PMC:17827 / 1058-12604 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/17827","sourcedb":"PMC","sourceid":"17827","source_url":"http://www.ncbi.nlm.nih.gov/pmc/17827","text":"Synopsis\n\nIntroduction:\nActivated SFB in the invasive pannus tissue appear to play a major role in the crippling destruction of cartilage and bone in the joints of patients with RA [1,2,3]. Several studies indicate that RA-SFB are morphologically altered [1,4], grow in an anchorage-independent fashion [5], and strongly express markers of activation, including major histocompatibility complex (MHC)-II and other surface molecules, proto-oncogenes, or matrix degrading enzymes (reviewed in [2]). Finally, activated RA-SFB elicit erosive arthritis upon intra-articular injection or engraftment into severe combined immunodeficiency mice [6,7].\n\nAims:\nTo investigate ex vivo the pathophysiological properties of activated FB from the RA synovial membrane (SM), it is desirable to obtain freshly isolated SFB with phenotypic features as close as possible to the configuration observed in vivo. Because the generation of cell lines requires a number of passages in vitro to eliminate contaminating cells (especially macrophages), the risk of substantial in vitro alteration/growth selection exists.\nWe attempted to isolate FB from primary cultures of synovial cells, using a negative separation technique to eliminate macrophages, to minimize these artifacts.\n\nMethods:\nSynovial tissue was obtained from a total of 16 patients fulfilling the American Rheumatism Association criteria for RA [8] and 21 patients with osteoarthritis (OA) under approval of the local Ethics Committees. The tissue was placed in cell culture medium at ambient temperature and subjected to tissue digestion within 2 h. Synovectomy samples of RA and OA SM were finely minced, digested for 30 min at 37°C in phosphate-buffered saline (PBS) containing 0.1% trypsin (Sigma, Deisenhofen, Germany), and thereafter digested in 0.1% collagenase P (Boehringer Mannheim, Mannheim, Germany) in Dulbecco's modified Eagle medium (DMEM)/10% fetal calf serum (FCS) for 2 h at 37°C, 5% CO2. The cell suspension was then filtered and the cells collected by centrifugation. Cells were kept in primary culture for 7 days (DMEM/10% FCS, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B [Gibco BRL, Eggenstein, Germany], including removal of non-adherent cells on days 1, 3, 5, and 7) and subsequently used for SFB isolation. The samples were randomly tested to exclude Mycoplasma contamination. For negative isolation of SFB from primary culture, adherent synovial cells were detached by short-term trypsinization for 2 min (0.25% trypsin/0.2% EDTA; Gibco) and 107/ml synovial cells were incubated with 4 × 107/ml Dynabeads® M-450 CD14 (clone RMO52; Dynal, Hamburg, Germany) in PBS/2% FCS for 1 h at 4°C. Nine milliliters of PBS/2% FCS were then added and the conjugated cells collected using the Dynal magnetic particle concentrator®. The compositions of magnetobead-conjugated cells and unconjugated cells were analyzed by flow cytometry. Phenotype analysis of the expression of FB markers, as well as that of SFB features previously reported at a tissue level, was conducted by flow cytometry in RA-SFB, either negatively isolated from primary culture or obtained from conventional fourth passage. The findings were compared with those of normal skin-FB (lineage control) and OA-SFB (disease control). The proliferation of RA-SFB, either negatively isolated from primary culture or obtained from conventional fourth passage, was assayed by [3H]-thymidine incorporation.\n\nResults:\nThe primary culture of RA synovial cells resulting from trypsin/collagenase digestion of the RASM contained large, spindle-shaped Thy-1+ SFB (CD90+; Fig. 1C) (monoclonal antibody [mAb] AS02; Dianova, Hamburg, Germany) and small, round CD14+ cells, most probably macrophages (Fig. 1D) (mAb Tyk4; Dako, Hamburg, Germany), 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; Immunotech, Hamburg, Germany) and CD144 (Fig. 1G) (mAb Cadherin 5; Immunotech), which clearly identified human umbilical vein endothelial cells (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; Prof. Schuppan, Berlin, Germany). An average of 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a) in flow cytometry (FACS) [10]; the average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a). There were \u003c1% T cells (CD3+) (mAb UCHT-1; ATCC, Manassas, VA, USA), B cells (CD19+/20+) (mAbs HD 37 and B-Ly 1; Dako), plasma cells (CD38+) (mAb AT 13/5; Dako), natural killer (NK) cells (CD56+) (mAb NKH/1; Immunotech), dendritic cells (CD83+) (mAb HB 15a; Immunotech), endothelial cells (CD144+), or PMN (CD15+) (mAb80H5; Immunotech), indicating that non-adherent cells had been efficiently removed during primary culture. The total yield of cells following 7 days of primary culture averaged (5.2 ± 1.1) × 107 cells (mean ± SEM; n = 7).