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Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation.

Hardy RS, Hülso C, Liu Y, Gasparini SJ, Fong-Yee C, Tu J, Stoner S, Stewart PM, Raza K, Cooper MS, Seibel MJ, Zhou H - Arthritis Res. Ther. (2013)

Bottom Line: FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P<0.05).TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P<0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169±29.7 versus TNF-α, 923±378.8 pg/ml/1×10⁵ cells; P<0.05).Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Fibroblast-like synoviocytes (FLS) play a central role in defining the stromal environment in inflammatory joint diseases. Despite a growing use of FLS isolated from murine inflammatory models, a detailed characterisation of these cells has not been performed.

Methods: In this study, FLS were isolated from inflamed joints of mice expressing both the T cell receptor transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) and their purity in culture determined by immunofluorescence and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Basal expression of proinflammatory genes was determined by real-time RT-PCR. Secreted interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA), and its regulation by tumor necrosis factor-alpha (TNF-α and corticosterone (the major glucocorticoid in rodents) measured relative to other mesenchymal cell populations.

Results: Purity of FLS culture was identified by positive expression of fibronectin, prolyl 4-hydroxylase, cluster of differentiation 90.2 (CD90.2) and 248 (CD248) in greater than 98% of the population. Cultured FLS were able to migrate and invade through matrigel, a process enhanced in the presence of TNF-α. FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P<0.05). This elevated expression was abrogated in the presence of corticosterone at 100 nmol/l. TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P<0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169±29.7 versus TNF-α, 923±378.8 pg/ml/1×10⁵ cells; P<0.05). Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven.

Conclusions: This study has established a well characterised set of key inflammatory genes for in vitro FLS culture, isolated from K/BxN mice and non-inflamed wild-type controls. Their response to both pro- and anti-inflammatory signalling has been assessed and shown to strongly resemble that which is seen in human FLS culture. Additionally, this study provides guidelines for the effective characterisation, duration and treatment of murine FLS culture.

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Inflammatory gene regulation by corticosteone and TNF-α in FLS. Fold change in mRNA expression of inflammatory genes in (fibroblast-like synoviocytes) FLS, determined by real-time RT-PCR. Expression of mRNA was measured at 16 hr for IL-6, chemokine ligand 2 (CCL-2), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) following pretreatment with either corticosterone (a-d) (100 nmol/l) or TNFα (e-h) (10 ng/ml) for 24 hr. Data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to untreated wild-type (WT) control FLS. *P < 0.05, **P < 0.001 versus respective untreated control; #P > 0.05 versus untreated WT control FLS. Results shown are the combined duplicates of three separate FLS lines and two WT control FLS lines.
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Figure 3: Inflammatory gene regulation by corticosteone and TNF-α in FLS. Fold change in mRNA expression of inflammatory genes in (fibroblast-like synoviocytes) FLS, determined by real-time RT-PCR. Expression of mRNA was measured at 16 hr for IL-6, chemokine ligand 2 (CCL-2), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) following pretreatment with either corticosterone (a-d) (100 nmol/l) or TNFα (e-h) (10 ng/ml) for 24 hr. Data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to untreated wild-type (WT) control FLS. *P < 0.05, **P < 0.001 versus respective untreated control; #P > 0.05 versus untreated WT control FLS. Results shown are the combined duplicates of three separate FLS lines and two WT control FLS lines.

Mentions: We examined the mRNA expression of the proinflammatory cytokine IL-6, the chemokine CCL-2 and the surface markers VCAM-1 and ICAM-1, to obtain a basic measure of the inflammatory profile of FLS isolated from K/BxN mice. Results shown are given as fold change relative to FLS isolated from normal joints in control wild-type mice (WT control FLS) to better assess basal activation of inflammatory markers. Basal expression of IL-6, CCL-2 and VCAM-1 were significantly higher in K/BxN FLS relative to WT control FLS (IL-6, 3.6 ± 0.25 fold; CCL-2, 11.2 ± 0.28 fold; VCAM-1, 9 ± 0.1 fold relative to untreated WT control FLS; P < 0.05), whilst no difference was observed with ICAM-1 (Figure 3a, b, c, d). Treatment with the anti-inflammatory glucocorticoid, corticosterone, resulted in a significant decrease in both IL-6 and VCAM-1 expression relative to their respective untreated controls (IL-6, 5.3 ± 0.04 fold; VCAM-1, 3.3 ± 0.07 fold; P < 0.05) and led to a strong trend towards decreased CCL-2 expression (1.9 ± 0.9 fold; P = 0.09) (Figure 3a, b, c, d). These decreases in IL-6, VCAM-1 and CCL-2 in K/BxN FLS resulted in their expression being comparable to that observed in untreated WT control FLS.


Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation.

