMENT: THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. METHODS: Assays or assessments carried out, together with statistical test applied. RESULTS: We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. CONCLUSIONS: These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.     

Cytokine production by human leukocytes with different expressions of natural antiviral immunity and the effect of antibodies against interferons and TNF-±

Introduction: Two activities of innate antiviral immunity were studied: the resistance of human peripheral blood mononuclear cells (PMBCs) ex vivo to viral infection and the production of cytokines. Materials and Methods: Samples of blood were taken from healthy blood donors and from persons with frequent infections of the upper respiratory system. PMBCs were isolated by gradient centrifugation. Vesicular stomatitis virus (VSV) was used as the indicatory virus to infect PMBCs. The cytokines: IFN, TNF, and IL-6 were titrated by biological methods and IL-10 by ELISA. Results: Blood donors were divided for two groups: those with VSV-resistant and those with VSV-sensitive PMBCs and secretion of cytokines by them was compared. The resistant PMBCs produced more cytokines than the sensitive ones. A statistically significant difference, was found only in the case of the IFNs. To examine the contribution of IFNs and TNF in maintaining resistance, leukocytes from both groups were treated with specific anti-cytokine antibodies. The authors’ previous study showed that the elimination of spontaneous IFN-α, IFN-β, IFN-γ, and TNF-α from resistant leukocytes resulted in increased VSV replication This indicates the important role of cytokines. In VSV-sensitive PMBCs, anti-IFN-α showed the opposite effect (decreased virus replication). In the absence of spontaneous IFN-α, disturbances in cytokine production were observed. Conclusions: Complete resistance of PMBC to VSV infection is accompanied by higher cytokine release, The paradoxical effect of anti-IFN-α on virus replication in leukocytes sensitive to viral infection may be attributed to changes in the cytokine profile balance, i.e. high TNF production by VSV-infected leukocytes and a complete reduction of IL-6 production.     

The most severe forms of type I autoimmune hepatitis are associated with genetically determined levels of TGF-β1

We previously reported that paediatric (PAH) and adult (AAH) forms of type I autoimmune hepatitis (AH) have different HLA-associations and clinical outcome. In the present study we investigated the role of TGF-β1 genetic polymorphisms in the different outcome of PAH and AAH. We found a significant increase of “high producer” 25GG genotype in PAH and 10CC in AAH. Low inflammation and low fibrosis in AAH was associated with the increase of codon 10CC (high producer) and codon 25CC (low producer) genotypes. The analysis in AAH of the two positions-haplotypes revealed that combined presence of 25GG and 10CC seems to neutralize the 10CC effect which remained in AAH having the 10CC⁺–25GG^? haplotype. Altogether these results may explain, at least partially, the different clinical outcome of AAH and PAH.     

Astragaloside inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes and ameliorates the progression of osteoarthritis in mice

Abstract

Background: Osteoarthritis (OA) is a chronic joint-degeneration disease and accounts for the most frequent arthritis in aging people. OA is characterized by the degeneration of articular cartilage, subchondral bone sclerosis and synovitis. Inflammation as an important role in OA progression, in that anti-inflammatory agents could effectively inhibit the development of OA with minimal side effects, therefore developing a nature anti-inflammatory compound will be a promising therapy for treating OA.

Methods: We treated patient-derived chondrocytes and mouse models of OA with astragaloside, an effective component of astragalus membranaceus, and measured its effect on pro-inflammatory cytokines and OA progression in mice.

Results: In vitro, astragaloside induced a dose-dependent inhibition of IL-1β-induced the production of inflammatory factors, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), expression of MMP 13 and ADAMTS-5, and the activation of NF-κB signaling. In vivo, astragaloside ameliorate the degeneration of cartilage in mouse model of OA.

Conclusion: Astragaloside potentially serve as a promising and effective therapeutic agent for treating OA patients.     

