Disease progression of RA, which is characterized by villous proliferation of synoviocytes, mainly of synovial fibroblasts, results in bone and joint destruction. The spontaneous arrest of the proliferation of synovial tissue is of particular interest, strongly suggesting the involvement of apoptosis in this process. Villous proliferation involves both the proliferation and apoptosis of synovial lining cells, the latter being affected by various T-cell types, primarily cytotoxic T-lymphocytes (CTLs), which infiltrate the synovial lining cell layer. The activated T cells attack and damage the synovial lining cells, causing apoptosis and leading to the growth of new synovial cells to replace the damaged cells (38–40). The synovial lining layer samples used for the microarray analysis were dissected synovial tissues without joint erosion and bone tissue.
We used LMD followed by a cDNA microarray to analyse gene-expression profiles in the synovial lining tissues in RA. A total of 14519 genes from among more than 48000 transcripts met the normalization criteria for microarray analysis and were divided into an RA group and an OA group. After filtering with the SAM method, detailed expression profiles of the 197 genes statistically selected from among all the cases were obtained. Cluster analysis produced two distinct molecular RA subgroups (high RA and low RA), which were closely associated with the histological and clinical activity of RA. The two molecular subgroups showed different expression levels of genes crucial for proliferative inflammation. The expressions of CCL5, CXCL9/10, STAT1, and IRF1 were found to be up-regulated in RA. Because of the small quantity of total RNA obtained from the dissected sample, quantitative real-time transcription polymerase chain reaction (qRT-PCR) assays were performed using the same cRNA samples as for the microarray analysis. The qRT-PCR measurements were always very similar to the expression levels of the microarray (data not shown). In our study, the results of immunohistochemical analysis of the up-regulated molecules, that is CCL5, CXCL9/10, STAT1, and IRF1, matched the results of the analysis of mRNA expression levels.
The various cytokines and chemokines present in the RA synovium create a complex situation with simultaneous activation of multiple signalling pathways that may influence STAT1 signalling. As a result, the synovium becomes inflamed. Yarilina et al (8
) reported that TNF initiated a type I IFNβ-mediated autocrine loop and that expression of inflammation-related genes (CXCL9, CXCL10, CCL5, etc.) was sustained and amplified by the sequential induction of IRF1, IFNβ, and STAT1. TNF-mediated production of IFNβ and the ensuing autocrine regulation of gene expression was shown to depend on IRF1 and on synergy between the small amounts of IFNβ produced and additional TNF-induced signals, such as activation of NF-k
B. The model used by Yarilina et al, the TNF-activated IRF1-IFNβ-JAK-STAT signalling pathway, contributes to the proinflammatory functions of TNF and the macrophage responses to endogenous inflammatory factors such as TNF (8
). In our study, strong expression of CCL5, CXCL9/10, STAT1, and IRF1 was observed in the RA synovial lining cells at mRNA and protein levels. IFNβ could not be detected by the normalization criteria, but it is possible that its expression at the mRNA level is slight, which make it possible to maintain the autocrine loop. Several studies have reported that the IFNβ protein was observed in synovial tissue of RA rather than of OA and was detected in all compartments of the synovium, especially in fibroblast-like synoviocytes (FLS) of the intimal lining layer (41
). In our study, the expression of IFNβ in RA synovial tissue was detected by means of immunohistochemical staining (data not shown). As shown in , the network created by IPA suggested that the TNF-activated IRF1-IFN-STAT1 pathway is located in the synovial lining cells of RA. This finding in our study supports the notion that the expressions of molecules contained in the model, that is, in the TNF-mediated autocrine and feedforward loop, are highly similar. In addition, the local signalling seemed to be impacted by synovial proliferation.
Nowadays, RA is often treated with TNF inhibitor therapy, but this treatment is associated with side-effects on the systemic immune system and may cause serious complications such as tuberculosis and malignancy (43
). However, we found in this study that IRF1 appears to be clinically efficacious for the inhibition of chronic and delayed inflammation of RA, and that target therapy using IRF1 may entail fewer immunogenic systemic immunodeficiency problems than do therapies using TNF antibodies. Moreover, our results indicate that treatment using the IRF1 antibody would help to inhibit the autocrine loop, which is associated with the TNF-activated IRF1-IFNβ-JAK-STAT signalling pathway. One study has in fact suggested that inhibition of the IRF1 transcription factor may represent a novel approach to controlling RA (45
Chemokines and their receptors play important roles in directing the migration of immunocompetent cells to sites of inflammation and determining the pathohistological outcome of chronic inflammation and synovial hyper-plasia (46
). Serum concentrations of CXCL9/10 may thus serve as sensitive markers for disease activity in patients with RA (48
). It has been reported that CXCR3, the receptor of CXCL9/10, was strongly expressed by mast cells within the sublining region of RA synovial tissues. These findings suggest that the presence of CXCR3 protein on mast cells in RA sublining synovial tissue plays a significant role in the pathophysiology of RA, and is accompanied by elevated levels of CXCL9/10 (49
). In other studies, CXCL9/10 have been shown to be highly expressed in RA synovial tissues and fluids (49
), and the concentration gradient of CXCL9/ 10, between the serum and synovial fluid, favours the migration of CXCR3 receptor-expressing cells from the blood into synovium in RA (50
). Our data show that expression of CXCL9/10 at the protein level was higher in the RA synovial lining region than in the sublining. The synovial lining cells thus play an important role in the production of CXCL9/10.
CXCL12 in RA has been shown to play multifunctional roles in the recruitment, retention, and survival of inflammatory cells as well as in angiogenesis and joint tissue destruction. In human synovial fibroblast cell lines, the CXCL12 gene is up-regulated at the transcriptional level by growth arrest, suggesting that it is associated with synovial proliferation (53
). Using cDNA microarray from bulk tissues, van der Pouw Kraan et al (5
) showed that the expression of the CXCL12 gene was up-regulated in RA synovium in comparison with OA tissues. By contrast, we found that the expression of the CXCL12 gene was down-regulated in the RA synovial lining region, suggesting that it was transcribed by sublining cells of RA at a higher level. In fact, an increase in CXCL12 immunos-taining has been observed in the RA sublining synovium and in pervascular inflammatory aggregates as compared with OA (54
Our study indicates that the findings from the micro-array analysis of RA synovial lining cells are similar to the gene expression patterns obtained from RA bulk tissues (5
); that is, STAT1, IRF1, CCL5, and CXCL9/10 were highly expressed in the synovial lining cells of RA. We also demonstrated the presence of molecularly distinct classes of the rheumatoid synovium.
In conclusion, we have analysed gene-expression profiles in the synovial lining tissues in RA and OA, followed by LMD and cDNA microarray analyses, and found that the molecular activities correspond to their clinical and histological counterparts. As for synovial proliferation, our findings point to the importance of a mechanism in which TNF-induced gene expression is sustained and amplified as a result of the sequential induction of IRF1, IFNβ, STAT1, and the chemokine pathway in the synovial lining layer of RA. Finally, the different expression profiles of several candidate genes identified in our study may provide useful information for future studies using the LMD technique concerning the diagnosis and prognosis of RA. Our study has made it clear that it is important in rheumatology to understand the local signalling of every structural component, such as the lining, sublining layer, and lymphoid follicles.