PMC:17827 / 47160-59988 JSONTXT

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isolation from primary culture\nNegative isolation of SFB from primary cultures of RASM using magnetobead-coupled anti-CD14 mAbs proved successful, providing satisfactory numbers of cells and avoiding repeated passaging and numerous cumulative population doublings. With culture times as short as 7 days, fresh SFB express surface and intracellular antigens typical of the FB lineage and/or SFB features as reported at a tissue level (Fig. 6 and Table 4) [1,2,12,13,14,15, 21,22,23,24,25,28]. This approach may also reduce the risk of in vitro growth selection. In the case of conventionally isolated RA-SFB, for example, the higher the number of passages, the higher the percentage of cells with trisomy 7 [33]. Some SFB functions (eg cell density at saturation and expression of transcription factors), in contrast, decrease at later passages [5,32,48].\nWhile the use of enzymes for the initial tissue digestion represents at least a transient stress for the cells, this method may also represent an alternative to tissue-outgrowth techniques, known to select for premitotic FB [26]. This is important since the relative contribution of pre-mitotic or postmitotic FB to the pathogenetic features of RA is presently unclear.\n\nAdvantages of negative isolation\nNegative isolation with magnetobead-coupled anti-CD14 mAbs shows the following advantages. There is minimal contamination with macrophages (\u003c2%) and other inflammatory cells (all \u003c1%). Thus, although CD14 may be expressed to a lower degree than CD68 on RA synovial lining macrophages [36,39], the degree of CD14-positivity appears more than sufficient to conduct the isolation procedure. The positive fraction, in turn, contains virtually no SFB, indicating minimal cell loss, and thereby also excluding major subset selection. Another advantage is that negative isolation with Dynabeads® M-450 CD14 avoids direct contact of SFB with mAbs and/or magnetobeads, thereby reducing possible functional alterations of the cells. Finally, the anti-Thy-1 mAb AS02 (used in the parallel attempt to positively isolate SFB) identifies 91% of normal skin-FB, but not more than 74% of RA-SFB (Tables 1 and 4). In addition, the prolyl-4-hydroxylase+ cells obtained by negative isolation with Dynabeads® M-450 CD14 contain both a Thy-1+ and a Thy-1– fraction. Thy-1-independent isolation thus circumvents the danger of selecting for Thy-1+ SFB subpopulations, especially relevant since Thy-1+ and Thy-1- FB diverge in several characteristics potentially relevant to RA; for example, cytokine and matrix production, as well as antigen presentation [49] (reviewed in [50,51,52]).\nThe negative isolation technique, however, also bears possible disadvantages: 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 fresh synovial specimens. On the other hand, 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.\n\nAdvantages of limited-passage isolation\nLimited-passage isolation shows the following advantages. It avoids phenotypic and functional in vitro alterations due to repeated passaging; indeed, even a low number of passages increases the proliferation rates in response to cytokine stimulation (Fig. 8). Passaging also alters 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 cells positive for procollagen I and III, and the proto-oncogenes c-Fos and Jun-D, on the other (Figs. 7 and 8, and Table 5). These results are relevant especially in view of the question whether proliferation and some phenotypic features of RA-SFB (in particular, the expression of proto-oncogenes) can be assimilated to those of dysregulated growth [1,2]. As these features seem to depend on the length of in vitro culture, caution should be applied to the interpretation of data from passaged cells [1,2,12,13,14,15, 23,24,25]. Another advantage is that this isolation procedure reduces the time of exposure to surviving macrophages to 7 days (in contrast to the weeks typical of conventional passaging), thus limiting long-term in vitro changes resulting from monokine secretion or cell-cell contact between SFB and synovial macrophages. Limited-passage isolation may therefore allow one to differentiate between FB-inherent changes (eg growth selection and cell senescence) and the changes mediated by the degree or length of contact with surviving macrophages during the initial passages. A high percentage of RA-SFB (and, to a lesser degree, also OA-SFB) from limited-passage isolation express MHC-II molecules without in vitro stimulation with cytokines (Table 4), which in conventionally isolated cells requires stimulation with interferon-gamma (used not only to explore the antigen-presentation potential of RA-SFB, but also to inhibit collagen biosynthesis [19,31,53]). In this aspect, the present in vitro system may be very useful in studies on the interrelationship of SFB with T cells or macrophages, in which the influence of exogenous mediators complicates the design and/or interpretation of the experiments.