Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

Introduction

The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.

Methods

Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF^4-23. Nitric oxide (NO) production, prostaglandin E₂ (PGE₂) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data.

Results

The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE₂ release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF^4-23 had the opposite effect and reduced NO and PGE₂ release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE₂ release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE₂ release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression.

Conclusions

Mechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.     

Low oxygen tension increased fibronectin fragment induced catabolic activities - response prevented with biomechanical signals

Introduction

The inherent low oxygen tension in normal cartilage has implications on inflammatory conditions associated with osteoarthritis (OA). Biomechanical signals will additionally contribute to changes in tissue remodelling and influence the inflammatory response. In this study, we investigated the combined effects of oxygen tension and fibronectin fragment (FN-f) on the inflammatory response of chondrocytes subjected to biomechanical signals.

Methods

Chondrocytes were cultured under free-swelling conditions at 1%, 5% and 21% oxygen tension or subjected to dynamic compression in an ex vivo 3D/bioreactor model with 29 kDa FN-f, interleukin-1beta (IL-1β) and/or the nitric oxide synthase (NOS) inhibitor for 6 and 48 hours. Markers for catabolic activity (NO, PGE₂), tissue remodelling (GAG, MMPs) and cytokines (IL-1β, IL-6 and TNFα) were quantified by biochemical assay. Aggrecan, collagen type II, iNOS and COX-2 gene expression were examined by real-time quantitative PCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data.

Results

Both FN-fs and IL-1β increased NO, PGE₂ and MMP production (all P < 0.001). FN-f was more active than IL-1β with greater levels of NO observed at 5% than 1% or 21% oxygen tension (P < 0.001). Whilst FN-f reduced GAG synthesis at all oxygen tension, the effect of IL-1β was significant at 1% oxygen tension. In unstrained constructs, treatment with FN-f or IL-1β increased iNOS and COX-2 expression and reduced aggrecan and collagen type II (all P < 0.001). In unstrained constructs, FN-f was more effective than IL-1β at 5% oxygen tension and increased production of NO, PGE₂, MMP, IL-1β, IL-6 and TNFα. At 5% and 21% oxygen tension, co-stimulation with compression and the NOS inhibitor abolished fragment or cytokine-induced catabolic activities and restored anabolic response.

Conclusions

The present findings revealed that FN-fs are more potent than IL-1β in exerting catabolic effects dependent on oxygen tension via iNOS and COX-2 upregulation. Stimulation with biomechanical signals abolished catabolic activities in an oxygen-independent manner and NOS inhibitors supported loading-induced recovery resulting in reparative activities. Future investigations will utilize the ex vivo model as a tool to identify key targets and therapeutics for OA treatments.     

Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Role of PTPα in the Destruction of Periodontal Connective Tissues

IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα^+/+ and PTPα^−/− mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5–3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα^−/− mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα^KO and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions.     

Regulation of Wound Healing by Granulocyte-Macrophage Colony-Stimulating Factor after Vocal Fold Injury

Objectives

Vocal fold (VF) scarring remains a therapeutic challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates epithelial wound healing, and recently, growth factor therapy has been applied to promote tissue repair. This study was undertaken to investigate the effect of GM-CSF on VF wound healing in vivo and in vitro.

Methods

VF scarring was induced in New Zealand white rabbits by direct injury. Immediately thereafter, either GM-CSF or PBS was injected into the VFs of rabbits. Endoscopic, histopathological, immunohistochemical, and biomechanical evaluations of VFs were performed at 3 months post-injury. Human vocal fold fibroblasts (hVFFs) were cultured with GM-CSF. Production of type I and III collagen was examined immunocytochemically, and the synthesis of elastin and hyaluronic acids was evaluated by ELISA. The mRNA levels of genes related to ECM components and ECM production-related growth factors, such as HGF and TGF-ß1, were examined by real time RT-PCR.

