Introduction

Rheumatoid arthritis (RA) is a systemic immune-mediated disease that primarily affects the diarthrodial joints . RA is the most common inflammatory arthritis; uncontrolled active RA results in joint damage, disability and decreased quality of life. Among the unanswered questions about the pathogenesis of RA is how the aberrant systemic immune reaction involving both adaptive and innate immune components translates into a destructive inflammatory process at the joints. As for many inflammatory diseases, increasing evidence indicates that the resident cells not only respond to the insult of the immune system but actively recruit, amplify and perpetuate the inflammatory reaction. In rheumatoid synovitis, the resident fibroblast-like synoviocytes (FLSs) have been recognised as the key tissue component supporting the destructive inflammatory process . FLSs are mesenchymal cells that share many characteristics of fibroblasts from distant sites. As in other tissues, they are responsible for much of the synthesis of extracellular matrix and express phenotypic characteristics of the fibroblast lineage. For the synovial tissue, however, these mesenchymal cells display distinct intrinsic capabilities that are vital to the structural and functional integrity of the diarthrodial joint . Recent evidence indicates previously unappreciated heterogeneity between fibroblasts from different organs . In the context of a systemic immune-mediated disease, these phenotypic differences between fibroblasts may provide a means that determines the pattern of organ involvement as well as the tissue response to inflammatory signals. Therefore, understanding of how FLSs are different from fibroblasts of other organs, their role in regulating immune reactions and the molecular means of their destructive potential will provide mechanistic insight into the pathogenesis of RA and may reveal novel targets for the therapeutic intervention in inflammatory arthritis, especially RA.

The normal synovium

The normal synovium is a thin highly organised structure that resides between the joint cavity and the fibrous joint capsule. At the cartilage–bone interface, the synovial membrane extends on to the surface of the cartilage for a short distance as a wedge . The synovium has two separate layers: a lining layer formed by condensed cells one- to four-cells thick and a loosely organised sublining layer composed of extracellular matrix, scattered fibroblasts, macrophages, mast cells, nerves, blood vessels and lymphatics . In contrast to the compact layer of the synovial lining layer, the sublining is more amorphous, consisting of loose connective tissue that forms a microanatomic base for the synovial lining. The sublining allows for the transfer of both molecular and cellular elements from circulating blood to the synovial lining and the synovial fluid space. Importantly, the sublining provides immunologic competent cells for infectious surveillance and a possible reservoir of FLS for continuous repopulation of the synovial lining. Moreover, mesenchymal progenitor cells have been isolated from adult human synovial membranes . These cells potentially differentiate into a range of cell types which share a common mesenchymal origin, including adipocytes, osteoblasts and chondroblasts, all of which are cellular components of the diarthrodial joint .

Unlike an epithelial layer, whose structure contains a discrete basement membrane and cells tightly connected to one another, the normal synovial lining lacks a true basement membrane and the cells form sporadic and discrete regions of cell-to-cell contact with wide intercellular spaces between interdigitating cellular processes, thus forming a continuous network of compacted cells rather than a continuous cellular lining layer . The lining cells are embedded within a specialised extracellular matrix. Both lining cells and matrix are organised into a functional ‘cellular basement membrane’ between the synovial fluid compartment and the highly enervated and vascularised synovial sublining. The reticular organisation of the lining facilitates passage of serum components to the joint cavity, a process thought to be critical for providing nutrients to the avascular articular cartilage. Further, the distinct organisation allows for deformable tissue architecture to the joint cavity that facilitates movement.

In healthy states, the synovial lining contains two morphologically distinct cell types: type A or macrophage-like synoviocytes (MLSs) and type B or FLS . MLSs are characterised by a large Golgi apparatus and numerous lysosomes; they are derived from bone-marrow myeloid precursors and express a surface receptor profile similar to other tissue-resident macrophage populations, including CD14, CD16 and CD68 . These phagocytic cells likely participate in clearance of debris from the joint space and may serve as sentinels for microbial encounters. By contrast, FLSs display abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few vacuoles; they are mesenchymal cells expressing markers common to fibroblasts in other anatomic sites including collagen, vimentin and CD90. However, in contrast to many other fibroblast populations, they express CD55 (decay-accelerating factor; DAF), CD106 (vascular cell adhesion molecule 1; VCAM-1), uridine diphosphoglucose dehydrogenase (UDPDG) and lubricin . Both CD55 and CD106 expressed on FLSs may provide a means for mediating heterophilic cellular interactions between FLSs and MLSs, the MLSs expressing the corresponding ligands, CD97 and VLA-4, respectively . The abundant rough endoplasmic reticulum indicates that the FLSs within the synovial lining are major producers of the molecular components of the synovial fluid. In addition to producing hyaluronan, which requires UDPDG, the lining FLSs synthesise the glycoprotein lubricin, both of which are essential for the lubricating ability of synovial fluid . Lubricin contributes to joint homeostasis by protecting cartilage and inhibiting the growth of FLS . Loss-of-function mutations in lubricin cause the human autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) which is characterised by progressive joint failure . In mice lacking lubricin, cartilage deteriorates over time and the synovium becomes hyperplastic . Of note, the fibroblast-like cells in the synovium reveal marked differences between the lining and sublining layers. Fibroblasts in the synovial sublining lack expression of CD55, CD106, UDPDG and lubricin suggesting functional differences between the FLS subpopulations in the synovial lining and sublining .

