PMC:17827 / 30045-47159 JSONTXT

{"project":"2_test","denotations":[{"id":"11178129-6366990-4386856","span":{"begin":401,"end":403},"obj":"6366990"},{"id":"11178129-7510485-4386857","span":{"begin":407,"end":409},"obj":"7510485"},{"id":"11178129-9056477-4386858","span":{"begin":900,"end":902},"obj":"9056477"},{"id":"11178129-8752941-4386859","span":{"begin":903,"end":905},"obj":"8752941"},{"id":"11178129-10571119-4386860","span":{"begin":906,"end":908},"obj":"10571119"},{"id":"11178129-10571119-4386861","span":{"begin":1001,"end":1003},"obj":"10571119"},{"id":"11178129-8151561-4386862","span":{"begin":1499,"end":1500},"obj":"8151561"},{"id":"11178129-8752676-4386863","span":{"begin":1501,"end":1502},"obj":"8752676"},{"id":"11178129-8849370-4386864","span":{"begin":1998,"end":2000},"obj":"8849370"},{"id":"11178129-8912499-4386865","span":{"begin":5686,"end":5687},"obj":"8912499"},{"id":"11178129-7632096-4386866","span":{"begin":5688,"end":5689},"obj":"7632096"},{"id":"11178129-2596570-4386867","span":{"begin":5690,"end":5692},"obj":"2596570"},{"id":"11178129-2694400-4386868","span":{"begin":5693,"end":5695},"obj":"2694400"},{"id":"11178129-2084514-4386869","span":{"begin":5696,"end":5698},"obj":"2084514"},{"id":"11178129-8003048-4386870","span":{"begin":5699,"end":5701},"obj":"8003048"},{"id":"11178129-6366990-4386871","span":{"begin":5703,"end":5705},"obj":"6366990"},{"id":"11178129-2442194-4386872","span":{"begin":5706,"end":5708},"obj":"2442194"},{"id":"11178129-7510485-4386873","span":{"begin":5712,"end":5714},"obj":"7510485"},{"id":"11178129-1411083-4386874","span":{"begin":5715,"end":5717},"obj":"1411083"},{"id":"11178129-1657009-4386875","span":{"begin":5718,"end":5720},"obj":"1657009"},{"id":"11178129-8082888-4386876","span":{"begin":9710,"end":9712},"obj":"8082888"},{"id":"11178129-1411083-4386877","span":{"begin":11744,"end":11746},"obj":"1411083"}],"text":"Results\n\nExpression of Thy-1 in RA synovial tissue\nThe 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].\n\nPhenotype analysis of RA synovial cells in primary culture\nRA synovial cells were subjected to extensive analysis to carefully characterize the starting population before negative isolation (ie the 7-day primary culture of synovial cells resulting from trypsin/collagenase digestion of the RASM). The primary culture contained large, spindle-shaped Thy-1+ SFB (Fig. 1C) and small, round CD14+ cells, most probably macrophages (Fig. 1D; mAb Tyk4), as detected by immunohistochemical staining [6,9]. Endothelial cells were absent, as confirmed by lack of staining for von Willebrand factor (Fig. 1F; mAb 4F9) and CD144 (Fig. 1G; mAb Cadherin 5), which clearly identified HUVEC (data not shown). The FB nature of the spindle-shaped cells was confirmed by intracellular staining for procollagen I and III (Fig. 1E,H; rabbit antibodies MP I and MP III). An average of approximately 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a and Fig. 4A) in FACS analysis [10]. The average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a and Fig. 4B). There were \u003c1% T cells (CD3+; mAb UCHT-1), B cells (CD19+/20+; mAbs HD 37 and B-Ly 1), plasma cells (CD38+; mAb AT 13/5), NK cells (CD56+; mAb NKH/1), dendritic cells (CD83+; mAb HB 15a), endothelial cells (CD144+), or PMN (CD15+; mAb 80H5), indicating that non-adherent cells had been efficiently removed during primary culture. Endothelial cells presumably did not adhere to the culture dish due to the absence of gelatin coating and unfavorable medium composition (see Materials and Methods).\nAdherent synovial cells were then detached by short-term trypsinization for 2 min (0.25% trypsin/0.2% EDTA; Gibco) and used for negative isolation. The total yield of cells following 7 days of RA primary culture averaged (5.2 ± 1.1) × 107 cells (mean ± SEM; n = 7; open synovectomy samples). The yield of cells from arthroscopic synovectomy samples ([3.1 ± 0.6] × 107 cells; n = 3) was comparable. There was no significant difference between the yield of cells from the RA or OA SM.\n\nFlow cytometry/histochemical analysis of cells negatively isolated from primary culture\nNegative isolation from primary culture using Dynabeads® M-450 CD14 (clone RMO52) resulted in cells 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) (mAb 3-2B12, Dianova), as shown by FACS analysis (Table 1b) and confirmed by immunohistochemistry in chamber slides. There were very few contaminating non-specific esterase+ (n = 3 RA and n = 3 OA patients; Fig. 2C), CD14+, CD68+, and/or CD11b+ macrophages (\u003c2%; Fig. 2B,D and Table 1b), as well as \u003c1% T cells (CD3+), B cells (CD19+/20+), plasma cells (CD38+), NK cells (CD56+), dendritic cells (CD83+), PMN (CD15), or endothelial cells (CD144+; von Wille-brand factor-positive).