\n\nSimilarities/discrepancies between the in situ and in vitro phenotype of RA-SFB\n\nSimilarities\nThe cells obtained by negative isolation generally showed features similar to those reported in in situ analyses. The expression of Thy-1, CD13 (aminopeptidaseN), and vimentin exemplify this. The expression of these molecules, particularly their local upregulation by cytokines [54,55] and/or heterogeneous expression at different anatomical sites [9,49], may be pathogenetically relevant, either in terms of defining functionally heterogeneous SFB subpopulations [49] (reviewed in [50,51,52]), by providing pro-inflammatory enzymes [17,47,56,57,58,59], or as targets of autoimmune reactions [60].\nMHC-II. The constitutive MHC-II expression in isolated RA-SFB and OA-SFB, confirming previous in situ observations in the RA and OA SM [13,21,22,28] and in systemic scleroderma [61], is probably related to the inflammatory microenvironment, since normal skin-FB (Table 4) and normal SFB [31] hardly express MHC-II. This is further supported by the a strong decrease of the percentage of MHC-II+ cells upon repeated passage (both conventional and following negative isolation), possibly due to lack of or a progressive decrease of external stimulation with pro-inflammatory mediators (Fig. 7 and Table 5). Notably, there was a significant, negative correlation between the percentage of MHC-II-positive SFB and treatment of the RA patients with Methotrexate (ρ=-0.866, P = 0.01, n = 7), indicating that effective antirheumatic therapy may be reflected by decreased MHC-II expression on RA-SFB [28].\nProcollagen III. The significant increase of the MFI for intracellular procollagen III in RA-SFB and OA-SFB (Table 4) suggests that the pattern of SFB activation may be functional (among other things) to the increase of collagen metabolism; this is supported by the increased expression of collagen α2I and α1III mRNA in the RASM in situ [2,23,24], in comparison with normal or non-RA synovial tissue. Also, in as much as procollagen III is the fetal form of collagen used in wound healing and tissue repair (reviewed in [62,63]), ongoing fibrosis may be a considerable component of the disease process in RA (and OA), in analogy to systemic scleroderma [61] or interstitial kidney fibrosis [11,26]. A striking increase of procollagen I and III expression in RA-SFB from primary culture to fourth passage (increase of positive cells by ≥ 38%; Fig. 7 and Table 5), on the other hand, indicates that primary-culture cells do not exploit their full potential of matrix production.\n\nDiscrepancies\nThe expression of VCAM-1 [1,15,64,65] and Jun and Fos proto-oncogenes [23,25,66,67] showed some in situ / in vitro discrepancies.\nVCAM-1. Surprisingly, only a moderate and variable percentage of RA-SFB (whether conventionally passaged or negatively isolated from primary culture) expressed the adhesion molecule VCAM-1 (Tables 4 and 5). There were also no significant differences between RA-SFB and OA-SFB or normal skin-FB (Table 4). This is in apparent contrast to the enhanced expression of VCAM-1 reported in the lining layer of RA and OA synovial tissue [64,65]; however, it is well compatible with the large variability in VCAM-1 expression observed in vitro (Table 4) [1,15,68] and in situ [1,15]. The significant, positive correlation between VCAM-1 expression in isolated primary-culture RA-SFB and the erythrocyte sedimentation rate in RA patients (ρ=1.00, P = 0.000, n = 4) indicates that the variability may depend on disease activity, as also suggested in recent reports [68,69,70].\nProto-oncogene expression. Negatively isolated RA-SFB expressed c-Fos, c-Jun, and Jun-D (Table 4), in analogy to the features of SFB in RASM [23,25,66]. The expression of c-Jun and Jun-D in the present study could be unequivocally demonstrated by FACS analysis (Table 4), while previous immunohistochemical studies had failed to detect these molecules in situ [23]. Notably, however, the degree of proto-oncogene expression (both percentage of positive cells and MFI) did not significantly differ from that of normal skin-FB (Fig. 6 and Table 4) or, as previously reported, of SFB from traumatic joint injury [67]. This finding supports in situ observations that, at a single-cell level, the degree of proto-oncogene expression in RA, OA, and joint trauma is similar [23], thereby questioning whether proto-oncogene expression in RA reflects per se cell transformation and/or severe metabolic abnormalities.\nNotably, the expression of the proto-oncogenes c-Fos and Jun-D was strikingly increased in conventional fourth-passage RA-SFB as compared with isolated primary-culture RA-SFB (increase of positive cells ≥ 38%; Fig. 7 and Table 5). This increase was limited to RA, since the percentage of cells positive for these molecules was significantly decreased in OA-SFB and numerically decreased in normal skin-FB upon passaging. As a consequence of these reciprocal changes, the percentage of positive cells and/or MFI for c-Fos and Jun-D in conventional fourth-passage RA-SFB was significantly higher than in OA-SFB, under these circumstances confirming previously published data [25].