TGF-β1 and IGF-1 influence the re-differentiation capacity of human chondrocytes in 3D pellet cultures in relation to different oxygen concentrations

To prevent de-differentiation of chondrocytes in vitro, the 3D environment, growth factors and different oxygen concentrations were considered. In this in vitro study, we quantified the influence of insulin-like growth factor (IGF)-1 and/or transforming growth factor (TGF)-β1 under differing oxygen (5/21% O(2)) levels on the proliferation and synthesis rates of hyaline extracellular matrix (ECM) components in chondrogenic pellet cultures. Human chondrocytes isolated from articular cartilage were transferred into conical tubes to form pellets. Pellets were stimulated with TGF-β1 and/or IGF-1. After 2 and 5 weeks of cultivation the DNA concentration and expression of pro-collagen type 1, type 2 and aggrecan were analysed. Under hypoxia the DNA content remained stable. In contrast, under normoxia, cells showed an increase of DNA concentration after stimulation with TGF-β1/IGF-1 and TGF-β1. Nevertheless, DNA contents under normoxia did not reach the values of hypoxic-cultivated cells. Under both culture conditions a reduced synthesis of pro-collagen type 1 could be determined. Although the expression of pro-collagen type 2 was significantly higher under normoxia, a decrease in the case of TGF-β1/IGF-1- and IGF-1-stimulated cells was observed. Under hypoxia pro-collagen type 2 contents remained stable or increased for TGF-β1/IGF-1-stimulated cells. Furthermore, incubation with growth factors resulted in aggrecan accumulation under hypoxia, while a reduced expression under normoxia could be determined for TGF-β1/IGF-1- and IGF-1-stimulated cells. Our results demonstrate that the treatment with growth factors causes differences in the expression of ECM compounds within pellet cultures. While under normoxia TGF-β1 alone leads to a positive effect of the expression of hyaline cartilage-specific ECM components, an additive effect of both growth factors was only determined under hypoxia.     

Increased levels of α6 integrins are associated with the metastatic phenotype of human breast cancer cells

Integrins play an important role in interactions between cells and the extracellular matrix, and thus have a potential role in metastasis. Expression levels of 6, 1 and 4 integrin sub-units were measured in a panel of human breast cancer cell lines by RT/PCR, immunoprecipitation and flow cytometry. All the lines expressed 6, with the highest levels in the MDA-MB-231 and MDA-MB-435 cells. These grew the most aggressively and were metastastic in nude mice. Low levels of 6 protein were measured in breast cancer cells that were poorly tumorigenic and non-metastatic in nude mice, and there was an inverse relationship between ER and 6 expression. RT/PCR revealed that all lines expressed the 2 isoforms of 6, with the 6A isoform generally more abundant than 6B isoform. Clones of MDA-MB-435 were isolated by sterile sorting for cells with high or low 6 expression, and two variants established from metastases in nude mice were found to differ in 6 expression. When injected into nude mice, the 6-high variants produced significantly more lung metastases than the 6-low variants. 1 was abundant in all lines, while 4 was not detected in MDA-MB-134 cells, and in the MDA-MB-435 cells an alternately spliced variant of 4 was identified. Sequencing of the alternate variant revealed a novel sequence from a splicing event in the cytoplasmic tail of 4. None of the cells with this variant mRNA expressed detectable levels of 4 protein. Our results suggest that high 6 expression in human breast cancer cells is associated with tumorigenicity and metastatic potential.     

IL-1β/IL-6 network in the tumor microenvironment of human colorectal cancer

Objectives

Recent studies suggest that the interaction between interleukin (IL)-1β and IL-6 in the microenvironment might be involved in the development and progression of human colorectal cancer (CRC). However, the expression of IL-1β/IL-6 network within the CRC microenvironment is not fully understood.

Interleukin-25 production is differently regulated by TNF-α and TGF-β1 in the human gut

An altered balance between effector and regulatory factors is supposed to sustain the tissue-damaging immune response in inflammatory bowel disease (IBD). We have recently shown that in IBD, there is a defective synthesis of the counter-regulatory cytokine, interleukin (IL)-25. In this study we investigated factors that control IL-25 production in the gut. IBD patients produced less IL-25 when compared with normal controls. Stimulation of normal intestinal explants with tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ) or IL-21, reduced IL-25 synthesis. Consistently, IL-25 production was enhanced by anti-TNF-α both in vitro and in vivo. Upregulation of IL-25 was also seen in normal colonic explants stimulated with transforming growth factor-β1 (TGF-β1). As in IBD, TGF-β1 activity is abrogated by Smad7, we next assessed whether inhibition of Smad7 with an antisense oligonucleotide enhanced IL-25 expression. Knockdown of Smad7 was accompanied by an increase in IL-25 production. Data show that IL-25 production is differently regulated by TNF-α and TGF-β1 in the human gut.     

Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit the production of IL-1β, IL-6 and IL-10 on human macrophages

Objective

Tea tree oil (TTO) is an essential oil with anti-inflammatory properties, steam distilled from the plant Melaleuca alternifolia. We investigated the immunomodulatory properties of TTO and its components (terpinen-4-ol and alpha-terpineol) using lipopolysaccharide (LPS)-stimulated macrophages.

Methods

The ability of TTO, terpinen-4-ol and alpha-terpineol to modulate the macrophage response to bacterial LPS stimulation was assessed by ELISA for tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 cytokine production and by western blotting for the activation of nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling, which are associated with the expression of pro-inflammatory cytokines. We used a human monocytic cell line (U937) differentiated into macrophages.

Results

LPS induced the production of all cytokines, and TTO and its components significantly reduced the production of IL-1β, IL-6 and IL-10. The production of TNF-α was not affected by either TTO or its major components. The modulation of cytokine production was not mediated by changes in NF-κB or p38 MAPK activation.

Conclusion

TTO, terpinen-4-ol and alpha-terpineol can suppress the production of inflammatory mediators in LPS-stimulated human macrophages; this inhibition was mediated by interfering with the NF-kB, p38 or ERK MAPK pathways.     

Differential induction of human monocyte transforming growth factorβ1 production and its regulation by interleukin 4

We have previously shown that trauma patients' monocytes which arein vivo activated by multiple injury-induced mediators have elevated transforming growth factor-beta (TGF) bioactivity. Interleukin-4 (IL-4), a Th₂ and B lymphocyte stimulatory factor, has been shown to inhibit monocyte production of a number of mediators both after lipopolysaccharide stimulation and after trauma-induced stimulation. However, IL-4 inhibitory effects appears to vary, depending on the mixture of inducing stimuli. Here we describe thein vitro IL-4 inhibition of human monocyte TGF bioactivity using several stimulation induction protocols: muramyl dipeptide stimulation alone, or after FcRI (CD64) cross-linking induction, interferon-gamma (IFN) priming, or trauma-generatedin vivo mediator induction. IL-4 suppressed both muramyl dipeptide-induced TGF bioactivity and TGF mRNA in a dose-dependent fashion and was most effective when IL-4 was administered at initiation of normal monocyte stimulation. Muramyl dipeptide (MDP)-induced increases in trauma patients' monocyte TGF bioactivity were also inhibited by high doses of IL-4 (25 ng/ml). FcRI cross-linking increased MDP-induced normal monocyte TGF bioactivity, but this increase could be consistently inhibited only by very high IL-4 concentrations (50 ng/ml). IL-4 did not consistently downregulate MDP-induced TGF bioactivity in IFN-primed monocytes. IL-4 can suppress monocyte TGF production, as well as other monocyte mediators, but its efficiency depends on the stimuli combination present in the microenvironment.     

The interaction of influenza H5N1 viral hemagglutinin with sialic acid receptors leads to the activation of human γδT cells

Highly pathogenic avian influenza H5N 1 epidemics are a significant public health hazard. Genetically engineered H5N 1 viruses with mammalian transmission activity highlight the potential risk of a human influenza H5N 1 pandemic. Understanding the underlying principles of the innate immune system in response to influenza H5N 1 viruses will lead to improved prevention and control of these potentially deadly viruses, γδT cells act as the first line of defense against microbial infection and help initiate adaptive immune responses during the early stages of viral infection. In this study, we investigated the molecular mechanisms of γδ T cells in response to influenza H5N1 viral infection, We found that recombinant hemagglutinin （rHA） derived from three different strains of influenza H5N 1 viruses elicited the activation of γδ T cells cultured in peripheral blood mononuclear cells （PBMCs）. Both the cell surface expression of CD69, an early activation marker on γδ T cells, and the production of interferon-y （IFN-y） were significantly increased. Notably, the rHA protein-induced γδ T-cell activation was not mediated by TCRγδ, NKG2D or pattern recognition receptors （PRRs） or NKp46 receptors. The interaction of rHA proteins with sialic acid receptors may play a critical role in γδ T-cell activation. Our data may provide insight into the mechanisms underlyingγδT-cell activation in response to infection with H5N1 viruses.