\nThe average yield of RA-SFB negatively isolated from primary culture was (2.8 ± 0.9) × 107 cells (mean ± SEM; n = 7; open synovectomy samples). A similar yield was observed upon isolation of cells from arthroscopic synovectomy samples ([1.7 ± 0.6] × 107 cells; n = 3). There was no significant difference between the yield from the primary culture of the RASM and that of the OA SM.\nInclusion of the trypsin component in the initial tissue digestion led to a considerably higher yield of cells following negative isolation from primary culture (1.2-fold to 5-fold; n = 3) than without trypsin or with DNAse instead of trypsin.\nThe composition of the cells was very similar to that obtained with primary-culture or first-passage normal skin-FB (Table 1g). Comparable results, although with a considerably lower percentage of Thy-1+ cells, were also obtained when OA-SFB were negatively isolated from primary culture (Table 1e). Conventional fourth-passage RA-SFB (Table 1d) or OA-SFB (Table 1f) showed FB markers on a high percentage of cells (\u003e98% prolyl-4-hydroxylase+ cells; \u003e80% Thy-1+ cells) and contained virtually no contaminating macrophages (\u003c2% CD14+-positive or CD68+-positive cells). The same was true for isolated primary RA-SFB kept in culture until fourth passage (Table 1c).\n\nIn vitro morphology of negatively isolated RA-SFB upon reculture\nNegatively isolated RA-SFB showed almost exclusively spindle-shaped or stellate, flat morphology when recultured (Fig. 5A). Recultured CD14+ cells (Fig. 5B) exhibited small, round morphology with attached Dynabeads® and contained only very few cells with FB morphology.\n\nPhenotype characterization of negatively isolated RA-SFB\nTo verify whether isolated RA-SFB displayed the features observed in situ in the RASM [1,2,12,13,14,15, 21,22,23,24,25,28], the expression of several surface or intracellular/nuclear molecules was investigated by FACS analysis (Fig. 6 and Table 4). The phenotype was compared with that of primary-culture normal skin-FB and isolated primary-culture OA-SFB (Fig. 6 and Table 4).\n\nSurface antigens\nApproximately 74% of RA-SFB expressed the Thy-1 antigen (Fig. 6G and Table 4), compared with 91% of normal skin-FB (Fig. 6A and Table 4). Approximately 77% of the cells expressed MHC-I molecules, compared with 90% of normal skin-FB (Table 4). Strikingly, approximately 66% of the cells expressed MHC-II molecules (Fig. 6H and Table 4), significantly more than OA-SFB (17%; Fig. 6E and Table 4) and normal skin-FB (2%; Fig. 6B and Table 4). At the same time, however, the MFI for MHC-II did not significantly differ between RA-SFB and normal skin-FB (Table 4).\nThe expression of CD13 (aminopeptidase N; EC 3.4.11.2) on 85% of RA-SFB was comparable with that of normal skin-FB (84%; Table 4).\nA low or moderate and variable percentage of normal skin-FB, as well as OA-SFB and RA-SFB, expressed VCAM-1 (using two different anti-VCAM-1 mAbs; see Table 3) without statistically significant differences among these three different FB preparations (Table 4).\n\nCytoplasmic antigens\nApproximately 85% of the RA-SFB expressed the FB marker prolyl-4-hydroxylase (Fig. 6M, and Tables 1 and 4), comparable with the expression in normal skin-FB (71%; Tables 1 and 4).\nRA-SFB showed a similar percentage but a higher MFI for the cytoskeletal protein vimentin (Fig. 6I) than normal skin-FB (Fig. 6C and Table 4), although the MFI difference did not attain statistical significance.\nA mean of approximately 45% and 50% of the cells, respectively (Table 4), expressed procollagen I (Fig. 6J) and procollagen III (Fig. 6K), as confirmed by double-staining (Fig. 6L). The percentage of cells expressing procollagen I and III was similar to that of normal skin-FB (68% and 55%, respectively; Table 4); however, the MFI for procollagen III was significantly higher in RA-SFB (26 versus 15; Table 4).\n\nCytoplasmic/intranuclear antigens\nApproximately 57% of RA-SFB expressed c-Fos (Fig. 6N and Table 4). Neither the percentage nor the MFI were significantly different from those of normal skin-FB.