Introduction
In patients with rheumatoid arthritis (RA), joint pathology is mediated by typical changes in the synovial tissue. Hyperplasia of the synovial lining layer, infiltration of mononuclear cells into the sublining layer, activation of fibroblast-like synoviocytes and the production of catabolic mediators such as IL-1β, TNF-α and matrix metalloproteinases are involved in the joint destruction of patients with RA [
1]. Although secondary, synovitis is also found in osteoarthritis (OA) as a response of cartilage degradation and irritation of the lining cells with cartilage matrix components. Eventually, this also induces thickening of the lining layer and aggravates the damage of articular cartilage by the release of inflammatory cytokines and destructive proteases [
2].
Increases in knowledge about inflammatory cytokines and cytokine networks in chronic joint diseases has promoted the development of a new generation of biological drugs now available as inhibitors of TNF, IL-1 and others. However, little is known about mechanisms that protect and regenerate joints, although it has been shown that the progress of chronic joint diseases is decisively determined by the balance of anabolic and catabolic activities [
3,
4].
Bone morphogenetic proteins (BMPs) are anabolic candidates with pleiotropic functions in the development, homeostasis and repair of various tissues. Current approaches focus mainly on their ability to regenerate bone and cartilage by the induction of differentiation, apoptosis and proliferation of undifferentiated cells as well as by the stimulation of extracellular matrix formation [
5,
6]. These stimulatory properties led to the clinical use of recombinant BMP-7 in the treatment of bone nonunions [
7]. In contrast, BMP signaling has been shown to be involved in the onset and progression of ankylosing enthesitis in spondyloarthropathies and in the induction of osteophytes in OA [
8,
9]. Antagonism of BMP signaling was therefore suggested as an attractive therapeutic principle [
8,
10].
These and other findings with opposing functional implications [
5,
11,
12] demonstrate that the exact role of individual BMPs in degenerative joint diseases is still insufficiently understood.
In this study we focused on the expression of BMP-4 and BMP-5 in the synovial tissue of chronic joint diseases. Both proteins have a fundamental role in embryogenesis and in the induction of cartilage and bone [
13,
14]. Genetic and expression data suggest that BMP-5 is a key molecule in initiating the formation of particular skeletal elements in mammals [
15].
In adult organisms, both BMP-4 and BMP-5, are sufficient to induce the heterotopic formation of bone and cartilage
in vivo [
16]. Moreover, diminished repair after bone fracture in BMP-5-null mutated short-ear mice suggests that BMP-5 might also be required for the growth and repair of skeletal structures after birth [
15]. BMP-4 stimulates the synthesis of extracellular matrix in chondrocytes and supports the healing of bone fractures. Overexpression of BMP-4 leads to increased cartilage formation and chondrocyte differentiation without disturbing joint formation [
17].
However, little is known about BMPs in synovial tissue. Lories and colleagues [
18] demonstrated that BMP-2, BMP-4, BMP-6 and BMP-7 are expressed in the synovial membrane of patients with RA. BMP-2 and BMP-6, but not BMP-4 and BMP-7, are induced in fibroblast-like synoviocytes by stimulation with IL-1β and TNF-α. Moreover, intra-articular injection of BMP-2 induced fibrosis of the synovium [
10], suggesting distinct effects of BMPs in synovial inflammation and joint pathology.
Here we have investigated the expression characteristics of BMP-4 and BMP-5, which were identified as differentially expressed BMPs in a comparative microarray study on synovial tissue from normal donors and patients with joint diseases. We confirmed the array data by semiquantitative PCR, in situ hybridization and immunohistochemistry. Decreased expression of these morphogens in the inflamed tissues and changes in their histomorphological distribution suggest that distinct members of the BMP family are involved in joint homeostasis. They may be attractive candidates for readjustment of an unbalanced intra-articular milieu dominated by destruction and lack of repair.
Discussion
Inflammation and destruction are leading pathomechanisms in chronic joint diseases. In recent years, however, aspects of regeneration and homeostasis have become more and more important. Members of the TGF-β family, especially BMPs, are pivotal factors in skeletal tissue development and may contribute to the repair of various other tissues. We investigated the expression of BMPs in the synovial tissue compartment under normal and pathologic conditions by using microarray technology. All BMPs from BMP-2 to BMP-11, BMP-14 and BMP-15 revealed low to very low signal levels. Of these experiments, BMP-4 and BMP-5 were significantly decreased in RA in comparison with ND. This difference was confirmed by semiquantitative PCR. In addition, PCR analysis revealed a reduced expression of BMP-4 and BMP-5 in OA tissue in comparison with normal tissue. This variance of BMP expression levels in OA tissue in comparison with normal or RA synovial tissue may be explained by technical differences in sensitivity and resolution between PCR and microarray hybridization. However, the groups analyzed by PCR and microarray were independent. BMP expression in OA may therefore be more variable than that in RA. Immunostaining in normal donors revealed the expression of both BMPs predominantly in the synovial lining layer, whereas in patients with RA the expression was more frequently found in the sublining layer. A decrease in BMP-4 and BMP-5 in RA and OA could be correlated with markers of systemic and in part with markers of local inflammation as well as with disease duration. A relation of BMP suppression to therapy with steroids and disease-modifying anti-rheumatic drugs administered only in RA was excluded because BMP expression in synovium of OA patients was affected similarly, although to a lesser extent.