Results

The GM-CSF-treated VFs showed reduced collagen deposition in comparison to the PBS-injected controls (P<0.05). Immunohistochemical staining revealed lower amounts of type I collagen and fibronectin in the GM-CSF-treated VFs (P<0.05 and P<0.01, respectively). Viscous and elastic shear moduli of VF samples were significantly lower in the GM-CSF group than in the PBS-injected group (P<0.001 and P<0.01, respectively). Mucosal waves in the GM-CSF group showed significant improvement when compared to the PBS group (P = 0.0446). GM-CSF inhibited TGF-β1-induced collagen synthesis by hVFFs (P<0.05) and the production of hyaluronic acids increased at 72 hours post-treatment (P<0.05). The expressions of HAS-2, tropoelastin, MMP-1, HGF, and c-Met mRNA were significantly increased by GM-CSF, although at different time points (P<0.05).

Conclusion

The present study shows that GM-CSF offers therapeutic potential for the remodeling of VF wounds and the promotion of VF regeneration.     

An Evaluation of the Effects of Cytokines on Intracellular Oxidative Production in Normal Neutrophils by Flow Cytometry

To evaluate the effects of inflammatory cytokines on oxidative production in normal neutrophils, seven kinds of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), interleukin-2 (IL-2), IL-6, IL-1α IL-1β, and interferon-β (IFN-β) were tested. The intracellular hydrogen peroxide (H₂O₂) in individual cells was determined by flow cytometry. According to the levels of intracellular H₂O₂ enhanced by cytokines, these seven cytokines were classified into three types: (1) prominently effective—GM-CSF; (2) moderately effective—G-CSF, IL-6, and IL-2; (3) weakly or ineffective—IFN-β, IL-1α, and IL-1β. Changes in cell size and cell surface structure after stimulation of those seven cytokines were simultaneously measured by flow cytometry. The most prominently effective cytokine, GM-CSF, initially caused enlargement of cell size and irregularity of the cell surface and subsequently increased H₂O₂ production by neutrophils. In contrast, the weakly or ineffective cytokines, like IL-1β, had no effects on cell size or cell surface. Our study indicates that some kinds of cytokines enhance oxidetive production and cause morphological changes in neutrophils.     

Transforming growth factor-β inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-β (TGF-β). In this paper, we show that TGF-β inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-β-treated chondrocytes show reduced ¹²⁵I-IL-1 binding that returns to a normal level when TGF-β is removed from the culture medium. The inhibitory effect of TGF-β is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor—ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of M_r =116 and 80 kDa and a minor band of M_r =100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-β treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-β to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1β, 6, and 8, tumor necrosis factor-α, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E₂ production. WISH cells were preincubated with cytokines (0.0001–10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE₂ production was measured by radioimmunoassay. EGF, IL-1β and TNF-α alone caused a dose-dependent increase in PGE₂ production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1β or TNF-α, there was a dose-dependent potentiation of EGF-induced PGE₂ production that was greater than the sum of EGF alone and IL-1β or TNF-α alone. In each case, the minimum dose of IL-1β or TNF-α which amplified EGF-induced PGE₂ production was 0.1 ng/ml (p < 0.05, Student's t-test). These data show that low concentrations of IL-1β or TNF-α may serve to amplify EGF-mediated PGE₂ biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsivecells.     

Mycobacterium tuberculosis-specific CD4+, IFNgamma+, and TNFalpha+ multifunctional memory T cells coexpress GM-CSF

Multifunctional T cells expressing several cytokines in parallel are thought to play a crucial role in protection against different infections. To characterize T cell cytokine patterns associated with disease and protection in Mycobacterium tuberculosis infection we determined the expression of IFNγ, IL-2, TNFα, and GM-CSF in T cell subpopulations from children with tuberculosis (TB) and healthy latently M. tuberculosis-infected children (LTBI) after short-term in vitro restimulation. We identified CD4⁺ effector memory T cells (T_EM) as the major source of all measured cytokines after antigen-specific restimulation. T_EM from children with TB expressed higher proportions of IFNγ, TNFα, and IL-2 after Mtb restimulation while no differences were detected for GM-CSF between both study groups. GM-CSF secretion strongly depended on antigen-specific stimulation. Analyses of multiple cytokine patterns revealed that the majority of GM-CSF-positive M. tuberculosis-specific memory T cells coexpressed IFNγ and TNFα therefore showing a characteristic feature of multifunctional T cells. We conclude that children with active TB possess higher proportions of IFNγ-, TNFα-, and/or IL-2-positive T_EM than children with LTBI while GM-CSF coexpression reveals a novel subpopulation within CD4⁺ memory T cells not increased in children with active TB.     

