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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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Regulation of IL-6 in FLS. Dose response analysis of IL-6 mRNA expression, determined by RT real-time PCR in (fibroblast-like synoviocytes) FLS isolated from K/BxN mice following treatment with (a) corticosterone (0, 1, 10, 100, 500, 1000 nmol/l) or (b) TNFα (0, 0.1, 1, 5, 10, 25 ng/ml). (c) Fold change in IL-6 mRNA expression in wild-type (WT) control FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by real-time RT-PCR. All mRNA data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to either untreated control or untreated WT con FLS. (d) IL-6 secretion into culture media (pg/ml/100000 cells, ± standard error) in WT con FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by specific ELISA. Both mRNA and conditioned media were collected at 16 hr following treatment with either control, TNFα (10 ng/ml) or corticosterone (100 nmol/l). *P < 0.05, **P < 0.001 versus respective untreated controls; #P < 0.05 versus untreated WT control FLS. Dose response experiments are the combined duplicates of two K/BxN FLS. All other data are the combined duplicates of three separate FLS lines, two WT control FLS lines, two C2C12 repeats and two MC3T3-E1 repeats.
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Figure 4: Regulation of IL-6 in FLS. Dose response analysis of IL-6 mRNA expression, determined by RT real-time PCR in (fibroblast-like synoviocytes) FLS isolated from K/BxN mice following treatment with (a) corticosterone (0, 1, 10, 100, 500, 1000 nmol/l) or (b) TNFα (0, 0.1, 1, 5, 10, 25 ng/ml). (c) Fold change in IL-6 mRNA expression in wild-type (WT) control FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by real-time RT-PCR. All mRNA data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to either untreated control or untreated WT con FLS. (d) IL-6 secretion into culture media (pg/ml/100000 cells, ± standard error) in WT con FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by specific ELISA. Both mRNA and conditioned media were collected at 16 hr following treatment with either control, TNFα (10 ng/ml) or corticosterone (100 nmol/l). *P < 0.05, **P < 0.001 versus respective untreated controls; #P < 0.05 versus untreated WT control FLS. Dose response experiments are the combined duplicates of two K/BxN FLS. All other data are the combined duplicates of three separate FLS lines, two WT control FLS lines, two C2C12 repeats and two MC3T3-E1 repeats.

Mentions: IL-6 mRNA expression was examined in response to the proinflammatory cytokine, TNF-α and the anti-inflammatory glucocorticoid, corticosterone. Time-course analysis of two FLS lines revealed maximal responses between 8 and 24 hr for TNF-α (10 ng/ml) and 8 and 16 hr for corticosterone (100 nmol/l) (Additional file 3). Consequently for all experiments in this study treatments were fixed at 16 hr. Dose response experiments were performed in two FLS lines (Figure 4a, b). TNF-α resulted in a significant increase in IL-6 mRNA expression at 1 ng/ml (3.9 ± 0.41 fold; P < 0.05). IL-6 mRNA expression continued to increase in a dose-dependant manner up to the maximum supraphysiological dose used in the study at 25 ng/ml (8 ± 0.23 fold; P < 0.001). Treatment with corticosterone resulted in a dose-dependant decrease in IL-6 mRNA expression that was significant at 10 nmol/l (1.9 ± 0.04 fold; P < 0.05) with maximal inhibition at 100 nmol/l (11.1 ± 0.02 fold; P < 0.05). For all experiments in this study TNF-α and corticosterone treatments were fixed at 10 ng/ml and 100 nmol/l respectively.


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)

Regulation of IL-6 in FLS. Dose response analysis of IL-6 mRNA expression, determined by RT real-time PCR in (fibroblast-like synoviocytes) FLS isolated from K/BxN mice following treatment with (a) corticosterone (0, 1, 10, 100, 500, 1000 nmol/l) or (b) TNFα (0, 0.1, 1, 5, 10, 25 ng/ml). (c) Fold change in IL-6 mRNA expression in wild-type (WT) control FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by real-time RT-PCR. All mRNA data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to either untreated control or untreated WT con FLS. (d) IL-6 secretion into culture media (pg/ml/100000 cells, ± standard error) in WT con FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by specific ELISA. Both mRNA and conditioned media were collected at 16 hr following treatment with either control, TNFα (10 ng/ml) or corticosterone (100 nmol/l). *P < 0.05, **P < 0.001 versus respective untreated controls; #P < 0.05 versus untreated WT control FLS. Dose response experiments are the combined duplicates of two K/BxN FLS. All other data are the combined duplicates of three separate FLS lines, two WT control FLS lines, two C2C12 repeats and two MC3T3-E1 repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Regulation of IL-6 in FLS. Dose response analysis of IL-6 mRNA expression, determined by RT real-time PCR in (fibroblast-like synoviocytes) FLS isolated from K/BxN mice following treatment with (a) corticosterone (0, 1, 10, 100, 500, 1000 nmol/l) or (b) TNFα (0, 0.1, 1, 5, 10, 25 ng/ml). (c) Fold change in IL-6 mRNA expression in wild-type (WT) control FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by real-time RT-PCR. All mRNA data were normalized for levels of the housekeeping gene 18S rRNA and presented as fold change in expression (± standard error) relative to either untreated control or untreated WT con FLS. (d) IL-6 secretion into culture media (pg/ml/100000 cells, ± standard error) in WT con FLS, C2C12, MC3T3-E1 and K/BxN FLS, determined by specific ELISA. Both mRNA and conditioned media were collected at 16 hr following treatment with either control, TNFα (10 ng/ml) or corticosterone (100 nmol/l). *P < 0.05, **P < 0.001 versus respective untreated controls; #P < 0.05 versus untreated WT control FLS. Dose response experiments are the combined duplicates of two K/BxN FLS. All other data are the combined duplicates of three separate FLS lines, two WT control FLS lines, two C2C12 repeats and two MC3T3-E1 repeats.
Mentions: IL-6 mRNA expression was examined in response to the proinflammatory cytokine, TNF-α and the anti-inflammatory glucocorticoid, corticosterone. Time-course analysis of two FLS lines revealed maximal responses between 8 and 24 hr for TNF-α (10 ng/ml) and 8 and 16 hr for corticosterone (100 nmol/l) (Additional file 3). Consequently for all experiments in this study treatments were fixed at 16 hr. Dose response experiments were performed in two FLS lines (Figure 4a, b). TNF-α resulted in a significant increase in IL-6 mRNA expression at 1 ng/ml (3.9 ± 0.41 fold; P < 0.05). IL-6 mRNA expression continued to increase in a dose-dependant manner up to the maximum supraphysiological dose used in the study at 25 ng/ml (8 ± 0.23 fold; P < 0.001). Treatment with corticosterone resulted in a dose-dependant decrease in IL-6 mRNA expression that was significant at 10 nmol/l (1.9 ± 0.04 fold; P < 0.05) with maximal inhibition at 100 nmol/l (11.1 ± 0.02 fold; P < 0.05). For all experiments in this study TNF-α and corticosterone treatments were fixed at 10 ng/ml and 100 nmol/l respectively.

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