\nThe proto-oncogenes c-Jun and Jun-D (both percentage of positive cells and MFI) were also comparably expressed in RA-SFB and normal skin-FB (Fig. 6O and Table 4).\nThe differences between RA-SFB and normal skin-FB were, in general, not specific to RA since they were also observed in the comparison between the disease control OA-SFB and normal skin-FB (Table 4). In fact, some of the differences in OA were even more pronounced than in RA, attaining statistical significance in the direct comparison between OA and RA (Table 4). Briefly, significantly higher MFI levels in OA-SFB were noted for MHC-I, MHC-II and procollagen I, as well as c-Fos. Opposite differences among the different FB preparations, when considering the percentage of positive cells or the MFI (eg for the comparison of MHC-II and procollagen I and III expression in RA-SFB versus OA-SFB), appeared due to large variations in one or both parameters.\n\nComparison of FACS analysis and immunohistochemistry\nTo verify whether the short-term trypsin digestion (2 min at 37°C) employed for removal of the cells from the culture dishes and subsequent FACS analysis altered the expression of the antigens, selected experiments were carried out to compare the expression of surface and intracellular antigens in FACS analysis (Table 1b,e, and Table 4) and immunohistochemistry on chamber slides. Negatively isolated SFB from n = 3 OA and n = 3 RA patients showed comparable percentages of positive cells for surface antigens (RA-Thy-1, 95.3% FACS versus 85% immunohistochemistry; CD14, 1.1% versus 0%; VCAM-1, 0.7% versus 0%) and intracellular antigens (RA-prolyl-4-hydroxylase, 94.7% versus 85%; CD68, 1.7% versus 0%) with both methods, confirming previously published results [47] and excluding the possibility that short-term trypsinization significantly influences antigen expression.\n\nPhenotype comparison of primary-culture and fourth-passage RA-SFB\nIn order to assess differences between the primary RA-SFB derived from the isolation technique developed in the present study (immediately following isolation or at fourth passage) and those obtained by conventional repeated passaging, the phenotypic features of the three preparations were compared.\n\nIsolated primary RA-SFB versus conventional fourth-passage RA-SFB\nThe 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 in comparison with isolated primary RA-SFB (Fig. 7C,D,K,L and Table 5). The percentages of cells positive for MHC-I, CD13, prolyl-4-hydroxylase, vimentin, procollagen I and III, c-Fos and Jun-D were, in contrast, significantly increased in conventional fourth passage (Fig. 7E,F,G,H,I,J and Table 5).\nThe upregulation of the proto-oncogenes c-Fos and Jun-D was limited to RA, since the percentages of cells positive for these molecules were significantly decreased upon passaging in OA-SFB (c-Fos: isolated primary-culture OA-SFB, 53.9 ± 10.4%; conventional fourth-passage OA-SFB, 15.0 ± 6.6%; Jun-D: isolated primary-culture OA-SFB, 37.6 ± 17.7%; conventional fourth-passage OA-SFB, 25.1 ± 4.2%; n =3 for both SFB preparations) and numerically decreased upon passaging in normal skin-FB (c-Fos: primary-culture/first-passage skin-FB, 87.2 ± 2.1%; conventional fourth-passage skin-FB, 59.4 ± 26.0%; Jun-D: primary-culture/first-passage skin-FB, 87.4 ± 1.5%; conventional fourth-passage skin-FB, 66.2 ± 18.6%; n = 3 for both). These decreases were also noted for the MFI (data not shown). The percentage of positive cells and/or MFI for c-Fos and Jun-D in conventional fourth-passage RA-SFB were significantly higher than in OA-SFB as a consequence of these reciprocal changes, under these circumstances confirming previously published data [25].\n\nIsolated primary RA-SFB versus isolated fourth-passage RA-SFB\nSome of the differences observed between isolated primary RA-SFB and conventional fourth passage also arose when the isolated primary RA-SFB were passaged until the fourth passage; this applied to the significantly increased percentage of cells positive for MHC-I, prolyl-4-hydroxylase, vimentin, and procollagen I and III, as well as to the decreased percentage of MHC-II-positive cells (Table 5). In addition, isolated fourth-passage RA-SFB showed a significantly decreased MFI (but a numerically increased percentage) for Thy-1 in comparison with isolated primary RA-SFB (Table 5).