RA is a heterogeneous autoimmune disease. However, these heterogeneous chronic diseases were recently able to be monitored in line with their gene expression patterns by microarray-based molecular studies [27
]. The histology of RA affected joints indicates chronic inflammation with hyperplasia in the synovial lining cells. It is now well established that FLSs actively participate in RA synovitis and that FLSs in RA joints aggressively proliferate to form a pannus, eventually destroying articular bone and cartilage [28
]. Several cytokines, such as IL-1β, TNF-α and IL-6, have been described in association with the proliferative response of FLSs. In trials of these therapeutic agents, however, responses were not achieved in a significant proportion of the patients, suggesting that some important factor(s) still remain to be discovered.
To our knowledge this report is the first demonstration that the MLN51 is essential for the hyperproliferation of RA FLSs in line with GM-CSF signaling in RA pathogenesis. Our results show that the SF-mediated growth of RA FLSs was markedly blocked by anti-GM-CSF neutralizing antibody, and additionally that growth-retarded RAFLSs recovered their proliferative capacity by the addition of GM-CSF. These results indicate that GM-CSF in SF is important in the hyperproliferation of RA FLSs. In contrast, in the microarray analysis, semiquantitative RT-PCR and Western blot analysis experiments, we found that the MLN51 was consistently overexpressed in the hyperactive RA FLSs at low passages or the RA FLSs cultured in the presence of SF. MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation capacity of RA FLSs, suggesting that the MLN51 gene is strongly involved in the pathogenesis of RA.
We extracted total RNA from RA FLSs and OA FLSs, which were labeled by cDNA synthesis and ultimately hybridized to a HI380 microarray containing 384 cDNA clones. The differential hybridization was performed with Cy-5-labeled RA cDNA and Cy-3-labeled OA cDNA probes. Through the microarray analysis, we found that MLN51, a novel gene in association with RA, was markedly upregulated among the many upregulated genes selected on the basis of their immunologic characteristics (Table ). MLN51 overexpression in RA FLSs was confirmed by RT-PCR analysis with three different RA FLS samples (Figure ) and by Western blot experiments with additional three different RA FLS samples (Figure ).
List of genes upregulated in rheumatoid arthritis/osteoarthritis microarray analysis (HI380)
Figure 1 MLN51 expression is upregulated in rheumatoid arthritis FLSs compared with osteoarthritis FLSs. (a) Total RNA sample (1 μg) was extracted from three rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) and one osteoarthritis (OA) FLS (more ...)
We next investigated whether SFs have an effect on the growth rate of RA FLSs, what kinds of factors in the SFs are involved in the proliferation of RA FLSs, and whether the factors have a role in the expression of MLN51. We determined the growth kinetics of FLSs at different passages and SF-treated RA FLSs. The RA FLSs at passage 11 showed obvious growth retardation (Figure , left panel); however, the same sample clearly recovered from its growth retardation when cultured in the presence of 10-fold-diluted SF (Figure , right panel). We next quantified inflammatory cytokine levels in SFs (Figure ). The results indicated that GM-CSF in all SFs from six patients with RA exists at nearly equal levels, in contrast with other inflammatory cytokines (such as IL-1β and TNF-α). We found that MLN51 expression in RA FLSs was upregulated in mRNA level (Figure , left panel) and protein level (Figure , left panel) by treatment of cultures not only with SFs but also with GM-CSF. The upregulation of MLN51 by GM-CSF treatment was also confirmed in six different RA FLS samples by RT-PCR (s-2, 2–6 and 2–14; Figure , right panel) and by Western blot analysis (2–18, 2–36, 2–38; Figure , right panel). Moreover, in both RA FLS samples (2–18 and 2–38), the MLN51 protein expression was enhanced by GM-CSF treatment in a dose-dependent manner (Figure ). These results strongly suggest that the growth rate recovery of RA FLSs by SF or GM-CSF is associated with the expression of MLN51.
Figure 2 The growth kinetics of RA FLSs at different passages or in SF-treated cultures. (a) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) at passages 3 (P#3), 5 (P#5) and 11 (P#11) were used to measure their growth kinetics. RA FLSs (2–14) (more ...)
Figure 3 Western blot analysis of hMLN51 in RA fibroblast-like synoviocytes (FLSs) treated with GM-CSF. Rheumatoid arthritis (RA) FLSs (2–18 and 2–38) isolated from the two patients with RA were seeded at 5 × 104 cells per well in a six-well (more ...)
There are many kinds of different cytokines and growth factors in the RA joint microenvironments. To identify factors having an effect on the growth of RA FLSs, we investigated the inflammatory cytokines and growth factors on the growth of RA FLSs (2–14) in vitro
. The results indicated that GM-CSF and TNF-α may have an effect on the growth rate recovery of the high-passage-number RA FLSs. GM-CSF and TNF-α treatment resulted on approximately 2.0-fold and 1.3-fold increases in the proliferation of RA FLSs, respectively, compared with that of untreated controls (Figure ). These results support the notion that resident joint cells (chondrocytes and synovial fibroblasts) produce GM-CSF in culture in response to TNF-α and IL-1β [30
]. However, our result showed that IL-1β did not induce active proliferation of RA FLSs, indicating that IL-1β may not be a key factor in active RA FLS proliferation. In contrast, the growth rate recovery of the high-passage-number RA FLSs was achieved in vitro
when the cells were treated with 100 ng/ml GM-CSF (a significant difference from the control cells), although GM-CSF concentrations measured in SFs of patients with RA were a maximum of 400 pg/ml and SF treatment induced active proliferation of RA FLSs. This suggested that the combinations of various proinflammatory cytokines or other factors together with GM-CSF in the SF may be involved in RA pathogenesis in vivo
. To address the effects of GM-CSF in SF on the growth of RA FLSs, we cultured the RA FLSs in culture media containing SF and anti-GM-CSF mAb or a recombinant GM-CSF. Incubation of the two different RA FLSs with SFs containing anti-GM-CSF mAb significantly impaired the SF-mediated proliferation efficacy of FLSs (Figure ). These results suggest that the GM-CSF in SF has a key role in the hyperproliferation of RA FLSs, further supporting the results above. The cell viability for all cultures (data not shown) was 98 to 99%. The recovery of the growth-retarded RA FLS proliferation capacity was obviously improved by GM-CSF in a dose-dependent manner.
