The present study demonstrates considerable alterations in the cytokine expression of repeated-passage RA-SFB upon extended in vitro culture, as shown by significantly lower protein concentrations for IL-6 and TNF-α and non-significant changes in the mRNA levels for almost all cytokines. These findings are important because most studies have been performed with fibroblasts at higher passage (3–10), which are prone to similar alteration in vitro. They also point to the suitability/usefulness of isolated early-passage cells for studies on the features/functional characteristics of SFB in RA.
Cytokine expression in RA-SFB
IL-6, a pleiotropic cytokine produced predominantly by synovial macrophages and fibroblasts, plays a major role in cellular activation. The detection of high levels of IL-6 mRNA/protein (see Fig. 2 and Table 4) agrees with previous observations in passaged SFB/synoviocytes [24–28]. In those studies, however, stimulation of cells with IL-1β or TNF-α was necessary to induce the production of 80–90 ng/ml of IL-6 . In the present study, in contrast, first-passage RA-SFB constitutively secreted IL-6. This indicates a high intrinsic activation status of the cells, i.e. retention of in vivo features present before removal from the synovial membrane . A clearly decreased IL-6 production in repeated-passage RA-SFB (see Table 4) may represent culture alterations due to the lack of cytokine stimulation.
IL-10 is produced by T cells, B cells, mast cells, and activated monocytes or macrophages. Its primary function appears to limit and terminate inflammatory responses (including those in experimental arthritis). In contrast to recent suggestions , the capability of RA-SFB to produce IL-10 remains uncertain. First, synovial macrophages, rather than SFB, are known to produce considerable amounts of IL-10 [31, reviewed in 32]. Second, the low amounts of IL-10 detected in primary-culture RA-SFB in the present study are probably the product of the few remaining macrophages (see Table 3). This hypothesis is supported by the lack of IL-10 protein/mRNA detected in most repeated-passage RA-SFB (only one positive sample each), which lack contaminating macrophages.
Similarly, the proinflammatory cytokine TNF-α is predominantly produced by synovial macrophages [25,33,34], and not by SFB [35–37]. In contrast to IL-10, however, TNF-α protein or mRNA were also detected in repeated-passage RA-SFB (lacking contaminating macrophages). TNF-α may therefore be a genuine product of RA-SFB, allowing these cells to contribute to synovial inflammation. Its significantly decreased production in repeated-passage RA-SFB may reflect either loss of macrophages and/or in vitro alterations of SFB due to withdrawal of cytokine stimulation.
In this study, no IL-1β protein (and only minimal amounts of IL-1β mRNA) was detected in primary-culture and repeated-passage RA-SFB. This result is in contrast to reports showing IL-1β mRNA or protein in nonstimulated RA-SFB [24,38]; however, it confirms findings that showed expression of IL-1β mRNA but lack of secretion of IL-1β protein by RA-SFB . As for TNF-α, the detection of IL-1β mRNA in repeated-passage RA-SFB suggests some IL-1β transcription.
IL-13 is an anti-inflammatory cytokine expressed in a subset of activated T cells. It downregulates macrophage activity and thereby reduces the production of proinflam-matory cytokines. IL-13 mRNA was not detectable in any of the FB preparations assayed, in good agreement with the lack of IL-13 protein production by passaged RA-SFB, whether nonstimulated or after stimulation with IL-1β, TNF-α, or lipopolysaccharide . Therefore, it appears that RA-SFB do not contribute to the local production of this potent anti-inflammatory cytokine [11,40].
IL-15, an IL-2-like cytokine produced by monocytes/ macrophages, dendritic cells, fibroblasts, and bone-marrow stroma cells, activates cell proliferation, cytotoxity, and cytokine production in natural killer cells and is a potent chemoattractant for T cells. Low levels of IL-15 mRNA were detected in all FB preparations; however, IL-15 protein was absent in the supernatants of first-passage or repeated-passage RA-SFB (see Fig. 2, Table 4). This finding is in contrast to reports showing IL-15 mRNA and protein in repeated-passage SFB cell lines (approximately 50 pg/ml; ). However, a lower expression of IL-15 mRNA in SFB cell lines in comparison with freshly isolated synovial tissue cells (containing synovial macrophages, the predominant producers of IL-15 [12,15]) strongly indicates that RA-SFB produce very little, if any, IL-15 .
IL-18 is produced by PBMC, dendritic cells, and activated endothelial cells and has pleiotropic effects on T cell functions. The failure to detect IL-18 mRNA or protein in non-stimulated, primary-culture, or repeated-passage RA-SFB confirms previous reports in nonstimulated, passaged RA-SFB lines . The capability of FB to express IL-18 mRNA  was confirmed by its detection in nonstimulated (see Fig. 2) and TNF-α/IL-1β-stimulated primary-culture normal-skin FB (dotted line in Fig. 2). However, we cannot exclude the possibility that contaminating endothe-lial cells were a source of IL-18 mRNA in these cultures .
Interestingly, the cytokines IL-1β, IL-6, IL-15, and TNF-α in RA-SFB showed a dissociated pattern of mRNA and protein expression, suggesting post-transcriptional or post-translational regulation. Such regulation (at the level of mRNA stability, translational efficacy, stability of the protein product or other post-translational modifications) has been demonstrated for IL-1β [41–43], IL-6 [44,45], IL-15 , and TNF-α . Its importance for RA-SFB functions remains to be analyzed.