Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture

Background  

Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels.     

Characterisation of the prostaglandin E2-ethanolamide suppression of tumour necrosis factor-α production in human monocytic cells

Background and purpose

Prostaglandin ethanolamides or prostamides are naturally occurring neutral lipid derivatives of prostaglandins that have been shown to be synthesised in vivo following COX-facilitated oxygenation of arachidonoyl ethanolamine (anandamide). Although the actions of prostaglandins have been extensively studied, little is known about the physiological or pathophysiological effects of prostamides. Since prostaglandin E₂ has potent immunosuppressive/immunomodulating actions, the aim of the present study was to determine whether the derivative, prostaglandin E₂ ethanolamide (PGE₂-EA), could modulate the production of the pro-inflammatory cytokine tumour necrosis factor-α in human blood and human monocytic cells and indicate whether this action involved the same receptor systems/signals as PGE₂.

Experimental approach

Whole human blood, monocytes isolated from the blood or the human monocytic cell line THP-1 was incubated with LPS and the level of TNF-α produced was measured by ELISA assay. The actions of PGE₂-EA were assessed on the LPS-induced TNF-α release. In addition, in order to ascertain the receptors involved, the levels of cyclic AMP in cells were measured in monocytes and THP-1 cells in response to PGE₂-EA and directly compared to those of PGE₂. The effect of PGE₂-EA on the binding of radiolabelled PGE₂ to cells was also measured. Cells were incubated with radiolabelled arachidonic acid and ethanolamine to estimate the production of PGE₂-EA.

Key results

PGE₂-EA potently suppressed TNF-α production in blood, monocytes and the cell line THP-1 in a concentration-dependent manner. This occurred via cyclic AMP pathways as indicated by agents which interfere with these pathways and also direct ligand binding experiments. It was also shown that the cells were able to endogenously produce PGE₂-EA.

Conclusions and implications

This study reports that PGE₂-EA can downregulate the production of TNF-α by human mononuclear cells in response to an immune stimulus, i.e. LPS-activated TLR4, and that this appears to occur via a cAMP-dependent mechanism that most likely involves binding to the EP₂ receptor.     

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

Background

Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) contribute to this effect.

Methods

Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing), were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β₁ concentrations were measured in culture supernatants by ELISA.

Results

Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM) of initial area vs. 48.0% ± 0.4 at 48 hours; P < 0.001 and 41.5% ± 0.6 vs. 60.6% ± 0.3 at 48 hours; P < 0.001, respectively). Fixed platelets had no effect in the system. Both TGF-β₁ and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β₁ release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction.

Conclusion

We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.     

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix

Summary Fibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E₂ (PGE₂), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE₂ inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.     

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.

Measurements and Main Results

In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.

Conclusions

We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.     

Increased production of tumor necrosis factor and prostaglandin E2 by monocytes in cancer patients and its unique modulation by their plasma

Summary We investigated the role of monocytes in the production of tumor necrosis factor (TNF) and prostaglandin E₂ (PGE₂) in 77 cancer patients with malignancies of the digestive tract, using 30 normal individuals and 18 noncancer patients as controls. Monocytes were incubated with lipopolysaccharide for 20 h, and TNF production and PGE₂ production were analyzed by bioassays. Elevated levels of TNF (>512 U/ml) and PGE₂ (>8 ng/ml) production were demonstrated in many cancer patients when these factors were induced in the medium with 10% fetal bovine serum. The elevated level of TNF was seen to be restricted for the most part to patients with malignancies. Thus, 51 out of 59 cancer patients (86%), consisting of 44 primary cancer patients and 15 recurrent cancer patients, showed an increased level of TNF. In contrast, almost all of 18 postoperative cancer patients showed TNF levels comparable to those of normal individuals. Furthermore, 16 primary cancer patients were also demonstrated to have reduced levels of TNF production by monocytes after curative operation. When 10% cancer-patient plasma was added to the induction culture, TNF production by monocytes was drastically suppressed in the cancer patients. Interestingly, the same addition of plasma induced a prominent enhancement of PGE₂ production in the cancer patients. The plasma of noncancer patients did not modulate production of these factors. No TNF activity was found in the plasma of cancer patients, but such plasma did contain an increased level of PGE₂ (100–300 pg/ml). Although PGE₂ (>2ng/ml) was able to suppress TNF production by monocytes, the addition of 10% plasma PGE₂ was not enough to induce suppression. An unknown factor(s) in the plasma of cancer patients may uniquely modulate the elevated TNF and PGE₂ production in these patients.     