Figure 4 Effects of GM-CSF and cytokines on the growth of high-passage-number RA FLSs. (a) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) 2–14 (at passage 11) were seeded at 1.5 × 104 cells per well in triplicate in a 24-well plate. (more ...)
We cultured RA FLSs (2–14) in SF-containing medium in the presence of anti-GM-CSF, anti-GM-CSF plus anti-IL-1β or anti-GM-CSF plus anti-TNF-α mAbs to investigate the effects of IL-1β or TNF-α in SF on recovery of the growth of RA FLSs. As shown in Figure , cultures treated with both anti-GM-CSF and anti-TNF-α mAbs showed slightly more suppression than the cultures treated with anti-GM-CSF mAb alone, in terms of SF-mediated FLS proliferation. Our results in Figures and suggest that not only GM-CSF, but also some other proinflammatory cytokines such as TNF-α, are likely to be involved in the hyperproliferation of RA FLSs. However, anti-IL-1β mAb did not have a significant effect on the SF-mediated proliferation of RA FLSs. These cytokine effects on the FLS proliferation were similar to those on the MLN51 gene expression level (data not shown).
Figure 5 Inhibitory effects of neutralizing antibodies to cytokines on the SF-mediated proliferation capacity of RA FLSs. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs; 2–14) at passage 12, at a concentration of 5 × 103 cells per (more ...)
To examine a specific requirement for the MLN51 gene in cell proliferation, siRNA prepared from the 5' region of human MLN51 cDNA was introduced into passage 5 of RA FLSs (2–14). The growth kinetics of the transfected RA FLSs was monitored for 5 days (Figure ) and the level of the corresponding MLN51 mRNA was measured by semiquantitative RT-PCR (Figure ). As shown in Figure , treatment of the FLSs with hMLN51-siRNA caused complete abrogation of RA FLS proliferation, whereas treatment with control siRNA was without effect. These results strongly suggest that the MLN51 gene has a crucial role in the hyperproliferation of RA FLSs.
Figure 6 Effects of MLN51-knock-down on the growth of RA FLSs. (a) Fibroblast-like synoviocytes (FLSs; 104 cells; rheumatoid arthritis (RA) 2–14, at passage 5) were transfected with 4 μg of hMLN51 siRNA or control siRNA. The transfected cells were (more ...)
We next generated the BmDCs from the DBA/1J mouse, which is a frequently used animal model for arthritis. It is known that the DBA/1 mouse strain has a H-2q
haplotype and readily develops arthritis after immunization with heterologous or autologous type II collagen of rat, bovine or chick CII origin [32
]. In addition, DCs are particularly relevant in the pathogenesis of most inflammatory arthropathies because of their potent antigen-presenting capacity and their unique ability to activate naïve T cells [33
]. In addition, DC populations have been described in line with synovitis in RA, although a functional contribution to the disease remains difficult to assess [36
]. The immature BmDCs were generated from bone marrow progenitors by culturing the progenitors in the presence of GM-CSF alone. Immature BmDCs were matured with lipopolysaccharide and anti-CD40. We then performed semiquantitative RT-PCR of mouse MLN51
gene expression with the aim of confirming the differences observed in cDNA microarray analysis. As shown in Figure , the MLN51
gene was highly expressed only in the immature BmDCs and barely detected in the bone marrow progenitor or mature BmDCs. These results suggest that the expression of MLN51
is associated with the GM-CSF treatment. We hypothesized that the MLN51
gene might have one or more important roles in immature DCs in line with their specific biological functions or with some aspects of cell viability. We investigated a function of MLN51
on the growth of DCs by using BC-1 cells (an immature DC cell line). BC-1 cells transfected with MLN51
siRNA were harvested daily, and cell proliferation and MLN51
mRNA expression were measured by RT-PCR. As shown in RA-FLSs (Figure ), the transfection of MLN51
-specific siRNA abrogated the proliferation of BC-1 cells (Figure ) resulting from the MLN51
knock-down (Figure ). These results indicate that the MLN51
gene, identified in breast cancers, is important in the proliferation of not only FLSs but also established DC cell lines.
Figure 7 MLN51 expression in dendritic cells (DCs) and its effect on the proliferation of immature DCs. (a) MLN51 expression is upregulated only in immature bone marrow-derived dendritic cells (BmDCs) in contrast with bone marrow progenitor cells or mature BmDCs. (more ...)
In summary, our results strongly suggest that the MLN51 gene, whose expression depends upon GM-CSF signaling, may have a crucial role in the hyperproliferation of FLSs in the pathogenesis of RA.