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

Introduction

Although IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.

Methods

Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1β-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1β signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes.

Results

SOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1β stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1β-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-β-activated kinase 1 (TAK1) expression.

Conclusions

Our results show that SOCS1 is induced by IL1-β in OA chondrocytes and suppresses the IL-1β-induced synthesis of matrix-degrading enzymes by inhibiting IL-1β signaling at multiple levels. It suggests that the IL-1β-inducible SOCS1 acts as a negative regulator of the IL-1β response in OA cartilage.     

Salmonella Induced IL-23 and IL-1β Allow for IL-12 Production by Monocytes and M?1 through Induction of IFN-γ in CD56+ NK/NK-Like T Cells

Background

The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-γ. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain.

Methodology/Principal Findings

We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mϕ1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1β and IL-18, but not IL-12. Supernatants of Salmonella-infected Mϕ1 contained more IL-18 and IL-1β as compared with supernatants of Mϕ1 stimulated with isolated TLR agonists, and induced IFN-γ production in human CD56⁺ cells in an IL-23 and IL-1β-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1β led to the production of GM-CSF in CD56⁺ cells. Both IFN-γ and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production.

Conclusions/Significance

The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-γ and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-γ production in the type-1 cytokine pathway.     

Macrophage responses to interferon-γ are dependent on cystatin C levels

The aim of the present investigation was to elucidate possible effects of cystatin C on inflammatory responses mediated by macrophages. Previously it has been shown that in vitro treatment of murine peritoneal macrophages with interferon-γ (IFN-γ) causes a down-regulation of cystatin C secretion. To investigate whether such changes in cystatin C expression in turn can affect inflammatory responses mediated by macrophages, we have compared effects of IFN-γ on macrophages isolated from wild-type (cysC^+/+) and cystatin C knockout (cysC^−/−) mice. It was shown that IFN-γ-primed cysC^−/− macrophages exhibit significantly higher interleukin-10 (IL-10) but lower tumor necrosis factor-α (TNF-α) expression, and reduced nuclear factor (NF)-κB p65 activation, compared to similarly primed cysC^+/+ cells. Exogenously added cystatin C enhanced IFN-γ-induced activation of NF-κB p65 and increased mRNA levels for inducible NO synthase (iNOS) in cysC^−/− macrophages as well as levels of nitric oxide and TNF-α in the cell culture medium, in agreement with an enhanced pro-inflammatory response. Accordingly, IFN-γ-induced IL-10 mRNA expression in cysC^−/− macrophages was down-regulated by exogenously added cystatin C. Taken together, our data provide evidence that changes in cystatin C levels alter macrophage responses to IFN-γ. The latter down-regulates the production of cystatin C, which leads to a suppressed inflammatory condition with enhanced IL-10 levels and down-regulated TNF-α and NF-κB. It is concluded that cystatin C through this effect can act as an immunomodulatory molecule.     

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-α in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-α. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-α or a combination of both (at 10 ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10 ng/ml TNF-α for 6 or 24 h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-α suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-α mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1β expression by TNF-α was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-α in chondrocytes.     