\n\nConventional fourth-passage RA-SFB versus isolated fourth-passage RA-SFB\nThe most important difference between these two preparations was the significantly increased percentage of cells positive for the proto-oncogenes c-Fos and Jun-D, observed only in conventional fourth-passage RA-SFB (Table 5). The MFI for Thy-1 was also significantly higher in conventional fourth-passage cells.\n\nProliferation rates of first- and fourth-passage FB\nThe proliferation rates of skin-FB and SFB were determined and the different preparations compared.\n\nIsolated first-passage FB versus conventional fourth-passage FB\nAt rest, the proliferation rates of conventional fourth-passage normal skin-FB did not significantly differ from those of the corresponding cells in first passage (Fig. 8A). The same was true for conventional fourth passage OA-SFB and RA-SFB (Fig. 8B,C). Upon stimulation with PDGF (2.5 and 5 U/ml for all cells, 10 U/ml only for OA-SFB), however, the proliferation rates of skin, RA, and OA conventional fourth-passage FB were significantly higher (maximum mean increase, 5.4-fold) than those observed in first-passage cells (Fig. 8A,B,C). Following stimulation with IL-1β, in contrast, only conventional fourth-passage RA-SFB (150 U/ml IL-1β) showed significantly higher proliferation rates than first-passage cells (Fig. 8C).\n\nIsolated first-passage versus isolated fourth-passage OA-SFB and RA-SFB\nThe proliferation rates of isolated fourth-passage cells in OA-SFB differed significantly from those of isolated first-passage cells only upon stimulation with IL-1β (50 and 150 U/ml; Fig. 8B). Such differences were not observed at rest or following stimulation with PDGF.\nIsolated fourth-passage RA-SFB, in contrast, showed significantly higher proliferation rates than first-passage cells upon stimulation with all concentrations of IL-1β and PDGF, but not at rest (Fig. 8C).\n\nConventional fourth-passage versus isolated fourth-passage OA-SFB and RA-SFB\nThe proliferation rates of isolated fourth-passage SFB in both OA and RA were comparable with those of conventional fourth-passage SFB, whether at rest or PDGF-stimulated (Fig. 8B,C). Stimulation with IL-1β (at all concentrations), in contrast, induced significantly higher proliferation in isolated fourth-passage RA-SFB than in conventional fourth-passage RA-SFB (Fig. 8C). This difference was specific for RA-SFB, since it was not observed in OA-SFB (Fig. 8B).\nAs a consequence of differential IL-1β sensitivity in RA-SFB and OA-SFB, there were significant differences between isolated fourth-passage RA-SFB and OA-SFB (P ≤ 0.05; RA\u003eOA, for 100 U/ml IL-1β), as well as between conventional fourth-passage RA-SFB and OA-SFB (P ≤ 0.05; OA\u003eRA, for 50 and 150 U/ml IL-1β; Fig. 8B,C).\n\nAlternative attempts to isolate RA-SFB\nThe following isolation techniques were also tested to compare the quality and reliability of negative isolation from primary culture with possible alternative methods. The first technique was positive isolation directly from the trypsin/collagenase digest using the FB-directed anti-Thy1/CD90 mAb AS02 and Dynabeads® M-450 goat anti-mouse IgG. This approach, however, considered legitimate on the basis of the immunostaining in RA synovial tissue (Fig. 3A,E), remained unsatisfactory (yield, only approximately 6% of the initial cell numbers). Another technique involved negative isolation directly from the trypsin/collagenase digest. This approach proved technically prohibitive for the amount of different anti-leukocyte and anti-endothelial mAbs and Dynabeads® M-450 goat anti-mouse IgG necessary for complete removal of all contaminating cell types. Positive isolation from primary culture with the anti-Thy-1 mAb AS02 and Dynabeads® M-450 goat anti-mouse IgG was also tested. This method was unsuitable because: first, the percentages of contaminating macrophages were too high/variable (2-15%); second, the isolated SFB showed a significantly reduced surface density for Thy-1 (MFI 19 versus 152 before isolation; n = 4, P ≤ 0.05), presumably due to contact with the mAb used for isolation; third, the magnetobeads remained attached to the positively isolated SFB for as long as 1 week, thus requiring further manipulation for their removal (eg with anti-idiotypic mAbs); fourth, a substantial proportion of SFB (approximately 30%) was lost into the negative fraction; and, finally, a Thy-1-dependent isolation technique bore the risk of selecting for SFB subpopulations, since the prolyl-4-hydroxylase+ cells obtained by negative isolation with Dynabeads® M-450 CD14 contained both a Thy-1+ fraction (RA, 49.3± 14.9%, n = 6; OA, 43.6 ± 12.0%, n = 4) and a Thy-1– fraction, as demonstrated by double-staining FACS analysis.\n"}