\nNegative 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 (\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 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.\nProliferation assays showed that, at rest, the proliferation rates of isolated first-passage RA-SFB did not significantly differ from those of the corresponding cells in conventional fourth passage. Upon stimulation with platelet-derived growth factor (PDGF) (2.5 and 5 U/ml) and IL-1β (150 U/ml), however, the mean proliferation rates of conventional fourth-passage RA-SFB were significantly higher than those observed in first-passage cells (3.9-fold and 4.2-fold, respectively, in the case of 2.5 and 5 U/ml PDGF; 2.1-fold in the case of 150 U/ml IL-1β).\n\nDiscussion:\nThe advantages of negative isolation are as follows: first, there was minimal contamination with other cells, especially macrophages (\u003c2%); second, negative isolation avoided direct contact of SFB with mAbs and/or magnetobeads, thereby limiting possible functional alterations of the cells; and third, the FB marker mAb AS02 (anti-Thy-1; used in the parallel attempt to positively isolate SFB) identified 91% of primary-culture/first-passage normal skin-FB but only 74% of isolated primary RA-SFB, probably due the variable density of Thy-1 molecules on the cell surface (Table 1). Thus, Thy-1-independent isolation appears to avoid selection of Thy-1+ SFB subpopulations, a critical point since Thy-1+ and Thy-1- FB diverge in several characteristics potentially relevant to RA.\nPotential disadvantages of the negative isolation method include the limited number of cells obtained (approximately 2.8 × 107), due to the lack of in vitro expansion, and the necessity for reliable access to freshly obtained synovial specimens. However, the applicability of this procedure not only to joint replacement samples, but also to arthroscopic synovectomy samples, augments the potential sources of fresh synovial tissue. The use of enzymes for tissue digestion may also represent at least a transient stress for the cells. On the other hand, this method may avoid selection of premitotic FB, a problem usually linked to tissue-outgrowth techniques [11].\nThe advantages of limited-passage isolation include the following. The present technique avoids a high number of passages in vitro; indeed, even a low number of passages increased the proliferation rates in response to cytokine stimulation and altered the cellular phenotype of RA-SFB (as exemplified by a strong decrease of the percentage of MHC-II+ cells, on one hand, and a striking increase of the percentage of procollagen I and III+, as well as c-Fos and Jun-D+ cells, on the other). Caution should thus be applied to the interpretation of data from passaged cells, especially in the case of proto-oncogenes. A second advantage is that this isolation procedure reduces the time of exposure (7 days) to surviving macrophages (unlike the weeks of exposure upon conventional isolation), thus possibly minimizing long-term in vitro changes due to monokine secretion or cell–cell contact between SFB and synovial macrophages. Finally, a high percentage of RA-SFB from limited-passage isolation expressed MHC-II molecules (in analogy to findings in synovial tissue) without in vitro stimulation with cytokines, which in conventionally isolated cells requires stimulation with interferon-gamma. Therefore, the present in vitro system may be particularly useful in studies on the inter-relationship of SFB with T cells or macrophages, in which the influence of exogenous mediators may complicate the design and/or interpretation of the experiments.\nNegative isolation of cells from synovial primary culture with magnetobead-conjugated anti-CD14 mAbs thus yielded highly enriched RA-SFB. This technique, by avoiding repeated passaging and numerous cumulative population doublings, may minimize the risk of in vitro alteration/growth selection; in RA-SFB, this includes increased proliferation rates and changes of several phenotypic features.