Carnosol Inhibits Pro-Inflammatory and Catabolic Mediators of Cartilage Breakdown in Human Osteoarthritic Chondrocytes and Mediates Cross-Talk between Subchondral Bone Osteoblasts and Chondrocytes

Aim

The aim of this work was to evaluate the effects of carnosol, a rosemary polyphenol, on pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes and via bone-cartilage crosstalk.

Materials and Methods

Osteoarthritic (OA) human chondrocytes were cultured in alginate beads for 4 days in presence or absence of carnosol (6 nM to 9 μM). The production of aggrecan, matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinase (TIMP)-1, interleukin (IL)-6 and nitric oxide (NO) and the expression of type II collagen and ADAMTS-4 and -5 were analyzed. Human osteoblasts from sclerotic (SC) or non-sclerotic (NSC) subchondral bone were cultured for 3 days in presence or absence of carnosol before co-culture with chondrocytes. Chondrocyte gene expression was analyzed after 4 days of co-culture.

Results

In chondrocytes, type II collagen expression was significantly enhanced in the presence of 3 μM carnosol (p = 0.008). MMP-3, IL-6, NO production and ADAMTS-4 expression were down-regulated in a concentration-dependent manner by carnosol (p<0.01). TIMP-1 production was slightly increased at 3 μM (p = 0.02) and ADAMTS-5 expression was decreased from 0.2 to 9 μM carnosol (p<0.05). IL-6 and PGE₂ production was reduced in the presence of carnosol in both SC and NSC osteoblasts while alkaline phosphatase activity was not changed. In co-culture experiments preincubation of NSC and SC osteoblasts wih carnosol resulted in similar effects to incubation with anti-IL-6 antibody, namely a significant increase in aggrecan and decrease in MMP-3, ADAMTS-4 and -5 gene expression by chondrocytes.

Conclusions

Carnosol showed potent inhibition of pro-inflammatory and catabolic mediators of cartilage breakdown in chondrocytes. Inhibition of matrix degradation and enhancement of formation was observed in chondrocytes cocultured with subchondral osteoblasts preincubated with carnosol indicating a cross-talk between these two cellular compartments, potentially mediated via inhibition of IL-6 in osteoblasts as similar results were obtained with anti-IL-6 antibody.     

Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

Background

Studies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM₁₀) in vitro increase their production of inflammatory mediators and that supernatants from PM₁₀-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.

Methods

The present study concerns co-culture of AM and HBEC exposed to PM₁₀ (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.

Results

AM/HBEC co-cultures exposed to 100 μg/ml of PM₁₀ for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM₁₀-induced increase in co-culture mRNA expression.

Conclusion

We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM₁₀-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.     

Prostaglandin E2 produced by inducible COX-2 and mPGES-1 promoting cancer cell proliferation in vitro and in vivo

Aim

Many cancers originate and flourish in a prolonged inflammatory environment. Our aim is to understand the mechanisms of how the pathway of prostaglandin E₂ (PGE₂) biosynthesis and signaling can promote cancer growth in inflammatory environment at cellular and animal model levels.

Main methods

In this study, a chronic inflammation pathway was mimicked with a stable cell line that over-expressed a novel human enzyme consisting of cyclooxygenase isoform-2 (COX-2) linked to microsomal (PGE₂ synthase-1 (mPGES-1)) for the overproduction of pathogenic PGE₂. This PGE₂-producing cell line was co-cultured and co-implanted with three human cancer cell lines including prostate, lung, and colon cancers in vitro and in vivo, respectively.