{"project":"Colil","denotations":[{"id":"T63","span":{"begin":900,"end":902},"obj":"9056477"},{"id":"T64","span":{"begin":903,"end":905},"obj":"8752941"},{"id":"T65","span":{"begin":906,"end":908},"obj":"10571119"},{"id":"T66","span":{"begin":1001,"end":1003},"obj":"10571119"},{"id":"T67","span":{"begin":401,"end":403},"obj":"6366990"},{"id":"T68","span":{"begin":407,"end":409},"obj":"7510485"},{"id":"T69","span":{"begin":1998,"end":2000},"obj":"8849370"},{"id":"T70","span":{"begin":1998,"end":2000},"obj":"8849370"},{"id":"T71","span":{"begin":1499,"end":1500},"obj":"8151561"},{"id":"T72","span":{"begin":1501,"end":1502},"obj":"8752676"},{"id":"T73","span":{"begin":1499,"end":1500},"obj":"8151561"},{"id":"T74","span":{"begin":1501,"end":1502},"obj":"8752676"},{"id":"T75","span":{"begin":9710,"end":9712},"obj":"8082888"},{"id":"T76","span":{"begin":5686,"end":5687},"obj":"8912499"},{"id":"T77","span":{"begin":5688,"end":5689},"obj":"7632096"},{"id":"T78","span":{"begin":5690,"end":5692},"obj":"2596570"},{"id":"T79","span":{"begin":5693,"end":5695},"obj":"2694400"},{"id":"T80","span":{"begin":5696,"end":5698},"obj":"2084514"},{"id":"T81","span":{"begin":5699,"end":5701},"obj":"8003048"},{"id":"T82","span":{"begin":5703,"end":5705},"obj":"6366990"},{"id":"T83","span":{"begin":5706,"end":5708},"obj":"2442194"},{"id":"T84","span":{"begin":5712,"end":5714},"obj":"7510485"},{"id":"T85","span":{"begin":5715,"end":5717},"obj":"1411083"},{"id":"T86","span":{"begin":5718,"end":5720},"obj":"1657009"},{"id":"T87","span":{"begin":11744,"end":11746},"obj":"1411083"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Results\n\nExpression of Thy-1 in RA synovial tissue\nThe 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].\n\nPhenotype analysis of RA synovial cells in primary culture\nRA synovial cells were subjected to extensive analysis to carefully characterize the starting population before negative isolation (ie the 7-day primary culture of synovial cells resulting from trypsin/collagenase digestion of the RASM). The primary culture contained large, spindle-shaped Thy-1+ SFB (Fig. 1C) and small, round CD14+ cells, most probably macrophages (Fig. 1D; mAb Tyk4), as detected by immunohistochemical staining [6,9]. Endothelial cells were absent, as confirmed by lack of staining for von Willebrand factor (Fig. 1F; mAb 4F9) and CD144 (Fig. 1G; mAb Cadherin 5), which clearly identified HUVEC (data not shown). The FB nature of the spindle-shaped cells was confirmed by intracellular staining for procollagen I and III (Fig. 1E,H; rabbit antibodies MP I and MP III). An average of approximately 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a and Fig. 4A) in FACS analysis [10]. The average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a and Fig. 4B). There were \u003c1% T cells (CD3+; mAb UCHT-1), B cells (CD19+/20+; mAbs HD 37 and B-Ly 1), plasma cells (CD38+; mAb AT 13/5), NK cells (CD56+; mAb NKH/1), dendritic cells (CD83+; mAb HB 15a), endothelial cells (CD144+), or PMN (CD15+; mAb 80H5), indicating that non-adherent cells had been efficiently removed during primary culture. Endothelial cells presumably did not adhere to the culture dish due to the absence of gelatin coating and unfavorable medium composition (see Materials and Methods).\nAdherent synovial cells were then detached by short-term trypsinization for 2 min (0.25% trypsin/0.2% EDTA; Gibco) and used for negative isolation. The total yield of cells following 7 days of RA primary culture averaged (5.2 ± 1.1) × 107 cells (mean ± SEM; n = 7; open synovectomy samples). The yield of cells from arthroscopic synovectomy samples ([3.1 ± 0.6] × 107 cells; n = 3) was comparable. There was no significant difference between the yield of cells from the RA or OA SM.\n\nFlow cytometry/histochemical analysis of cells negatively isolated from primary culture\nNegative isolation from primary culture using Dynabeads® M-450 CD14 (clone RMO52) resulted in cells 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) (mAb 3-2B12, Dianova), as shown by FACS analysis (Table 1b) and confirmed by immunohistochemistry in chamber slides. There were very few contaminating non-specific esterase+ (n = 3 RA and n = 3 OA patients; Fig. 2C), CD14+, CD68+, and/or CD11b+ macrophages (\u003c2%; Fig. 2B,D and Table 1b), as well as \u003c1% T cells (CD3+), B cells (CD19+/20+), plasma cells (CD38+), NK cells (CD56+), dendritic cells (CD83+), PMN (CD15), or endothelial cells (CD144+; von Wille-brand factor-positive).