Hyperosmotic Stress as a Stimulant for Proinflammatory Cytokine Production

The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH₂-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC)in vitro.There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330–410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1α, IL-1β, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280–295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P< 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1α and IL-1β increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1α, IL-1β, TNF-α, and IL-8.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

12/15-Lipoxygenase Is Required for the Early Onset of High Fat Diet-Induced Adipose Tissue Inflammation and Insulin Resistance in Mice

Background

Recent understanding that insulin resistance is an inflammatory condition necessitates searching for genes that regulate inflammation in insulin sensitive tissues. 12/15-lipoxygenase (12/15LO) regulates the expression of proinflammatory cytokines and chemokines and is implicated in the early development of diet-induced atherosclerosis. Thus, we tested the hypothesis that 12/15LO is involved in the onset of high fat diet (HFD)-induced insulin resistance.

Methodology/Principal Findings

Cells over-expressing 12/15LO secreted two potent chemokines, MCP-1 and osteopontin, implicated in the development of insulin resistance. We assessed adipose tissue inflammation and whole body insulin resistance in wild type (WT) and 12/15LO knockout (KO) mice after 2–4 weeks on HFD. In adipose tissue from WT mice, HFD resulted in recruitment of CD11b⁺, F4/80⁺ macrophages and elevated protein levels of the inflammatory markers IL-1β, IL-6, IL-10, IL-12, IFNγ, Cxcl1 and TNFα. Remarkably, adipose tissue from HFD-fed 12/15LO KO mice was not infiltrated by macrophages and did not display any increase in the inflammatory markers compared to adipose tissue from normal chow-fed mice. WT mice developed severe whole body (hepatic and skeletal muscle) insulin resistance after HFD, as measured by hyperinsulinemic euglycemic clamp. In contrast, 12/15LO KO mice exhibited no HFD-induced change in insulin-stimulated glucose disposal rate or hepatic glucose output during clamp studies. Insulin-stimulated Akt phosphorylation in muscle tissue from HFD-fed mice was significantly greater in 12/15LO KO mice than in WT mice.

Conclusions

These results demonstrate that 12/15LO mediates early stages of adipose tissue inflammation and whole body insulin resistance induced by high fat feeding.     

Interleukin-1β induces the synthesis and activity of cytosolic phospholipase A2 and the release of prostaglandin E2 in human amnion-derived WISH cells

The objective of this study was to examine the expression and activity of cytosolic phospholipase A₂ (cPLA₂) in relation to prostaglandin E₂ (PGE₂) synthesis in human amnion-derived WISH cells in response to stimulation by interleukin-1β (IL-1β). cPLA₂ activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃). Treatment of WISH cells with IL-1β (0.01–1 ng/mL) for up to 24 h resulted in a significant increase in PGE₂ release in a concentration- and time-dependent manner accompanied by increases both in total cellular cPLA₂ activity and in cPLA₂ protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA₂ activity and cPLA₂ protein level indicates that IL-1β may induce the synthesis of CPLA₂. Incubation of the cells with 10 μM AACOCF₃ for 24 h significantly inhibited IL-1β-induced PGE₂ production strongly suggesting that cPLA₂ mediates IL-1β-induced PGE₂ formation. In unstimulated cells, there is appreciable total cellular cPLA₂ activity and protein, but these cells produce low amounts of PGE₂ until stimulated by IL-1β, suggesting that cPLA₂ translocation from cytosol to the membrane is necessary for its bioactivity. In contrast to IL-1β, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10⁻¹⁰−10⁻⁶ M) for 24 h significantly inhibited total cellular cPLA₂ activity in a concentration-dependent manner. The amount of total cellular cPLA₂ protein seen on Western blot remained unchanged following TPA treatment. These data suggest that in WISH cells, IL-1β induces both translocation to the membrane and de novo synthesis of cPLA₂ protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only cause cPLA₂ translocation but no increase in cPLA₂ protein synthesis, resulting in limited PFG synthesis. Our results provide a mechanism for the effect of IL-1β on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA₂ in the mechanism initiating human parturition.     

Human Osteoarthritic Cartilage Shows Reduced In Vivo Expression of IL-4, a Chondroprotective Cytokine that Differentially Modulates IL-1β-Stimulated Production of Chemokines and Matrix-Degrading Enzymes In Vitro

Background

In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β.

Methodology/Principal Findings

The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13.

Conclusions/Significance

Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.