\n","divisions":[{"label":"Title","span":{"begin":0,"end":8}},{"label":"Section","span":{"begin":10,"end":643}},{"label":"Title","span":{"begin":10,"end":23}},{"label":"Section","span":{"begin":645,"end":1256}},{"label":"Title","span":{"begin":645,"end":650}},{"label":"Section","span":{"begin":1258,"end":3464}},{"label":"Title","span":{"begin":1258,"end":1266}},{"label":"Section","span":{"begin":3466,"end":8246}},{"label":"Title","span":{"begin":3466,"end":3474}},{"label":"Section","span":{"begin":8248,"end":11545}},{"label":"Title","span":{"begin":8248,"end":8259}}],"tracks":[{"project":"2_test","denotations":[{"id":"11178129-8912499-4386791","span":{"begin":182,"end":183},"obj":"8912499"},{"id":"11178129-7632096-4386792","span":{"begin":184,"end":185},"obj":"7632096"},{"id":"11178129-2109776-4386793","span":{"begin":186,"end":187},"obj":"2109776"},{"id":"11178129-8912499-4386794","span":{"begin":256,"end":257},"obj":"8912499"},{"id":"11178129-6851475-4386795","span":{"begin":258,"end":259},"obj":"6851475"},{"id":"11178129-2784799-4386796","span":{"begin":304,"end":305},"obj":"2784799"},{"id":"11178129-7632096-4386797","span":{"begin":492,"end":493},"obj":"7632096"},{"id":"11178129-8151561-4386798","span":{"begin":638,"end":639},"obj":"8151561"},{"id":"11178129-8909250-4386799","span":{"begin":640,"end":641},"obj":"8909250"},{"id":"11178129-3358796-4386800","span":{"begin":1388,"end":1389},"obj":"3358796"},{"id":"11178129-8151561-4386801","span":{"begin":3840,"end":3841},"obj":"8151561"},{"id":"11178129-8752676-4386802","span":{"begin":3842,"end":3843},"obj":"8752676"},{"id":"11178129-8849370-4386803","span":{"begin":4440,"end":4442},"obj":"8849370"},{"id":"11178129-2042649-4386804","span":{"begin":9701,"end":9703},"obj":"2042649"}],"attributes":[{"subj":"11178129-8912499-4386791","pred":"source","obj":"2_test"},{"subj":"11178129-7632096-4386792","pred":"source","obj":"2_test"},{"subj":"11178129-2109776-4386793","pred":"source","obj":"2_test"},{"subj":"11178129-8912499-4386794","pred":"source","obj":"2_test"},{"subj":"11178129-6851475-4386795","pred":"source","obj":"2_test"},{"subj":"11178129-2784799-4386796","pred":"source","obj":"2_test"},{"subj":"11178129-7632096-4386797","pred":"source","obj":"2_test"},{"subj":"11178129-8151561-4386798","pred":"source","obj":"2_test"},{"subj":"11178129-8909250-4386799","pred":"source","obj":"2_test"},{"subj":"11178129-3358796-4386800","pred":"source","obj":"2_test"},{"subj":"11178129-8151561-4386801","pred":"source","obj":"2_test"},{"subj":"11178129-8752676-4386802","pred":"source","obj":"2_test"},{"subj":"11178129-8849370-4386803","pred":"source","obj":"2_test"},{"subj":"11178129-2042649-4386804","pred":"source","obj":"2_test"}]},{"project":"Colil","denotations":[{"id":"T1","span":{"begin":638,"end":639},"obj":"8151561"},{"id":"T2","span":{"begin":640,"end":641},"obj":"8909250"},{"id":"T3","span":{"begin":492,"end":493},"obj":"7632096"},{"id":"T4","span":{"begin":182,"end":183},"obj":"8912499"},{"id":"T5","span":{"begin":184,"end":185},"obj":"7632096"},{"id":"T6","span":{"begin":186,"end":187},"obj":"2109776"},{"id":"T7","span":{"begin":638,"end":639},"obj":"8151561"},{"id":"T8","span":{"begin":640,"end":641},"obj":"8909250"},{"id":"T9","span":{"begin":256,"end":257},"obj":"8912499"},{"id":"T10","span":{"begin":258,"end":259},"obj":"6851475"},{"id":"T11","span":{"begin":304,"end":305},"obj":"2784799"},{"id":"T12","span":{"begin":1388,"end":1389},"obj":"3358796"},{"id":"T13","span":{"begin":9701,"end":9703},"obj":"2042649"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"attributes":[{"subj":"T1","pred":"source","obj":"Colil"},{"subj":"T2","pred":"source","obj":"Colil"},{"subj":"T3","pred":"source","obj":"Colil"},{"subj":"T4","pred":"source","obj":"Colil"},{"subj":"T5","pred":"source","obj":"Colil"},{"subj":"T6","pred":"source","obj":"Colil"},{"subj":"T7","pred":"source","obj":"Colil"},{"subj":"T8","pred":"source","obj":"Colil"},{"subj":"T9","pred":"source","obj":"Colil"},{"subj":"T10","pred":"source","obj":"Colil"},{"subj":"T11","pred":"source","obj":"Colil"},{"subj":"T12","pred":"source","obj":"Colil"},{"subj":"T13","pred":"source","obj":"Colil"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93aa","default":true},{"id":"Colil","color":"#93c4ec"}]}]}}