\nThe average yield of RA-SFB negatively isolated from primary culture was (2.8 ± 0.9) × 107 cells (mean ± SEM; n = 7; open synovectomy samples). A similar yield was observed upon isolation of cells from arthroscopic synovectomy samples ([1.7 ± 0.6] × 107 cells; n = 3). There was no significant difference between the yield from the primary culture of the RASM and that of the OA SM.\nInclusion of the trypsin component in the initial tissue digestion led to a considerably higher yield of cells following negative isolation from primary culture (1.2-fold to 5-fold; n = 3) than without trypsin or with DNAse instead of trypsin.\nThe composition of the cells was very similar to that obtained with primary-culture or first-passage normal skin-FB (Table 1g). Comparable results, although with a considerably lower percentage of Thy-1+ cells, were also obtained when OA-SFB were negatively isolated from primary culture (Table 1e). Conventional fourth-passage RA-SFB (Table 1d) or OA-SFB (Table 1f) showed FB markers on a high percentage of cells (\u003e98% prolyl-4-hydroxylase+ cells; \u003e80% Thy-1+ cells) and contained virtually no contaminating macrophages (\u003c2% CD14+-positive or CD68+-positive cells). The same was true for isolated primary RA-SFB kept in culture until fourth passage (Table 1c).\n\nIn vitro morphology of negatively isolated RA-SFB upon reculture\nNegatively isolated RA-SFB showed almost exclusively spindle-shaped or stellate, flat morphology when recultured (Fig. 5A). Recultured CD14+ cells (Fig. 5B) exhibited small, round morphology with attached Dynabeads® and contained only very few cells with FB morphology.\n\nPhenotype characterization of negatively isolated RA-SFB\nTo verify whether isolated RA-SFB displayed the features observed in situ in the RASM [1,2,12,13,14,15, 21,22,23,24,25,28], the expression of several surface or intracellular/nuclear molecules was investigated by FACS analysis (Fig. 6 and Table 4). The phenotype was compared with that of primary-culture normal skin-FB and isolated primary-culture OA-SFB (Fig. 6 and Table 4).\n\nSurface antigens\nApproximately 74% of RA-SFB expressed the Thy-1 antigen (Fig. 6G and Table 4), compared with 91% of normal skin-FB (Fig. 6A and Table 4). Approximately 77% of the cells expressed MHC-I molecules, compared with 90% of normal skin-FB (Table 4). Strikingly, approximately 66% of the cells expressed MHC-II molecules (Fig. 6H and Table 4), significantly more than OA-SFB (17%; Fig. 6E and Table 4) and normal skin-FB (2%; Fig. 6B and Table 4). At the same time, however, the MFI for MHC-II did not significantly differ between RA-SFB and normal skin-FB (Table 4).\nThe expression of CD13 (aminopeptidase N; EC 3.4.11.2) on 85% of RA-SFB was comparable with that of normal skin-FB (84%; Table 4).\nA low or moderate and variable percentage of normal skin-FB, as well as OA-SFB and RA-SFB, expressed VCAM-1 (using two different anti-VCAM-1 mAbs; see Table 3) without statistically significant differences among these three different FB preparations (Table 4).\n\nCytoplasmic antigens\nApproximately 85% of the RA-SFB expressed the FB marker prolyl-4-hydroxylase (Fig. 6M, and Tables 1 and 4), comparable with the expression in normal skin-FB (71%; Tables 1 and 4).\nRA-SFB showed a similar percentage but a higher MFI for the cytoskeletal protein vimentin (Fig. 6I) than normal skin-FB (Fig. 6C and Table 4), although the MFI difference did not attain statistical significance.\nA mean of approximately 45% and 50% of the cells, respectively (Table 4), expressed procollagen I (Fig. 6J) and procollagen III (Fig. 6K), as confirmed by double-staining (Fig. 6L). The percentage of cells expressing procollagen I and III was similar to that of normal skin-FB (68% and 55%, respectively; Table 4); however, the MFI for procollagen III was significantly higher in RA-SFB (26 versus 15; Table 4).\n\nCytoplasmic/intranuclear antigens\nApproximately 57% of RA-SFB expressed c-Fos (Fig. 6N and Table 4). Neither the percentage nor the MFI were significantly different from those of normal skin-FB.\nThe proto-oncogenes c-Jun and Jun-D (both percentage of positive cells and MFI) were also comparably expressed in RA-SFB and normal skin-FB (Fig. 6O and Table 4).