Key findings

Increases in cell doubling rates for the three cancer cell types in the presence of the PGE₂-producing cell line were clearly observed. In addition, one of the four human PGE₂ subtype receptors, EP₁, was used as a model to identify PGE₂-signaling involved in promoting the cancer cell growth. This finding was further proven in vivo by co-implanting the PGE₂-producing cells line and the EP₁-positive cancer cells into the immune deficient mice, after that, it was observed that the PGE₂-producing cells promoted all three types of cancer formation in the mice.

Significance

This study clearly demonstrated that the human COX-2 linked to mPGES-1 is a pathway that, when mediated by the EP, is linked to promoting cancer growth in a chronic inflammatory environment. The identified pathway could be used as a novel target for developing and advancing anti-inflammation and anti-cancer interventions.     

CD14 monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells

Here, the effect of CD14⁺ monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-γ (IFN-γ) secretion capacities of CD4⁺ and CD8⁺ T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E₂ (PGE₂) as an important soluble mediator. CD14⁺ monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1β, either exogenously added or produced by CD14⁺ monocytes in culture, could trigger expression of high levels of PGE₂ by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE₂ expression, but also reversed the promotional effect of CD14⁺ monocytes and partially restored CD4⁺ and CD8⁺ T cell proliferation and IFN-γ secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.     

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.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Autocrine prostaglandin E2 signaling promotes tumor cell survival and proliferation in childhood neuroblastoma

Background

Prostaglandin E₂ (PGE₂) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE₂ production, the expression pattern and localization of PGE₂ receptors and intracellular signal transduction pathways activated by PGE₂.

Principal Findings

A high expression of the PGE₂ receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE₂ and stimulation of serum-starved neuroblastoma cells with PGE₂ increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE₂ (dmPGE₂) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE₂. Similarly, PGE₂ receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner.

Conclusions

These findings demonstrate that PGE₂ acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE₂ signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.     

Prostaglandin E2 Receptors of Monocytes/Macrophages: Regulation by Insulin and Interleukin-1α

The regulation of receptors for prostaglandin E₂ (PGE₂) in monocyte/macrophage-like cells, P388D₁, by interleukin-1α (IL-1α) and insulin has been investigated. Many of the effects of IL-1, such as fever and other inflammatory activities, are linked to the stimulation of PGE₂ synthesis. On the other hand, PGE₂ exhibits suppressive effects on many steps in the immune response, including IL-1 production. The binding of PGE₂ to monocytes is reported to be essential for the inhibition of IL-1 production and activity. This inhibition occurs through the stimulation of cyclic AMP synthesis by the activation of PGE₂ receptor-linked adenylate cyclase. Although IL-1α stimulates PGE₂ synthesis in monocytes/macrophages during immunoactivation, it inhibits the binding of PGE₂ to these cells and may thereby exert a countervailing effect on the immunosuppressive action of this prostanoid. By contrast, insulin at physiological concentrations enhances the PGE₂ binding to these cells. This suggests that insulin at physiological concentrations may enhance the immunosuppressive action of PGE₂. Since the stimulation of cAMP synthesis in cells is regulated by PGE₂ binding, it is possible that these hormonal factors may control the immune response by modulating the PGE₂ receptor activity of monocytes/macrophages. This article focuses on the interactions of insulin and IL-1 with PGE₂ receptors of monocytes/macrophages.     

Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle

Background  

Tissue engineering of vascularised skeletal muscle is a promising method for the treatment of soft tissue defects in reconstructive surgery. In this study we explored the characteristics of novel collagen and fibrin matrices for skeletal muscle tissue engineering. We analyzed the characteristics of newly developed hybrid collagen-I-fibrin-gels and collagen nanofibers as well as collagen sponges and OPLA^?-scaffolds. Collagen-fibrin gels were also tested with genipin as stabilizing substitute for aprotinin.     

