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. 1997 Jul;56(7):426–431. doi: 10.1136/ard.56.7.426

Transforming growth factor β1 and interleukin 4 induced α smooth muscle actin expression and myofibroblast-like differentiation in human synovial fibroblasts in vitro: modulation by basic fibroblast growth factor

D Mattey 1, P Dawes 1, N Nixon 1, H Slater 1
PMCID: PMC1752407  PMID: 9486005

Abstract

OBJECTIVE—To discover if α smooth muscle actin expression and myofibroblastic differentiation are induced in synovial fibroblasts by cytokines found in the inflamed RA joint.
METHODS—Immunofluorescent microscopy and western blotting were used to examine different cultures of human synovial fibroblasts for expression of α actin in the presence of the cytokines transforming growth factor β (TGFβ1), interleukin 1α (IL1α), IL4, IL6, tumour necrosis factor α (TNFα), and basic fibroblast growth factor (FGF).
RESULTS—A small but significant population of cells (14.4 ± 12.9%) expressed α actin under standard culture conditions. Upon treatment with TGFβ1 there was a pronounced increase in the number of cells expressing α actin (68.1 ± 5.49%), accompanied by a change in morphology to a myofibroblast-like phenotype. Other cytokines found within the inflamed joint such as IL1, TNFα , IL6, and basic FGF failed to induce α actin expression. However, IL4, which is normally absent or only present at low concentrations in the RA joint had a similar effect to TGFβ1. It was also found that basic FGF inhibited the induction of α actin expression by TGFβ1 and IL4.
CONCLUSION—In the presence of TGFβ1 or IL4, fibroblasts derived from synovial tissue or synovial fluid are induced to differentiate into myofibroblast-like cells containing the α smooth muscle form of actin. This differentiation is inhibited by basic FGF. It is suggested that the balance between these particular cytokines may be important in the modulation of fibroblast behaviour, which could have significant effects on joint repair mechanisms and the generation of fibrous tissue within the rheumatoid joint.



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Figure 1  .

Figure 1  

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Representative photographs to show immunofluorescent staining of α actin in cultures of synovial fibroblasts. (A) In control cultures the majority of cells show no α actin expression and have an elongated, spindle shaped appearance. Occasional cells possess positively stained α actin filaments (arrow). (B) In RA synovial fibroblasts incubated with TGF 1 (5 ng/ml) for three days the majority of cells are stained for α actin. (C) OA synovial fibroblasts incubated with IL4 (1 ng/ml) for three days. Expression of α actin is characterised by the appearance of numerous bundles of α actin filament (stress fibres), and is accompanied by a change to a flattened myofibroblast type morphology. (D) RA synovial fibroblasts incubated with TGF1 (5 ng/ml) in the presence of basic FGF (10 ng/ml ) show no increase in the expression of α actin, and remain mainly spindle shaped, or take on a dendritic morphology. (Bar in (A), (B), (D) represents 4 µm, bar in (C) represents 1 µm).

Figure 2  .

Figure 2  

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Disruption of the α actin cytoskeleton in cells pre-treated with TGFβ1(5 ng/ml) for three days before addition of bFGF (10 ng/ml) for three days. Note that many cells have also taken on a smaller, more dendritic morphology. (Bar represents 2 µm).

Figure 3  .

Figure 3  

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(A) Immunofluorescent staining of α actin in a cryostat section of RA synovium. Little or no staining is present in the lining layer (arrow) but intense staining is found around blood vessels, where smooth muscle cells are situated. (B) α Actin expression in a cryostat section of RA synovium showing a wider zone of staining around the blood vessels. The lining layer is mostly negative (arrow). (Bar represents 8 µm).

Figure 4  .

Figure 4  

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Western blots to show α actin expression in synovial fibroblasts with and without cytokine treatment. (A) OA synovial fibroblasts after three days in control medium (lane 1), IL4 (lane 2), TGFβ (lane 3). (B) RA synovial fibroblasts after three days in control medium (lane1), TGFβ1 (lane 2), basic FGF (lane 3), TGFβ1 and bFGF (lane 4). Cytokines were used at the following concentrations ; IL4, 1 ng/ml, TGFβ, 5 ng/ml, bFGF, 10 ng/ml. Molecular weight markers are shown in kilodaltons.

Selected References

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