\nThe differences between RA-SFB and normal skin-FB were, in general, not specific to RA since they were also observed in the comparison between the disease control OA-SFB and normal skin-FB (Table 4). In fact, some of the differences in OA were even more pronounced than in RA, attaining statistical significance in the direct comparison between OA and RA (Table 4). Briefly, significantly higher MFI levels in OA-SFB were noted for MHC-I, MHC-II and procollagen I, as well as c-Fos. Opposite differences among the different FB preparations, when considering the percentage of positive cells or the MFI (eg for the comparison of MHC-II and procollagen I and III expression in RA-SFB versus OA-SFB), appeared due to large variations in one or both parameters.\n\nComparison of FACS analysis and immunohistochemistry\nTo verify whether the short-term trypsin digestion (2 min at 37°C) employed for removal of the cells from the culture dishes and subsequent FACS analysis altered the expression of the antigens, selected experiments were carried out to compare the expression of surface and intracellular antigens in FACS analysis (Table 1b,e, and Table 4) and immunohistochemistry on chamber slides. Negatively isolated SFB from n = 3 OA and n = 3 RA patients showed comparable percentages of positive cells for surface antigens (RA-Thy-1, 95.3% FACS versus 85% immunohistochemistry; CD14, 1.1% versus 0%; VCAM-1, 0.7% versus 0%) and intracellular antigens (RA-prolyl-4-hydroxylase, 94.7% versus 85%; CD68, 1.7% versus 0%) with both methods, confirming previously published results [47] and excluding the possibility that short-term trypsinization significantly influences antigen expression.\n\nPhenotype comparison of primary-culture and fourth-passage RA-SFB\nIn order to assess differences between the primary RA-SFB derived from the isolation technique developed in the present study (immediately following isolation or at fourth passage) and those obtained by conventional repeated passaging, the phenotypic features of the three preparations were compared.\n\nIsolated primary RA-SFB versus conventional fourth-passage RA-SFB\nThe 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 in comparison with isolated primary RA-SFB (Fig. 7C,D,K,L and Table 5). The percentages of cells positive for MHC-I, CD13, prolyl-4-hydroxylase, vimentin, procollagen I and III, c-Fos and Jun-D were, in contrast, significantly increased in conventional fourth passage (Fig. 7E,F,G,H,I,J and Table 5).\nThe upregulation of the proto-oncogenes c-Fos and Jun-D was limited to RA, since the percentages of cells positive for these molecules were significantly decreased upon passaging in OA-SFB (c-Fos: isolated primary-culture OA-SFB, 53.9 ± 10.4%; conventional fourth-passage OA-SFB, 15.0 ± 6.6%; Jun-D: isolated primary-culture OA-SFB, 37.6 ± 17.7%; conventional fourth-passage OA-SFB, 25.1 ± 4.2%; n =3 for both SFB preparations) and numerically decreased upon passaging in normal skin-FB (c-Fos: primary-culture/first-passage skin-FB, 87.2 ± 2.1%; conventional fourth-passage skin-FB, 59.4 ± 26.0%; Jun-D: primary-culture/first-passage skin-FB, 87.4 ± 1.5%; conventional fourth-passage skin-FB, 66.2 ± 18.6%; n = 3 for both). These decreases were also noted for the MFI (data not shown). The percentage of positive cells and/or MFI for c-Fos and Jun-D in conventional fourth-passage RA-SFB were significantly higher than in OA-SFB as a consequence of these reciprocal changes, under these circumstances confirming previously published data [25].\n\nIsolated primary RA-SFB versus isolated fourth-passage RA-SFB\nSome of the differences observed between isolated primary RA-SFB and conventional fourth passage also arose when the isolated primary RA-SFB were passaged until the fourth passage; this applied to the significantly increased percentage of cells positive for MHC-I, prolyl-4-hydroxylase, vimentin, and procollagen I and III, as well as to the decreased percentage of MHC-II-positive cells (Table 5). In addition, isolated fourth-passage RA-SFB showed a significantly decreased MFI (but a numerically increased percentage) for Thy-1 in comparison with isolated primary RA-SFB (Table 5).\n\nConventional fourth-passage RA-SFB versus isolated fourth-passage RA-SFB\nThe most important difference between these two preparations was the significantly increased percentage of cells positive for the proto-oncogenes c-Fos and Jun-D, observed only in conventional fourth-passage RA-SFB (Table 5). The MFI for Thy-1 was also significantly higher in conventional fourth-passage cells.