\nFinally, a significant, positive correlation between the percentage of c-fos+ and procollagen I and III+ isolated primary-culture SFB in both RA and OA patients (RA: c-fos/procollagen I, ρ=1.000, P = 0.00, n = 7; c-fos/procollagen III, ρ=0.964, P = 0.00, n = 7) suggests a link between c-fos expression and augmented matrix production by SFB in rheumatic disorders, in line with the known regulation of collagen expression by the activator protein-1 transcription factor (reviewed in [13]).\n\nSuitability of fibroblast markers for unequivocal identification of SFB\nNone of the markers used to recognize SFB [9,23,24,62] proved to be universal (Table 4) or stable upon passaging (Fig. 7 and Table 5). Cellular uridine diphosphoglucose dehydrogenase activity, a useful histochemical marker for intimal SFB [37], while clearly detectable by immunohistochemistry in the lining layer of RASM tissue sections, was also only positive in a small subpopulation of cultured SFB (90% negative, 9% weakly positive, 1% strongly positive). Thus, despite its parallel recognition of endothelial cells [62], prolyl-4-hydroxylase at present remains the most suitable FB marker (see Tables 1,4 and 5).\nThe variable degree of positivity for different markers observed in the present study supports the existence of different SFB subpopulations or maturational stages (Tables 4 and 5, and Fig. 6E,H,I) [1,15]. By providing isolated SFB that do not undergo major subset selection, the present technique may allow, on one hand, the further characterization of these subpopulations and, on the other, the identification of more universal and/or less regulated pan-FB markers.\nIn summary, the present technique allows the isolation of highly enriched SFB with very limited in vitro culture time. These cells clearly show different phenotypic and functional properties in comparison with conventional fourth-passage RA-SFB. In conjunction with simultaneously derived synovial macrophages from the same patient (eg the positive fraction of the CD14 negative isolation), normally lost upon repeated passage due to their inability to divide in vitro (reviewed in [1]), and non-adherent, viable cells from the supernatant of the primary culture, recreation of a simplified 'rheumatoid' system can be attempted to investigate the relative contribution of the different cells to joint pathology [18,71].\n"}

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isolation from primary culture\nNegative isolation of SFB from primary cultures of RASM using magnetobead-coupled anti-CD14 mAbs proved successful, providing satisfactory numbers of cells and avoiding repeated passaging and numerous cumulative population doublings. With culture times as short as 7 days, fresh SFB express surface and intracellular antigens typical of the FB lineage and/or SFB features as reported at a tissue level (Fig. 6 and Table 4) [1,2,12,13,14,15, 21,22,23,24,25,28]. This approach may also reduce the risk of in vitro growth selection. In the case of conventionally isolated RA-SFB, for example, the higher the number of passages, the higher the percentage of cells with trisomy 7 [33]. Some SFB functions (eg cell density at saturation and expression of transcription factors), in contrast, decrease at later passages [5,32,48].\nWhile the use of enzymes for the initial tissue digestion represents at least a transient stress for the cells, this method may also represent an alternative to tissue-outgrowth techniques, known to select for premitotic FB [26]. This is important since the relative contribution of pre-mitotic or postmitotic FB to the pathogenetic features of RA is presently unclear.\n\nAdvantages of negative isolation\nNegative isolation with magnetobead-coupled anti-CD14 mAbs shows the following advantages. There is minimal contamination with macrophages (\u003c2%) and other inflammatory cells (all \u003c1%). Thus, although CD14 may be expressed to a lower degree than CD68 on RA synovial lining macrophages [36,39], the degree of CD14-positivity appears more than sufficient to conduct the isolation procedure. The positive fraction, in turn, contains virtually no SFB, indicating minimal cell loss, and thereby also excluding major subset selection. Another advantage is that negative isolation with Dynabeads® M-450 CD14 avoids direct contact of SFB with mAbs and/or magnetobeads, thereby reducing possible functional alterations of the cells. Finally, the anti-Thy-1 mAb AS02 (used in the parallel attempt to positively isolate SFB) identifies 91% of normal skin-FB, but not more than 74% of RA-SFB (Tables 1 and 4). In addition, the prolyl-4-hydroxylase+ cells obtained by negative isolation with Dynabeads® M-450 CD14 contain both a Thy-1+ and a Thy-1– fraction. Thy-1-independent isolation thus circumvents the danger of selecting for Thy-1+ SFB subpopulations, especially relevant since Thy-1+ and Thy-1- FB diverge in several characteristics potentially relevant to RA; for example, cytokine and matrix production, as well as antigen presentation [49] (reviewed in [50,51,52]).