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The effect of the pro-inflammatory cytokine tumor necrosis factor-alpha on human joint capsule myofibroblasts.

Mattyasovszky SG, Hofmann A, Brochhausen C, Ritz U, Kuhn S, Wollstädter J, Schulze-Koops H, Müller LP, Watzer B, Rommens PM - Arthritis Res. Ther. (2010)

Bottom Line: This effect was associated with downregulation of alpha-SMA and collagen type I by TNF-alpha application.Furthermore, we found a significant time-dependent upregulation of prostaglandin E2 synthesis upon TNF-alpha treatment.The effect of TNF-alpha on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Trauma and Orthopaedic Surgery, Johannes Gutenberg University School of Medicine, Langenbeckstr, 1, 55101 Mainz, Germany. Stefan.Mattyasovszky@gmx.de

ABSTRACT

Introduction: Previous studies have shown that the number of myoblastically differentiated fibroblasts known as myofibroblasts (MFs) is significantly increased in stiff joint capsules, indicating their crucial role in the pathogenesis of post-traumatic joint stiffness. Although the mode of MFs' function has been well defined for different diseases associated with tissue fibrosis, the underlying mechanisms of their regulation in the pathogenesis of post-traumatic joint capsule contracture are largely unknown.

Methods: In this study, we examined the impact of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on cellular functions of human joint capsule MFs. MFs were challenged with different concentrations of TNF-alpha with or without both its specifically inactivating antibody infliximab (IFX) and cyclooxygenase-2 (COX2) inhibitor diclofenac. Cell proliferation, gene expression of both alpha-smooth muscle actin (alpha-SMA) and collagen type I, the synthesis of prostaglandin derivates E2, F1A, and F2A, as well as the ability to contract the extracellular matrix were assayed in monolayers and in a three-dimensional collagen gel contraction model. The alpha-SMA and COX2 protein expressions were evaluated by immunofluorescence staining and Western blot analysis.

Results: The results indicate that TNF-alpha promotes cell viability and proliferation of MFs, but significantly inhibits the contraction of the extracellular matrix in a dose-dependent manner. This effect was associated with downregulation of alpha-SMA and collagen type I by TNF-alpha application. Furthermore, we found a significant time-dependent upregulation of prostaglandin E2 synthesis upon TNF-alpha treatment. The effect of TNF-alpha on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac.

Conclusions: Our results provide evidence that TNF-alpha specifically modulates the function of MFs through regulation of prostaglandin E2 synthesis and therefore may play a crucial role in the pathogenesis of joint capsule contractures.

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Related in: MedlinePlus

The experimental setup to study the effect of tumor necrosis factor-alpha (TNF-α) on human joint capsule myofibroblasts. Seven different groups (a-g) were chosen in the study. Group (a) as the control was cultured without any cytokine or inhibitor. The cytokine or the inhibitor or both were added after 3 days of culture. On day 6, the MTT assay was performed and the three-dimensional (3D) collagen gels were released from the culture plate. After 48 hours, gel surfaces were calculated as indicated in Materials and methods. Diclo, diclofenac; IFX, infliximab; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT-PCR, real-time polymerase chain reaction.
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Figure 1: The experimental setup to study the effect of tumor necrosis factor-alpha (TNF-α) on human joint capsule myofibroblasts. Seven different groups (a-g) were chosen in the study. Group (a) as the control was cultured without any cytokine or inhibitor. The cytokine or the inhibitor or both were added after 3 days of culture. On day 6, the MTT assay was performed and the three-dimensional (3D) collagen gels were released from the culture plate. After 48 hours, gel surfaces were calculated as indicated in Materials and methods. Diclo, diclofenac; IFX, infliximab; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT-PCR, real-time polymerase chain reaction.

Mentions: MFs were seeded into 96-well plates (Greiner Bio-One GmbH) at a density of 10 × 104 cells/well and incubated in 150 μL of serum-supplemented DMEM (5% FCS, 1% penicillin/streptomycin) for 48 hours. Thereafter, the cell layers were washed with PBS and incubated for 24 hours in 150 μL of serum-free DMEM supplemented with 1% bovine serum albumin (BSA). After 24 hours, the cells were washed with PBS and incubated for 72 hours in 150 μL of serum-free DMEM containing 1 or 10 ng/mL of TNF-α (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) and/or the chimeric monoclonal antibody to TNF-α (10 μg/mL IFX, generously provided by Centocor B.V., Leiden, The Netherlands), and/or 10 μg/mL of the COX2 inhibitor diclofenac (Figure 1). MFs cultured in 150 μL of serum-free DMEM without any cytokine or inhibitor were used as controls and named as the control group. Cell viability and proliferation were measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Promega GmbH, Mannheim, Germany). After 72 hours, 30 μL of 0.5% MTT solution was added to each well and incubated for 2 hours. The medium was removed, and the dye was resolved with 100 μL of isopropanol (Hedinger GmbH & Co. KG, Stuttgart, Germany). The optical density was measured at 570 nm (650 nm background) using an enzyme-linked immunosorbent assay reader (Sunrise; Tecan Deutschland GmbH, Crailsheim, Germany).