FLSs are major participants in all aspects of the active, continuous process of maintaining the synovial extracellular matrix. Histochemical studies show the production of matrix components (collagens, fibronectin, vitronectin, laminin, tenascin, the proteoglycans and elastin) by FLSs . These constituents are not present randomly within the synovium; rather, their distribution likely results from a tightly regulated process with specific matrix components present in distinct regions of the synovium. While fibronectin, laminin and hyaluronan, regular constituents of basement membranes at other anatomic sites, are relatively enriched in the lining matrix, collagens are present in relatively low abundance in the lining as compared to the sublining matrix . In contrast to the epithelial basement membranes, the extracellular matrix of the synovial lining layer lacks entactin, a cross-linking matrix component. The lack of entactin may provide a possible explanation for the less tightly organised synovial lining layer matrix when compared to the highly organised basement membranes underlying epithelial cells . The maintenance of the synovial lining functional integrity requires continuous matrix remodelling, a process resulting from a delicate balance between matrix degradation, matrix synthesis and matrix assembly all of which are substantially influenced by FLSs. In addition to the synthesis of extracellular matrix molecules, FLSs are instrumental in matrix component assembly via cell-surface expression of integrin species such as α5β1 and αVβ3 which direct fibrillogenesis of matrix components . Indeed, the normal synovial lining layer reveals reticular fibres that are associated with and orientated linearly along the compacted lining FLSs . Reticular fibres are supra-molecular structures that are comprised of a core of collagen bundles enveloped by proteoglycans . As in other tissues, this elaborate network of FLS and specialised matrix fibres likely contributes to the structural and functional integrity of the synovial lining.

FLSs also manufacture degradative enzymes that are required for matrix remodelling. Serine proteases, cathepsins, metalloproteinases (MMPs) and the membrane-type metalloproteinases (MT-MMPs) are expressed by FLSs, markedly in abundance by lining FLS . Thus, FLSs, especially FLSs of the lining layer, actively contribute to diarthrodial joint homeostasis by the maintenance of synovial lining flexibility and integrity, synthesis of lubricating substances and cartilage nutrition.

Recently, using a three-dimensional (3D) synovial micromass culture system, the inherent function of FLS in establishing a tissue structure that strikingly resembles the in vivo situation of the synovium was demonstrated . Primary FLSs were placed into a preformed matrix and the mixture of matrix and cells was cultured as a floating sphere. The 3D matrix sphere provides a scaffold for re-establishing tissue-like cellular organisation; the outer surface models the interface between the synovial connective tissue and the fluid-filled joint cavity. After several weeks of culture, histological examination of the 3D micromass revealed that FLSs organised a lining layer-like structure with compacted cells at the surface of the micromass. Beneath the lining layer, few, scattered FLSs within the matrix with wide acellular spaces were found. Thus, the cultured FLS spontaneously formed a tissue structure that recapitulates the synovial architecture with two distinct layers, the compacted lining layer and the loosely organised sublining layer. Intriguingly, dermal fibroblasts failed to establish a synovial-like architecture suggesting that lining layer formation is a unique feature of FLSs. For FLSs, the micromass lining layer resembled the in vivo situation, in that it manufactured extracellular matrix structures commonly associated with the synovial lining; numerous reticular fibres surrounded the compacted FLS of the lining. Moreover, lining FLSs but not cells of the sublining area displayed expression of lubricin, indicating that the distinct microanatomy of the lining layer brings forth specialised activity of lining FLSs. Importantly, when co-cultured with peripheral blood monocytes, FLSs supported the survival of monocytes and orchestrated the co-compaction of macrophages within the lining of the micromass culture. The molecular basis of FLS function in support of MLSs is not defined yet. However, these studies helped to clarify longstanding questions regarding FLS function in dictating synovial tissue behaviour; numerous characteristics of the synovial tissue can be attributed to inherent, autonomous activities of this specialised mesenchymal lineage.

The observations made by culturing FLS in a 3D micromass system highlight the importance of the distinct synovial micro-architecture for the function of lining FLS in elaborating a reticular fibre network, producing the synovial fluid protein lubricin and supporting the co-compaction of macrophages. Until recently, however, the molecular basis for the formation of the sophisticated structure of the synovial lining was not known. While electron microscopic analyses of the synovial lining layer revealed lining discontinuity with intercellular matrix space, they also demonstrated interlacing cellular connections . In other tissues, such as epithelia, the critical importance of intercellular adhesive interactions for the formation and maintenance of a tissue structure has long been recognised. The molecule that specifies homophilic FLS cell-to-cell adhesion, however, is different from that in epithelial tissues.

The identification of synovial expression of cadherin-11 by FLS has opened new avenues for exploring the mechanisms of synovial lining formation, structural organisation and integrity . The homophilic adhesion properties of cadherin-11 specify FLS-to-FLS adhesion; they also allow for establishing the synovial lining . Indeed, treatment of FLSs with a cadherin-11 fusion protein in the 3D micromass culture system described above efficiently abrogated lining formation. Lining formation was also seen when immortalised fibroblasts (L-cells) stably transfected with cadherin-11 were placed into the sphere. By contrast, empty vector control fibroblasts or E-cadherin-expressing fibroblasts failed to establish a lining-like structure indicating that this effect is a distinct characteristic of fibroblasts expressing cadherin-11. Importantly, when cadherin-11-deficient mice were examined, these mice displayed marked hypoplasia of the synovial lining with decreased lining compaction and decreased synovial membrane folds . These observations indicate that the synovial lining is attenuated in cadherin-11-deficient mice compared to wild-type mice. Further, when cadherin-11-deficient FLSs from these mice were cultured in the in vitro 3D synovial micromass system, the establishment of the lining/sublining architecture was largely absent with almost no surface cellular compaction, thus mimicking the in vivo effect. Together, these in vitro and in vivo studies indicate that cadherin-11 expressed by FLS is a critical determinant for synovial lining formation. In addition to its dominant role in establishing the synovial lining, cadherin-11, together with the cytoplasmic catenins provides a molecular means for regulating the function of FLS with dramatic implications for the synovial pathology in inflammatory arthritis.