\nThe negative isolation technique, however, also bears possible disadvantages: 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 fresh synovial specimens. On the other hand, 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.\n\nAdvantages of limited-passage isolation\nLimited-passage isolation shows the following advantages. It avoids phenotypic and functional in vitro alterations due to repeated passaging; indeed, even a low number of passages increases the proliferation rates in response to cytokine stimulation (Fig. 8). Passaging also alters 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 cells positive for procollagen I and III, and the proto-oncogenes c-Fos and Jun-D, on the other (Figs. 7 and 8, and Table 5). These results are relevant especially in view of the question whether proliferation and some phenotypic features of RA-SFB (in particular, the expression of proto-oncogenes) can be assimilated to those of dysregulated growth [1,2]. As these features seem to depend on the length of in vitro culture, caution should be applied to the interpretation of data from passaged cells [1,2,12,13,14,15, 23,24,25]. Another advantage is that this isolation procedure reduces the time of exposure to surviving macrophages to 7 days (in contrast to the weeks typical of conventional passaging), thus limiting long-term in vitro changes resulting from monokine secretion or cell-cell contact between SFB and synovial macrophages. Limited-passage isolation may therefore allow one to differentiate between FB-inherent changes (eg growth selection and cell senescence) and the changes mediated by the degree or length of contact with surviving macrophages during the initial passages. A high percentage of RA-SFB (and, to a lesser degree, also OA-SFB) from limited-passage isolation express MHC-II molecules without in vitro stimulation with cytokines (Table 4), which in conventionally isolated cells requires stimulation with interferon-gamma (used not only to explore the antigen-presentation potential of RA-SFB, but also to inhibit collagen biosynthesis [19,31,53]). In this aspect, the present in vitro system may be very useful in studies on the interrelationship of SFB with T cells or macrophages, in which the influence of exogenous mediators complicates the design and/or interpretation of the experiments.\n\nSimilarities/discrepancies between the in situ and in vitro phenotype of RA-SFB\n\nSimilarities\nThe cells obtained by negative isolation generally showed features similar to those reported in in situ analyses. The expression of Thy-1, CD13 (aminopeptidaseN), and vimentin exemplify this. The expression of these molecules, particularly their local upregulation by cytokines [54,55] and/or heterogeneous expression at different anatomical sites [9,49], may be pathogenetically relevant, either in terms of defining functionally heterogeneous SFB subpopulations [49] (reviewed in [50,51,52]), by providing pro-inflammatory enzymes [17,47,56,57,58,59], or as targets of autoimmune reactions [60].\nMHC-II. The constitutive MHC-II expression in isolated RA-SFB and OA-SFB, confirming previous in situ observations in the RA and OA SM [13,21,22,28] and in systemic scleroderma [61], is probably related to the inflammatory microenvironment, since normal skin-FB (Table 4) and normal SFB [31] hardly express MHC-II. This is further supported by the a strong decrease of the percentage of MHC-II+ cells upon repeated passage (both conventional and following negative isolation), possibly due to lack of or a progressive decrease of external stimulation with pro-inflammatory mediators (Fig. 7 and Table 5). Notably, there was a significant, negative correlation between the percentage of MHC-II-positive SFB and treatment of the RA patients with Methotrexate (ρ=-0.866, P = 0.01, n = 7), indicating that effective antirheumatic therapy may be reflected by decreased MHC-II expression on RA-SFB [28].\nProcollagen III. The significant increase of the MFI for intracellular procollagen III in RA-SFB and OA-SFB (Table 4) suggests that the pattern of SFB activation may be functional (among other things) to the increase of collagen metabolism; this is supported by the increased expression of collagen α2I and α1III mRNA in the RASM in situ [2,23,24], in comparison with normal or non-RA synovial tissue. Also, in as much as procollagen III is the fetal form of collagen used in wound healing and tissue repair (reviewed in [62,63]), ongoing fibrosis may be a considerable component of the disease process in RA (and OA), in analogy to systemic scleroderma [61] or interstitial kidney fibrosis [11,26]. A striking increase of procollagen I and III expression in RA-SFB from primary culture to fourth passage (increase of positive cells by ≥ 38%; Fig. 7 and Table 5), on the other hand, indicates that primary-culture cells do not exploit their full potential of matrix production.