The effect of the pro-inflammatory cytokine tumor necrosis factor-alpha on human joint capsule myofibroblasts.

Mattyasovszky SG, Hofmann A, Brochhausen C, Ritz U, Kuhn S, Wollstädter J, Schulze-Koops H, Müller LP, Watzer B, Rommens PM - Arthritis Res. Ther. (2010)

The experimental setup to study the effect of tumor necrosis factor-alpha (TNF-α) on human joint capsule myofibroblasts. Seven different groups (a-g) were chosen in the study. Group (a) as the control was cultured without any cytokine or inhibitor. The cytokine or the inhibitor or both were added after 3 days of culture. On day 6, the MTT assay was performed and the three-dimensional (3D) collagen gels were released from the culture plate. After 48 hours, gel surfaces were calculated as indicated in Materials and methods. Diclo, diclofenac; IFX, infliximab; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT-PCR, real-time polymerase chain reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875629&req=5

Figure 1: The experimental setup to study the effect of tumor necrosis factor-alpha (TNF-α) on human joint capsule myofibroblasts. Seven different groups (a-g) were chosen in the study. Group (a) as the control was cultured without any cytokine or inhibitor. The cytokine or the inhibitor or both were added after 3 days of culture. On day 6, the MTT assay was performed and the three-dimensional (3D) collagen gels were released from the culture plate. After 48 hours, gel surfaces were calculated as indicated in Materials and methods. Diclo, diclofenac; IFX, infliximab; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RT-PCR, real-time polymerase chain reaction.
Mentions: MFs were seeded into 96-well plates (Greiner Bio-One GmbH) at a density of 10 × 104 cells/well and incubated in 150 μL of serum-supplemented DMEM (5% FCS, 1% penicillin/streptomycin) for 48 hours. Thereafter, the cell layers were washed with PBS and incubated for 24 hours in 150 μL of serum-free DMEM supplemented with 1% bovine serum albumin (BSA). After 24 hours, the cells were washed with PBS and incubated for 72 hours in 150 μL of serum-free DMEM containing 1 or 10 ng/mL of TNF-α (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) and/or the chimeric monoclonal antibody to TNF-α (10 μg/mL IFX, generously provided by Centocor B.V., Leiden, The Netherlands), and/or 10 μg/mL of the COX2 inhibitor diclofenac (Figure 1). MFs cultured in 150 μL of serum-free DMEM without any cytokine or inhibitor were used as controls and named as the control group. Cell viability and proliferation were measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Promega GmbH, Mannheim, Germany). After 72 hours, 30 μL of 0.5% MTT solution was added to each well and incubated for 2 hours. The medium was removed, and the dye was resolved with 100 μL of isopropanol (Hedinger GmbH & Co. KG, Stuttgart, Germany). The optical density was measured at 570 nm (650 nm background) using an enzyme-linked immunosorbent assay reader (Sunrise; Tecan Deutschland GmbH, Crailsheim, Germany).

Bottom Line: This effect was associated with downregulation of alpha-SMA and collagen type I by TNF-alpha application.Furthermore, we found a significant time-dependent upregulation of prostaglandin E2 synthesis upon TNF-alpha treatment.The effect of TNF-alpha on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Trauma and Orthopaedic Surgery, Johannes Gutenberg University School of Medicine, Langenbeckstr, 1, 55101 Mainz, Germany. Stefan.Mattyasovszky@gmx.de

ABSTRACT

Introduction: Previous studies have shown that the number of myoblastically differentiated fibroblasts known as myofibroblasts (MFs) is significantly increased in stiff joint capsules, indicating their crucial role in the pathogenesis of post-traumatic joint stiffness. Although the mode of MFs' function has been well defined for different diseases associated with tissue fibrosis, the underlying mechanisms of their regulation in the pathogenesis of post-traumatic joint capsule contracture are largely unknown.

Methods: In this study, we examined the impact of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on cellular functions of human joint capsule MFs. MFs were challenged with different concentrations of TNF-alpha with or without both its specifically inactivating antibody infliximab (IFX) and cyclooxygenase-2 (COX2) inhibitor diclofenac. Cell proliferation, gene expression of both alpha-smooth muscle actin (alpha-SMA) and collagen type I, the synthesis of prostaglandin derivates E2, F1A, and F2A, as well as the ability to contract the extracellular matrix were assayed in monolayers and in a three-dimensional collagen gel contraction model. The alpha-SMA and COX2 protein expressions were evaluated by immunofluorescence staining and Western blot analysis.

Results: The results indicate that TNF-alpha promotes cell viability and proliferation of MFs, but significantly inhibits the contraction of the extracellular matrix in a dose-dependent manner. This effect was associated with downregulation of alpha-SMA and collagen type I by TNF-alpha application. Furthermore, we found a significant time-dependent upregulation of prostaglandin E2 synthesis upon TNF-alpha treatment. The effect of TNF-alpha on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac.

Conclusions: Our results provide evidence that TNF-alpha specifically modulates the function of MFs through regulation of prostaglandin E2 synthesis and therefore may play a crucial role in the pathogenesis of joint capsule contractures.

Show MeSH
Related in: MedlinePlus