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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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FACS/histochemical analysis of the negative fraction following isolation of RA-SFB from primary culture using Dynabeads® M-450 CD14. The AS02/Thy-1+ RA-SFB (A) were almost free of contaminating CD14+(B) (mAb Tyk4) or CD68+ macrophages (D) (mAb PG-M1). The RA-SFB were accordingly negative for the macrophage marker non-specific esterase (histochemistry (C)). Original magnification: (C) 184 ×. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
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Figure 2: FACS/histochemical analysis of the negative fraction following isolation of RA-SFB from primary culture using Dynabeads® M-450 CD14. The AS02/Thy-1+ RA-SFB (A) were almost free of contaminating CD14+(B) (mAb Tyk4) or CD68+ macrophages (D) (mAb PG-M1). The RA-SFB were accordingly negative for the macrophage marker non-specific esterase (histochemistry (C)). Original magnification: (C) 184 ×. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.

Mentions: Negative isolation of SFB from primary culture resulted in RA-SFB that were Thy-1+ (on average, approximately 74%; n = 9; Fig. 2A and Table 1b) and, more importantly, prolyl-4-hydroxylase+ (on average, approximately 85%; n = 9; Table 1b) (mAb3-2B12; Dianova), as shown by FACS analysis and confirmed by immunohistochemistry in chamber slides. There were very few contaminating non-specific esterase+ (three RA patients; Fig. 2C), CD14+, CD68+, and/or CD11b+ macrophages (<2%; Fig. 2B,D and Table 1b), as well as <1% T cells (CD3+), B cells (CD19+/20+), plasma cells (CD38+), NK cells (CD56+), dendritic cells (CD83+), PMN (CD15), or endothelial cells (CD144+; von Willebrand factor-positive). The positive fraction, in turn, contained virtually no SFB, indicating minimal cell loss, and thereby also excluding major subset selection. The average yield of RA-SFB negatively isolated from primary culture was (2.8 ± 0.9) × 107 cells (mean ± SEM; n = 7). Phenotype analyses yielded two main results. The first, regarding expression of SFB features previously reported at a tissue level, was that, strikingly, approximately 66% of the cells expressed MHC-II molecules (normal skin-FB, 2%; OA-SFB, 17%). A low or moderate and variable percentage of RA-SFB, but also normal skin-FB and OA-SFB, expressed vascular cell adhesion molecule-1 (VCAM-1) (using two different anti-VCAM-1 mAbs) without statistically significant differences between the three different FB preparations. RA-SFB showed a higher (although non-significant) mean fluorescence intensity (MFI) for the cytoskeletal protein vimentin than normal skin-FB. Approximately 45% and 50% of the cells, respectively, expressed procollagen I and procollagen III (similar to normal skin-FB); however, the MFI for procollagen III was significantly higher in RA-SFB. Approximately 57% of RA-SFB expressed c-Fos. Neither the percentage nor the MFI were significantly different from those of normal skin-FB (approximately 87%) or OA-SFB (approximately 54%). In general, the differences between RA-SFB and normal skin-FB were not specific to RA, since they were also observed in the comparison between the disease control OA-SFB and normal skin-FB. The second outcome of phenotype analysis was that the percentages of RA-SFB positive for MHC-II as well as the MFI for VCAM-1 and c-Jun were significantly decreased in conventional fourth passage compared with isolated primary RA-SFB. In contrast, the percentages of cells positive for MHC-I, CD13, prolyl-4-hydroxylase, vimentin, procollagen I and III, c-Fos, and Jun-D were significantly increased in conventional fourth passage.


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)

FACS/histochemical analysis of the negative fraction following isolation of RA-SFB from primary culture using Dynabeads® M-450 CD14. The AS02/Thy-1+ RA-SFB (A) were almost free of contaminating CD14+(B) (mAb Tyk4) or CD68+ macrophages (D) (mAb PG-M1). The RA-SFB were accordingly negative for the macrophage marker non-specific esterase (histochemistry (C)). Original magnification: (C) 184 ×. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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Figure 2: FACS/histochemical analysis of the negative fraction following isolation of RA-SFB from primary culture using Dynabeads® M-450 CD14. The AS02/Thy-1+ RA-SFB (A) were almost free of contaminating CD14+(B) (mAb Tyk4) or CD68+ macrophages (D) (mAb PG-M1). The RA-SFB were accordingly negative for the macrophage marker non-specific esterase (histochemistry (C)). Original magnification: (C) 184 ×. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
Mentions: Negative isolation of SFB from primary culture resulted in RA-SFB that were Thy-1+ (on average, approximately 74%; n = 9; Fig. 2A and Table 1b) and, more importantly, prolyl-4-hydroxylase+ (on average, approximately 85%; n = 9; Table 1b) (mAb3-2B12; Dianova), as shown by FACS analysis and confirmed by immunohistochemistry in chamber slides. There were very few contaminating non-specific esterase+ (three RA patients; Fig. 2C), CD14+, CD68+, and/or CD11b+ macrophages (<2%; Fig. 2B,D and Table 1b), as well as <1% T cells (CD3+), B cells (CD19+/20+), plasma cells (CD38+), NK cells (CD56+), dendritic cells (CD83+), PMN (CD15), or endothelial cells (CD144+; von Willebrand factor-positive). The positive fraction, in turn, contained virtually no SFB, indicating minimal cell loss, and thereby also excluding major subset selection. The average yield of RA-SFB negatively isolated from primary culture was (2.8 ± 0.9) × 107 cells (mean ± SEM; n = 7). Phenotype analyses yielded two main results. The first, regarding expression of SFB features previously reported at a tissue level, was that, strikingly, approximately 66% of the cells expressed MHC-II molecules (normal skin-FB, 2%; OA-SFB, 17%). A low or moderate and variable percentage of RA-SFB, but also normal skin-FB and OA-SFB, expressed vascular cell adhesion molecule-1 (VCAM-1) (using two different anti-VCAM-1 mAbs) without statistically significant differences between the three different FB preparations. RA-SFB showed a higher (although non-significant) mean fluorescence intensity (MFI) for the cytoskeletal protein vimentin than normal skin-FB. Approximately 45% and 50% of the cells, respectively, expressed procollagen I and procollagen III (similar to normal skin-FB); however, the MFI for procollagen III was significantly higher in RA-SFB. Approximately 57% of RA-SFB expressed c-Fos. Neither the percentage nor the MFI were significantly different from those of normal skin-FB (approximately 87%) or OA-SFB (approximately 54%). In general, the differences between RA-SFB and normal skin-FB were not specific to RA, since they were also observed in the comparison between the disease control OA-SFB and normal skin-FB. The second outcome of phenotype analysis was that the percentages of RA-SFB positive for MHC-II as well as the MFI for VCAM-1 and c-Jun were significantly decreased in conventional fourth passage compared with isolated primary RA-SFB. In contrast, the percentages of cells positive for MHC-I, CD13, prolyl-4-hydroxylase, vimentin, procollagen I and III, c-Fos, and Jun-D were significantly increased in conventional fourth passage.

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