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Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

Zimmermann T, Kunisch E, Pfeiffer R, Hirth A, Stahl HD, Sack U, Laube A, Liesaus E, Roth A, Palombo-Kinne E, Emmrich F, Kinne RW - Arthritis Res. (2000)

Bottom Line: To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies.This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta.This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

View Article: PubMed Central - HTML - PubMed

Affiliation: Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, Germany.

ABSTRACT
To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

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Immunohistochemical staining of the RA synovial membrane with the anti-Thy-1 mAb AS02. (A) This mAb strongly stained cells in the connective tissue layer beneath the lining layer (APAAP, red staining; arrows), while largely sparing cells of the lining layer (arrowhead). Endothelial cells (asterisk) and connective tissue cells in diffuse infiltrates were also stained, more weakly in the latter case. In serial sections, the anti-CD14 mAb MoS39 (kindly provided by Prof GR Burmester, Berlin, Germany) stained macrophages in the lining layer (arrowhead in (C)) and diffuse inflammatory infiltrates (peroxidase, brown staining; arrow in (C), (E)); the mAb against von Willebrand factor stained endothelial cells (peroxidase, brown staining; (D), (F), (G)). In double-staining experiments, the anti-Thy-1 mAb AS02 showed no overlap with anti-CD14 staining (E). In contrast, endothelial cells were double-stained with both the antibody against von Willebrand factor and the anti-Thy-1 mAb AS02 [arrowheads in (F)(G)]. There was no positive reaction with control isotype-matched mAbs (APAAP and peroxidase) (B), employed as controls for double-staining [(E), (F), (G)]. Original magnification: (A)-(D) and (F), 92 ×; (E) and (G), 184 ×.
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Figure 3: Immunohistochemical staining of the RA synovial membrane with the anti-Thy-1 mAb AS02. (A) This mAb strongly stained cells in the connective tissue layer beneath the lining layer (APAAP, red staining; arrows), while largely sparing cells of the lining layer (arrowhead). Endothelial cells (asterisk) and connective tissue cells in diffuse infiltrates were also stained, more weakly in the latter case. In serial sections, the anti-CD14 mAb MoS39 (kindly provided by Prof GR Burmester, Berlin, Germany) stained macrophages in the lining layer (arrowhead in (C)) and diffuse inflammatory infiltrates (peroxidase, brown staining; arrow in (C), (E)); the mAb against von Willebrand factor stained endothelial cells (peroxidase, brown staining; (D), (F), (G)). In double-staining experiments, the anti-Thy-1 mAb AS02 showed no overlap with anti-CD14 staining (E). In contrast, endothelial cells were double-stained with both the antibody against von Willebrand factor and the anti-Thy-1 mAb AS02 [arrowheads in (F)(G)]. There was no positive reaction with control isotype-matched mAbs (APAAP and peroxidase) (B), employed as controls for double-staining [(E), (F), (G)]. Original magnification: (A)-(D) and (F), 92 ×; (E) and (G), 184 ×.

Mentions: The mAb AS02 was tested by immunohistochemistry on cryostat sections of RASM to verify the feasibility of this anti-Thy-1 mAb for positive identification/isolation of SFB. The mAb AS02 stained connective tissue cells, while largely sparing the lining layer (see Fig. 3A for details), therefore reproducing the known distribution of FB within the SM [21,23,24]. The mAb AS02 showed no overlap with an anti-CD14 mAb in double-staining experiments, thereby excluding crossreactivity with monocytes/macrophages (Fig. 3C,E). However, the mAb AS02 stained endothelial cells, as demonstrated by double-staining with rabbit Ig against von Willebrand factor (Fig. 3D,F,G). Cultured, non-stimulated HUVEC, in contrast, did not express Thy-1 (data not shown). These results, in line with cytokine induction of Thy-1 expression on endothelial cells in culture [44,45,46], suggest that Thy-1 expression on RASM endothelial cells may reflect ongoing inflammation [46].


Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

Zimmermann T, Kunisch E, Pfeiffer R, Hirth A, Stahl HD, Sack U, Laube A, Liesaus E, Roth A, Palombo-Kinne E, Emmrich F, Kinne RW - Arthritis Res. (2000)

Immunohistochemical staining of the RA synovial membrane with the anti-Thy-1 mAb AS02. (A) This mAb strongly stained cells in the connective tissue layer beneath the lining layer (APAAP, red staining; arrows), while largely sparing cells of the lining layer (arrowhead). Endothelial cells (asterisk) and connective tissue cells in diffuse infiltrates were also stained, more weakly in the latter case. In serial sections, the anti-CD14 mAb MoS39 (kindly provided by Prof GR Burmester, Berlin, Germany) stained macrophages in the lining layer (arrowhead in (C)) and diffuse inflammatory infiltrates (peroxidase, brown staining; arrow in (C), (E)); the mAb against von Willebrand factor stained endothelial cells (peroxidase, brown staining; (D), (F), (G)). In double-staining experiments, the anti-Thy-1 mAb AS02 showed no overlap with anti-CD14 staining (E). In contrast, endothelial cells were double-stained with both the antibody against von Willebrand factor and the anti-Thy-1 mAb AS02 [arrowheads in (F)(G)]. There was no positive reaction with control isotype-matched mAbs (APAAP and peroxidase) (B), employed as controls for double-staining [(E), (F), (G)]. Original magnification: (A)-(D) and (F), 92 ×; (E) and (G), 184 ×.
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Related In: Results  -  Collection

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Figure 3: Immunohistochemical staining of the RA synovial membrane with the anti-Thy-1 mAb AS02. (A) This mAb strongly stained cells in the connective tissue layer beneath the lining layer (APAAP, red staining; arrows), while largely sparing cells of the lining layer (arrowhead). Endothelial cells (asterisk) and connective tissue cells in diffuse infiltrates were also stained, more weakly in the latter case. In serial sections, the anti-CD14 mAb MoS39 (kindly provided by Prof GR Burmester, Berlin, Germany) stained macrophages in the lining layer (arrowhead in (C)) and diffuse inflammatory infiltrates (peroxidase, brown staining; arrow in (C), (E)); the mAb against von Willebrand factor stained endothelial cells (peroxidase, brown staining; (D), (F), (G)). In double-staining experiments, the anti-Thy-1 mAb AS02 showed no overlap with anti-CD14 staining (E). In contrast, endothelial cells were double-stained with both the antibody against von Willebrand factor and the anti-Thy-1 mAb AS02 [arrowheads in (F)(G)]. There was no positive reaction with control isotype-matched mAbs (APAAP and peroxidase) (B), employed as controls for double-staining [(E), (F), (G)]. Original magnification: (A)-(D) and (F), 92 ×; (E) and (G), 184 ×.
Mentions: The mAb AS02 was tested by immunohistochemistry on cryostat sections of RASM to verify the feasibility of this anti-Thy-1 mAb for positive identification/isolation of SFB. The mAb AS02 stained connective tissue cells, while largely sparing the lining layer (see Fig. 3A for details), therefore reproducing the known distribution of FB within the SM [21,23,24]. The mAb AS02 showed no overlap with an anti-CD14 mAb in double-staining experiments, thereby excluding crossreactivity with monocytes/macrophages (Fig. 3C,E). However, the mAb AS02 stained endothelial cells, as demonstrated by double-staining with rabbit Ig against von Willebrand factor (Fig. 3D,F,G). Cultured, non-stimulated HUVEC, in contrast, did not express Thy-1 (data not shown). These results, in line with cytokine induction of Thy-1 expression on endothelial cells in culture [44,45,46], suggest that Thy-1 expression on RASM endothelial cells may reflect ongoing inflammation [46].

Bottom Line: To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies.This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta.This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

View Article: PubMed Central - HTML - PubMed

Affiliation: Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, Germany.

ABSTRACT
To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Show MeSH
Related in: MedlinePlus