Hardy RS, Hülso C, Liu Y, Gasparini SJ, Fong-Yee C, Tu J, Stoner S, Stewart PM, Raza K, Cooper MS, Seibel MJ, Zhou H - Arthritis Res. Ther. (2013)

Inflammatory gene regulation by corticosteone and TNF-α in FLS. Fold change in mRNA expression of inflammatory genes in (fibroblast-like synoviocytes) FLS, determined by real-time RT-PCR. Expression of mRNA was measured at 16 hr for IL-6, chemokine ligand 2 (CCL-2), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) following pretreatment with either corticosterone (a-d) (100 nmol/l) or TNFα (e-h) (10 ng/ml) for 24 hr. Data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to untreated wild-type (WT) control FLS. *P < 0.05, **P < 0.001 versus respective untreated control; #P > 0.05 versus untreated WT control FLS. Results shown are the combined duplicates of three separate FLS lines and two WT control FLS lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3672796&req=5

Figure 3: Inflammatory gene regulation by corticosteone and TNF-α in FLS. Fold change in mRNA expression of inflammatory genes in (fibroblast-like synoviocytes) FLS, determined by real-time RT-PCR. Expression of mRNA was measured at 16 hr for IL-6, chemokine ligand 2 (CCL-2), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) following pretreatment with either corticosterone (a-d) (100 nmol/l) or TNFα (e-h) (10 ng/ml) for 24 hr. Data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to untreated wild-type (WT) control FLS. *P < 0.05, **P < 0.001 versus respective untreated control; #P > 0.05 versus untreated WT control FLS. Results shown are the combined duplicates of three separate FLS lines and two WT control FLS lines.
Mentions: We examined the mRNA expression of the proinflammatory cytokine IL-6, the chemokine CCL-2 and the surface markers VCAM-1 and ICAM-1, to obtain a basic measure of the inflammatory profile of FLS isolated from K/BxN mice. Results shown are given as fold change relative to FLS isolated from normal joints in control wild-type mice (WT control FLS) to better assess basal activation of inflammatory markers. Basal expression of IL-6, CCL-2 and VCAM-1 were significantly higher in K/BxN FLS relative to WT control FLS (IL-6, 3.6 ± 0.25 fold; CCL-2, 11.2 ± 0.28 fold; VCAM-1, 9 ± 0.1 fold relative to untreated WT control FLS; P < 0.05), whilst no difference was observed with ICAM-1 (Figure 3a, b, c, d). Treatment with the anti-inflammatory glucocorticoid, corticosterone, resulted in a significant decrease in both IL-6 and VCAM-1 expression relative to their respective untreated controls (IL-6, 5.3 ± 0.04 fold; VCAM-1, 3.3 ± 0.07 fold; P < 0.05) and led to a strong trend towards decreased CCL-2 expression (1.9 ± 0.9 fold; P = 0.09) (Figure 3a, b, c, d). These decreases in IL-6, VCAM-1 and CCL-2 in K/BxN FLS resulted in their expression being comparable to that observed in untreated WT control FLS.

Bottom Line: FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P<0.05).TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P<0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169±29.7 versus TNF-α, 923±378.8 pg/ml/1×10⁵ cells; P<0.05).Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Fibroblast-like synoviocytes (FLS) play a central role in defining the stromal environment in inflammatory joint diseases. Despite a growing use of FLS isolated from murine inflammatory models, a detailed characterisation of these cells has not been performed.

Methods: In this study, FLS were isolated from inflamed joints of mice expressing both the T cell receptor transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) and their purity in culture determined by immunofluorescence and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Basal expression of proinflammatory genes was determined by real-time RT-PCR. Secreted interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA), and its regulation by tumor necrosis factor-alpha (TNF-α and corticosterone (the major glucocorticoid in rodents) measured relative to other mesenchymal cell populations.

Results: Purity of FLS culture was identified by positive expression of fibronectin, prolyl 4-hydroxylase, cluster of differentiation 90.2 (CD90.2) and 248 (CD248) in greater than 98% of the population. Cultured FLS were able to migrate and invade through matrigel, a process enhanced in the presence of TNF-α. FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P<0.05). This elevated expression was abrogated in the presence of corticosterone at 100 nmol/l. TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P<0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169±29.7 versus TNF-α, 923±378.8 pg/ml/1×10⁵ cells; P<0.05). Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven.

Conclusions: This study has established a well characterised set of key inflammatory genes for in vitro FLS culture, isolated from K/BxN mice and non-inflamed wild-type controls. Their response to both pro- and anti-inflammatory signalling has been assessed and shown to strongly resemble that which is seen in human FLS culture. Additionally, this study provides guidelines for the effective characterisation, duration and treatment of murine FLS culture.

Show MeSH
Related in: MedlinePlus