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

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.

Figure 1

Immunohistochemical staining of primary-culture RA synovial cells in chamber slides: (A), (B), (D)-(H) phase contrast, and (C) lightfield. While (A) control isotype-matched mAbs (peroxidase) or (B) rabbit serum (APAAP) showed no positive reaction, (C) the anti-Thy-1 mAb AS02 stained large, flat, spindle-shaped or stellate-shaped cells (ie cells with FB morphology; alkaline phosphatase, purple staining) and (D) the anti-CD14 mAb RMO52 identified small, round cells, conceivably macrophages (peroxidase, brown staining). The RA-SFB clearly expressed (E) procollagen I and (H) procollagen III (APAAP, red staining). The primary culture contained no endothelial cells, as documented by the lack of staining for (F) von Willebrand factor (APAAP) and (G) CD144 (APAAP). Original magnification: (A), (B), (F), and (G), 184 ×; (C)-(E) and (H) 368 ×.

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.

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 ×.

Figure 4

FACS analysis of primary-culture RA synovial cells (following 7 days of culture). The primary culture contained virtually only cells staining either with the FB-directed anti-Thy-1 mAb AS02 (A) or with the monocyte/macrophage-specific anti-CD14 mAb Tyk4 (B). FITC, fluoresceine isothiocyanate.

Figure 5

Morphology of negatively isolated RA-SFB and macrophages upon reculture. When recultured, the RA-SFB obtained by negative isolation with Dynabeads^® M-450 CD14 showed almost exclusively spindle-shaped, flat or stellate morphology (A). Recultured CD14⁺ cells (B) exhibited small, round, macrophage-like morphology with attached magnetobeads, and contained only very few cells with FB morphology. Original maginification: 368 ×.

Figure 6

Phenotype of isolated primary-culture RA-SFB (lower panels) in comparison with primary-culture normal skin-FB (upper panel) and isolated primary-culture OA-SFB. The double-staining experiments were performed with the anti-Thy-1 mAb AS02. (A)-(C) The expression of Thy-1 (A), MHC-II/Thy-1 (B) (double-staining), and vimentin/Thy-1 (C) (double-staining) in normal skin-FB; (D)-(F) the expression of Thy-1 (D), MHC-II/Thy-1 (E) (double-staining), and vimentin/Thy-1 (F) (double-staining) in OA-SFB; (G)-(I) the expression of the antigens Thy-1 (G), MHC-II/Thy-1 (H) (double-staining), vimentin/Thy-1 (I) (double-staining) on RA-SFB; (J)-(L) the expression of the cytoplasmic antigens procollagen I (J) and procollagen III/Thy-1 (K) (single-staining for procollagen III) and (L) (double-staining) in RA-SFB. Expression of prolyl-4-hydroxylase (M) and the proto-oncogenes c-Fos (N), and c-Jun (O) in RA-SFB is also shown. See Results and Table 4 for mean values and statistical comparison among the different FB types. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.

Figure 7

Phenotype comparison of isolated primary-culture (left column) and conventional fourth-passage RA-SFB (right column) from one representative patient. Expression of Thy-1 (A, B), MHC-II (C, D), prolyl-4-hydroxylase (E, F), procollagen III (G, H), c-Fos (I, J), and c-Jun (K, L). While the percentages of cells positive for MHC-II (C) and the MFI for c-Jun (K) were significantly higher in isolated primary-culture RA-SFB, the percentages of cells positive for prolyl-4-hydroxylase (F), procollagen III (H), and c-Fos (J) were significantly higher in conventional fourth passage (see Table 5 for details). PE, phycoerythrine; FITC, fluoresceine isothiocyanate.

Figure 8

Proliferation rates, as assessed by incorporation of [³H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB (n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB (n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB (n = 4, 3, and 5, respectively) (C), at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. *P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.