\n\nProliferation rates of first- and fourth-passage FB\nThe proliferation rates of skin-FB and SFB were determined and the different preparations compared.\n\nIsolated first-passage FB versus conventional fourth-passage FB\nAt rest, the proliferation rates of conventional fourth-passage normal skin-FB did not significantly differ from those of the corresponding cells in first passage (Fig. 8A). The same was true for conventional fourth passage OA-SFB and RA-SFB (Fig. 8B,C). Upon stimulation with PDGF (2.5 and 5 U/ml for all cells, 10 U/ml only for OA-SFB), however, the proliferation rates of skin, RA, and OA conventional fourth-passage FB were significantly higher (maximum mean increase, 5.4-fold) than those observed in first-passage cells (Fig. 8A,B,C). Following stimulation with IL-1β, in contrast, only conventional fourth-passage RA-SFB (150 U/ml IL-1β) showed significantly higher proliferation rates than first-passage cells (Fig. 8C).\n\nIsolated first-passage versus isolated fourth-passage OA-SFB and RA-SFB\nThe proliferation rates of isolated fourth-passage cells in OA-SFB differed significantly from those of isolated first-passage cells only upon stimulation with IL-1β (50 and 150 U/ml; Fig. 8B). Such differences were not observed at rest or following stimulation with PDGF.\nIsolated fourth-passage RA-SFB, in contrast, showed significantly higher proliferation rates than first-passage cells upon stimulation with all concentrations of IL-1β and PDGF, but not at rest (Fig. 8C).\n\nConventional fourth-passage versus isolated fourth-passage OA-SFB and RA-SFB\nThe proliferation rates of isolated fourth-passage SFB in both OA and RA were comparable with those of conventional fourth-passage SFB, whether at rest or PDGF-stimulated (Fig. 8B,C). Stimulation with IL-1β (at all concentrations), in contrast, induced significantly higher proliferation in isolated fourth-passage RA-SFB than in conventional fourth-passage RA-SFB (Fig. 8C). This difference was specific for RA-SFB, since it was not observed in OA-SFB (Fig. 8B).\nAs a consequence of differential IL-1β sensitivity in RA-SFB and OA-SFB, there were significant differences between isolated fourth-passage RA-SFB and OA-SFB (P ≤ 0.05; RA\u003eOA, for 100 U/ml IL-1β), as well as between conventional fourth-passage RA-SFB and OA-SFB (P ≤ 0.05; OA\u003eRA, for 50 and 150 U/ml IL-1β; Fig. 8B,C).\n\nAlternative attempts to isolate RA-SFB\nThe following isolation techniques were also tested to compare the quality and reliability of negative isolation from primary culture with possible alternative methods. The first technique was positive isolation directly from the trypsin/collagenase digest using the FB-directed anti-Thy1/CD90 mAb AS02 and Dynabeads® M-450 goat anti-mouse IgG. This approach, however, considered legitimate on the basis of the immunostaining in RA synovial tissue (Fig. 3A,E), remained unsatisfactory (yield, only approximately 6% of the initial cell numbers). Another technique involved negative isolation directly from the trypsin/collagenase digest. This approach proved technically prohibitive for the amount of different anti-leukocyte and anti-endothelial mAbs and Dynabeads® M-450 goat anti-mouse IgG necessary for complete removal of all contaminating cell types. Positive isolation from primary culture with the anti-Thy-1 mAb AS02 and Dynabeads® M-450 goat anti-mouse IgG was also tested. This method was unsuitable because: first, the percentages of contaminating macrophages were too high/variable (2-15%); second, the isolated SFB showed a significantly reduced surface density for Thy-1 (MFI 19 versus 152 before isolation; n = 4, P ≤ 0.05), presumably due to contact with the mAb used for isolation; third, the magnetobeads remained attached to the positively isolated SFB for as long as 1 week, thus requiring further manipulation for their removal (eg with anti-idiotypic mAbs); fourth, a substantial proportion of SFB (approximately 30%) was lost into the negative fraction; and, finally, a Thy-1-dependent isolation technique bore the risk of selecting for SFB subpopulations, since the prolyl-4-hydroxylase+ cells obtained by negative isolation with Dynabeads® M-450 CD14 contained both a Thy-1+ fraction (RA, 49.3± 14.9%, n = 6; OA, 43.6 ± 12.0%, n = 4) and a Thy-1– fraction, as demonstrated by double-staining FACS analysis.\n"}