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Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5.

Yoshida S, Arakawa F, Higuchi F, Ishibashi Y, Goto M, Sugita Y, Nomura Y, Niino D, Shimizu K, Aoki R, Hashikawa K, Kimura Y, Yasuda K, Tashiro K, Kuhara S, Nagata K, Ohshima K - Scand. J. Rheumatol. (2012)

Bottom Line: In addition, the expression of significant genes was confirmed immunohistochemically.Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA.Further understanding of the local signalling in structural components is important in rheumatology.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Kurume University, Japan.

ABSTRACT

Objectives: The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control.

Methods: Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically.

Results: The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD.

Conclusions: Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology.

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Related in: MedlinePlus

Microdissection of the synovial lining in RA. (A) After toluidine blue staining, a section was subjected to LMD. The sample was observed on the PC monitor and the lining area was marked. (B) The sample was cut with a laser beam along the defined line. (C) After microdissection, the sample was collected by gravity into a tube cap. In this manner, we obtained more than 5000 cells from the RA or OA synovial lining sections.
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fig1: Microdissection of the synovial lining in RA. (A) After toluidine blue staining, a section was subjected to LMD. The sample was observed on the PC monitor and the lining area was marked. (B) The sample was cut with a laser beam along the defined line. (C) After microdissection, the sample was collected by gravity into a tube cap. In this manner, we obtained more than 5000 cells from the RA or OA synovial lining sections.

Mentions: The tissue samples were immediately frozen in acetone/dry ice and stored at −80°C for microdissection. The synovial samples were embedded in an optical cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen. Cryosections (20-μm-thick) were mounted on 2.0-μm-thick PEN-Membrane slides (MicroDissect GmbH, Herborn, Germany). After fixation in 100% ethanol, the slides were stained rapidly with toluidine blue O (Chroma-Gesellschaft Schmid GmbH & Co, Köngen, Germany) and then washed in diethylpyro-carbonate (DEPC)-treated water and air-dried with a fan. The frozen sections were microdissected with a Leica LMD6000 laser microdissection system following the company's protocol (Leica, Wetzlar, Germany). The synovial lining regions were microdissected from the tissue sections with LMD (Figure 1), and the dissected cells were collected in 0.5 mL tube caps filled with 50 μL lysis buffer for RNA extraction (29, 30).


Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5.

Yoshida S, Arakawa F, Higuchi F, Ishibashi Y, Goto M, Sugita Y, Nomura Y, Niino D, Shimizu K, Aoki R, Hashikawa K, Kimura Y, Yasuda K, Tashiro K, Kuhara S, Nagata K, Ohshima K - Scand. J. Rheumatol. (2012)

Microdissection of the synovial lining in RA. (A) After toluidine blue staining, a section was subjected to LMD. The sample was observed on the PC monitor and the lining area was marked. (B) The sample was cut with a laser beam along the defined line. (C) After microdissection, the sample was collected by gravity into a tube cap. In this manner, we obtained more than 5000 cells from the RA or OA synovial lining sections.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3400100&req=5

fig1: Microdissection of the synovial lining in RA. (A) After toluidine blue staining, a section was subjected to LMD. The sample was observed on the PC monitor and the lining area was marked. (B) The sample was cut with a laser beam along the defined line. (C) After microdissection, the sample was collected by gravity into a tube cap. In this manner, we obtained more than 5000 cells from the RA or OA synovial lining sections.
Mentions: The tissue samples were immediately frozen in acetone/dry ice and stored at −80°C for microdissection. The synovial samples were embedded in an optical cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen. Cryosections (20-μm-thick) were mounted on 2.0-μm-thick PEN-Membrane slides (MicroDissect GmbH, Herborn, Germany). After fixation in 100% ethanol, the slides were stained rapidly with toluidine blue O (Chroma-Gesellschaft Schmid GmbH & Co, Köngen, Germany) and then washed in diethylpyro-carbonate (DEPC)-treated water and air-dried with a fan. The frozen sections were microdissected with a Leica LMD6000 laser microdissection system following the company's protocol (Leica, Wetzlar, Germany). The synovial lining regions were microdissected from the tissue sections with LMD (Figure 1), and the dissected cells were collected in 0.5 mL tube caps filled with 50 μL lysis buffer for RNA extraction (29, 30).

Bottom Line: In addition, the expression of significant genes was confirmed immunohistochemically.Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA.Further understanding of the local signalling in structural components is important in rheumatology.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Medicine, Kurume University, Japan.

ABSTRACT

Objectives: The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control.

Methods: Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically.

Results: The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD.

Conclusions: Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology.

Show MeSH
Related in: MedlinePlus