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Background

Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β₂, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.

Methods

Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.

Results

IL-13 induced increasing levels of MUC5AC protein, and TGF-β₂, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β₂, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β₂ neutralizing antibody reversed the increase in collagen content and SHG signal.

Conclusion

Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β₂ at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β₂ secretion from the airway epithelium.     

Downregulation of nuclear factor-kappa B (NF-κB) pathway by silibinin in human monocytes challenged with Paracoccidioides brasiliensis

AimsSilibinin is the major active component of silymarin, a polyphenolic plant flavonoid that has anti-inflammatory effects. The modulatory effect of silibinin on monocyte function against Paracoccidioides brasiliensis (Pb18) has not yet been demonstrated. The present study investigated whether the effect of silibinin on nuclear factor-kappa B (NF-κB) pathways may affect the production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), transforming growth factor beta (TGF-β1), prostaglandin E₂ (PGE₂), nitric oxide (NO) and fungicidal activity of human monocytes challenged in vitro with Pb18.Main methodsPeripheral blood monocytes from healthy individuals were treated with silibinin and challenged with Pb18 for 18 h. TNF-α, IL-10, TGF-β1 and PGE₂ expression were determined by immunoenzymatic assay (ELISA) and NO release was determined by the accumulation of nitrite in culture supernatants. Fungicidal activity of monocytes was analyzed after treatment with interferon-gamma plus silibinin and challenge with Pb18. NF-κB activation in cultured monocytes was evaluated by flow cytometry and ELISA.Key findingsSilibinin partially inhibited p65NF-κB activation as the number of cells expressing this factor was reduced and the concentration of nuclear p65NF-κB was low, compared to untreated controls. The addition of silibinin also resulted in suppression of TNF-α, IL-10, TGF-β1, PGE₂ and NO production but did not affect the fungicidal activity of monocytes against Pb18.SignificanceSilibinin exerts anti-inflammatory and anti-fibrotic effects on CD14± human monocytes challenged by Pb18 by partial inhibition of p65NF-κB activation.     

Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Objective

To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system.

Methods

Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using ³⁵SO₄²⁻-labelling and the dimethylmethylene blue assay. ³H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured.

Results

The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days. ³H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the ³⁵SO₄²⁻ incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts.

Conclusion

The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

Berberine inhibits the chemotherapy‐induced repopulation by suppressing the arachidonic acid metabolic pathway and phosphorylation of FAK in ovarian cancer

Objectives

Cytotoxic chemotherapy is an effective and traditional treatment of ovarian cancer. However, chemotherapy‐induced apoptosis may also trigger and ultimately accelerate the repopulation of the small number of adjacent surviving cells. This study mainly focused on the tumour cell repopulation caused by chemotherapy in ovarian cancer and the adjunctive/synergistic effect of Berberine on the prevention of tumour repopulation.

Materials and methods

The transwell system was used to mimic the co‐culture of surviving ovarian cancer cells in the microenvironment of cytotoxic chemotherapy‐treated dying cells. Tumour cell proliferation was observed by crystal violet staining. AA and PGE₂ levels were measured by ELISA, and changes of protein expression were analysed by Western blot.

Results

Chemotherapy drug VP16 treatment triggered AA pathway, leading to the elevated PGE₂ level, and ultimately enhanced the repopulation of ovarian cancer cells. Berberine can block the caspase 3‐iPLA₂‐AA‐COX‐2‐PGE₂ pathway by inhibiting the expression of iPLA₂ and COX‐2. Berberine can also reverse the increased phosphorylation of FAK caused by abnormal PGE₂ level and thus reverse the repopulation of ovarian cancer cells after VP16 treatment.

Conclusions

Our observation suggested that Berberine could inhibit the chemotherapy‐induced repopulation of ovarian cancer cells by suppressing the AA pathway and phosphorylation of FAK. And these findings implicated a novel combined use of Berberine and chemotherapeutics, which might prevent ovarian cancer recurrence by abrogating early tumour repopulation.