\n\nDiscrepancies\nThe expression of VCAM-1 [1,15,64,65] and Jun and Fos proto-oncogenes [23,25,66,67] showed some in situ / in vitro discrepancies.\nVCAM-1. Surprisingly, only a moderate and variable percentage of RA-SFB (whether conventionally passaged or negatively isolated from primary culture) expressed the adhesion molecule VCAM-1 (Tables 4 and 5). There were also no significant differences between RA-SFB and OA-SFB or normal skin-FB (Table 4). This is in apparent contrast to the enhanced expression of VCAM-1 reported in the lining layer of RA and OA synovial tissue [64,65]; however, it is well compatible with the large variability in VCAM-1 expression observed in vitro (Table 4) [1,15,68] and in situ [1,15]. The significant, positive correlation between VCAM-1 expression in isolated primary-culture RA-SFB and the erythrocyte sedimentation rate in RA patients (ρ=1.00, P = 0.000, n = 4) indicates that the variability may depend on disease activity, as also suggested in recent reports [68,69,70].\nProto-oncogene expression. Negatively isolated RA-SFB expressed c-Fos, c-Jun, and Jun-D (Table 4), in analogy to the features of SFB in RASM [23,25,66]. The expression of c-Jun and Jun-D in the present study could be unequivocally demonstrated by FACS analysis (Table 4), while previous immunohistochemical studies had failed to detect these molecules in situ [23]. Notably, however, the degree of proto-oncogene expression (both percentage of positive cells and MFI) did not significantly differ from that of normal skin-FB (Fig. 6 and Table 4) or, as previously reported, of SFB from traumatic joint injury [67]. This finding supports in situ observations that, at a single-cell level, the degree of proto-oncogene expression in RA, OA, and joint trauma is similar [23], thereby questioning whether proto-oncogene expression in RA reflects per se cell transformation and/or severe metabolic abnormalities.\nNotably, the expression of the proto-oncogenes c-Fos and Jun-D was strikingly increased in conventional fourth-passage RA-SFB as compared with isolated primary-culture RA-SFB (increase of positive cells ≥ 38%; Fig. 7 and Table 5). This increase was limited to RA, since the percentage of cells positive for these molecules was significantly decreased in OA-SFB and numerically decreased in normal skin-FB upon passaging. As a consequence of these reciprocal changes, the percentage of positive cells and/or MFI for c-Fos and Jun-D in conventional fourth-passage RA-SFB was significantly higher than in OA-SFB, under these circumstances confirming previously published data [25].\nFinally, a significant, positive correlation between the percentage of c-fos+ and procollagen I and III+ isolated primary-culture SFB in both RA and OA patients (RA: c-fos/procollagen I, ρ=1.000, P = 0.00, n = 7; c-fos/procollagen III, ρ=0.964, P = 0.00, n = 7) suggests a link between c-fos expression and augmented matrix production by SFB in rheumatic disorders, in line with the known regulation of collagen expression by the activator protein-1 transcription factor (reviewed in [13]).\n\nSuitability of fibroblast markers for unequivocal identification of SFB\nNone of the markers used to recognize SFB [9,23,24,62] proved to be universal (Table 4) or stable upon passaging (Fig. 7 and Table 5). Cellular uridine diphosphoglucose dehydrogenase activity, a useful histochemical marker for intimal SFB [37], while clearly detectable by immunohistochemistry in the lining layer of RASM tissue sections, was also only positive in a small subpopulation of cultured SFB (90% negative, 9% weakly positive, 1% strongly positive). Thus, despite its parallel recognition of endothelial cells [62], prolyl-4-hydroxylase at present remains the most suitable FB marker (see Tables 1,4 and 5).\nThe variable degree of positivity for different markers observed in the present study supports the existence of different SFB subpopulations or maturational stages (Tables 4 and 5, and Fig. 6E,H,I) [1,15]. By providing isolated SFB that do not undergo major subset selection, the present technique may allow, on one hand, the further characterization of these subpopulations and, on the other, the identification of more universal and/or less regulated pan-FB markers.\nIn summary, the present technique allows the isolation of highly enriched SFB with very limited in vitro culture time. These cells clearly show different phenotypic and functional properties in comparison with conventional fourth-passage RA-SFB. In conjunction with simultaneously derived synovial macrophages from the same patient (eg the positive fraction of the CD14 negative isolation), normally lost upon repeated passage due to their inability to divide in vitro (reviewed in [1]), and non-adherent, viable cells from the supernatant of the primary culture, recreation of a simplified 'rheumatoid' system can be attempted to investigate the relative contribution of the different cells to joint pathology [18,71].\n"}