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

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.

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.

Figure 2

Expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) in hip joint and contracted elbow joint capsules. Biopsy sections were stained as indicated in Materials and methods. (a) Hematoxylin-eosin staining of hip joint capsules revealed parallel orientation of the collagen fibers and small spindle-like fibrocytes. (b) The immunohistochemical detection of α-SMA in hip joint capsules showed that only smooth muscle cells associated with blood vessels were positive for this marker (arrows). (c, d) Arrows indicate multiple positive cells in the immunohistochemical staining for α-SMA (brown dye) in contracted elbow joint capsule which were not linked to blood vessels. Scale bars = 100 μm.

Figure 3

Phenotype of the cells used in this study. (a) Early cultures of fibroblasts were characterized by a typical spindle-like shape (arrows) and gradually matured into myofibroblasts (b) revealing typical stellate-shaped morphology (arrows) over the course of culture. (c, d) The myofibroblast cell marker alpha-smooth muscle actin was detected in confluent cell cultures as indicated in Materials and methods. Scale bars = 100 μm.

Figure 4

Cell viability and proliferative capacity of myofibroblasts upon tumor necrosis factor-alpha (TNF-α) treatment. The effect of the cytokine TNF-α (1 and 10 ng/mL) in the presence or absence of the TNF-α inhibitor infliximab (IFX) (10 μg/mL) or the cyclooxygenase inhibitor diclofenac (Diclo) on myofibroblasts was analyzed by using the MTT cell viability assay. Data are representative of nine (Table 1) independently performed TNF-α ± IFX experiments and seven independently performed TNF-α ± diclofenac experiments with four replicate measurements from each individual patient sample (n = 68 for the 'control' and 'TNF-α 10 ng/mL' groups, n = 28 for the 'TNF-α 10 ng/mL+diclofenac' group, and n = 36 for all other groups). Results are plotted as fold changes of the respective samples in the control group according to the paired non-parametric Wilcoxon test used for the statistical analysis. *P < 0.05, ***P < 0.001. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 5

The effect of tumor necrosis factor-alpha (TNF-α) on collagen gel contraction. (a) Images of the three-dimensional collagen gel contraction assay shown are representative of all (Table 1) independently performed experiments. Three collagen gels of each group are shown before (day 6) and after (day 8) releasing the gels from the culture plates, revealing distinct differences in gel contraction in the groups upon TNF-α and infliximab (IFX) or diclofenac (Diclo) coincubation. White circles in the first rows of the plates illustrate the margins of the gels comprising the gel surface areas. (b) Gel surfaces of the floating gels in the presence or absence of TNF-α (1 and 10 ng/mL), the TNF-α inhibitor IFX (10 μg/mL), or diclofenac (10 μg/mL) were scanned and calculated as described in Materials and methods. Data are representative of 10 (Table 1) independently performed TNF-α ± IFX experiments and three independently performed TNF-α ± diclofenac experiments with four replicate measurements from each individual patient sample (n = 52 for the 'control' and 'TNF-α 10 ng/mL' groups, n = 12 for the 'TNF-α 10 ng/mL+diclofenac' group, and n = 40 for all other groups). Results are plotted as fold changes of the respective samples in the control group according to the paired non-parametric Wilcoxon test used for the statistical analysis. **P < 0.01, ***P < 0.001.

Figure 6

Tumor necrosis factor-alpha (TNF-α) downregulates gene expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) and the extracellular matrix protein collagen type I. The mRNA expression of α-SMA (a) and collagen type I (b) was determined for every group (Figure 1) by quantitative real-time polymerase chain reaction. The mRNA level of α-SMA of every group examined was normalized to the housekeeping gene 18S. Data are representative of four (Table 1) independently performed TNF-α ± infliximab (IFX) experiments and TNF-α ± diclofenac (Diclo) experiments with duplicate measurements from each individual patient sample (n = 16 for the 'control' and 'TNF-α 10 ng/mL' groups and n = 8 for all other groups). Results are plotted as fold changes of the respective samples in the control group according to the paired non-parametric Wilcoxon test used for the statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Tumor necrosis factor-alpha (TNF-α) modulates the synthesis of prostaglandin E₂ (PGE₂) in alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs). (a) Immunofluorescent staining of cyclooxygenase-2 (COX2) (first panel, green, fluorescein isothiocyanate filter) and α-SMA (second panel, red, Texas Red filter) in MF cultures with or without the COX2 inhibitor diclofenac (Diclo). The third panel illustrates merged images of Höchst 33248-stained nuclei (blue, DAPI filter) as well as immunofluorescence for COX2 and α-SMA. α-SMA-positive MFs derived from hip joint capsules express the enzyme COX2. The fourth panel shows merged images of the negative controls. Scale bars are shown in the lower right corner of each panel. (b) MFs of three different donors were exposed to 10 μg/mL diclofenac. The expression of α-SMA (45 kDa), COX2 (72 kDa), and β-actin (42 kDa) as a loading control was evaluated by using Western blots. A characteristic double band for the COX2 protein corresponding to the expected molecular weight represents different glycosylated forms of the enzyme. (c) Gas chromatographic/mass spectrometric analysis revealed a time-dependent increase of PGE₂ concentration in MF cultures upon TNF-α stimulation with a peak after 24 and 48 hours. This effect was completely blocked by diclofenac. DAPI, 4'-6-diamidino-2-phenylindole.

Figure 8

Illustrative concept of myofibroblast (MF) modulation by tumor necrosis factor-alpha (TNF-α). TNF-α inhibits extracellular matrix (ECM) contraction by the downregulation of alpha-smooth muscle actin (α-SMA) and collagen type I expression in MFs presumably by promoting prostaglandin E₂ (PGE₂) synthesis. Both infliximab (IFX) by blocking TNF-α and diclofenac by inhibiting cyclooxygenase-2 (COX2) might enhance ECM contraction. NF-κB, nuclear factor-kappa-B.