Expression of BMPs in synovial tissues was investigated recently by Lories and colleagues [
18]. They compared synovium from RA and spondyloarthropathies with synovium from traumatic joint diseases and found BMP-2 and BMP-6 to be expressed most consistently with a calculated relative expression in the range 0.002 to 0.2% compared with β-actin. This confirms our own observations of a low expression level in the synovial tissue compartment. Similarly to their results, we could not detect differential expression of BMP-2 and BMP-6 mRNA in RA compared with normal tissue.
In vitro, however, Lories and colleagues found an increase in BMP-2 and BMP-6 expression on stimulation of cultivated synovial fibroblasts with TNF-α and IL-1β. These data seem in part controversial to our observation that in synovial tissue the expression of the BMPs investigated (BMP-4 and BMP-5) was decreased. In addition, BMPs were negatively correlated with local or systemic parameters of inflammation as well as the duration of the disease. This discrepancy might depend on differences in the biological function and regulation of individual members of the BMP family. In fact, Lories and colleagues [
18] also reported that BMP-4, in contrast to BMP-2 and BMP-6, was not increased by stimulation with IL-1β or TNF-α. Furthermore, local differences between stimulatory and inhibitory mechanisms for BMP production could explain our observed differences in the histomorphological distribution of BMP-expressing cells in RA compared with controls. A similar distribution and predominant expression of different BMPs in fibroblastoid and macrophagocytic cells was also shown by Lories and colleagues [
18] and van Lent and colleagues [
26].
That BMPs might provide a beneficial effect on joint repair can be assumed from their role in joint development [
27], their induction of chondrogenic differentiation in adult mesenchymal stem cells [
28,
29] and their effect on cartilage formation in tissue engineering with chondrocytes [
5]. Similarly, the decrease in BMP-7 expression and the increase in BMP antagonists found in osteoarthritic cartilage suggests that a loss of BMP signal might reduce the regenerative capacity of cartilage [
12,
30]. However, the role of BMPs in the homeostasis of joints and the regeneration of cartilage is still unclear. BMP-2 was found to be increased in osteoarthritic cartilage and stimulated in culture with the proinflammatory cytokines IL-1 and TNF [
31]. In contrast, other BMPs were unchanged [
32]. Furthermore, the expression of BMP-6 and BMP-7 was also decreased in articular cartilage of TNF-transgenic mice, suggesting that loss of BMP expression could be also involved in chronic inflammatory and not only degenerative joint diseases [
33]. The overall decrease in BMP-4 and BMP-5 in the synovial membrane therefore presents a new and additional aspect in the imbalance of joint homeostasis in chronic joint diseases.
As well as a possibly beneficial effect of BMPs on arthritic joints, intra-articular TGF-β injection was shown to induce osteophyte formation, a typical morphological change in OA [
34]. Moreover, recent studies suggested that other factors such as BMP-2 and BMP-4 might be involved as downstream mediators of the TGF-β effect and that these BMPs might be released by macrophages of the synovial lining layer [
26]. However, these data are derived from a mouse model with TGF-β injected into normal joints. Furthermore, the dosage of TGF-β applied was at least 1,000-fold higher than the TGF-β concentration found in normal or even osteoarthritic joint synovia [
35]. Nevertheless, these data demonstrate that uncontrolled high levels of morphogens may exert a negative influence. It is intriguing that inhibition of BMP signalling in a papain-induced OA mouse model could prevent osteophyte formation and synovial fibrosis but at the same time increased the loss of proteoglycan from the cartilage matrix, thereby certainly promoting the damage of the joint surface [
10].
Thus, regenerative triggers in the treatment of joint diseases will depend on a balanced action of stimulators and inhibitors of BMP signalling with precise modulation of specific BMPs. The histomorphological distribution may be also important. Expression in deeper layers as seen in the samples of our RA and OA patients may influence predominantly cells of the surrounding tissue, thereby contributing to synovial fibrosis. In contrast, expression in the synovial lining layer may be more relevant for stable or increased levels of BMP in the synovial fluid, where these morphogens may potentially influence articular cartilage. As BMP-4 and BMP-5 were found to be decreased in the synovium and their expression was attributed to the synovial lining layer in normal joints, they could be favorable candidates for therapeutic application. Nevertheless, it will be important to understand precisely the network of morphogen action and regulation in the joint, because injection of BMP-2 induced osteophyte formation in a murine model [
9]. Thus, the interaction of BMPs and inhibitors not only in the synovium but also in cartilage has to be elucidated. Although studies in developmental biology have contributed considerably to the understanding of the BMP network [
27], the role of these morphogens in adult tissues is still unclear.
Acknowledgements
The authors thank Johanna Golla and Thomas Rudolph for excellent technical assistance, Martin Sparmann MD (Department of Orthopedics, Immanuel Krankenhaus, Berlin, Germany) for synovial tissue samples, and Carola Werner (Department of Medical Statistics, University of Göttingen) for statistical assistance. This work was supported by the German Science Foundation (DFG Ha2267/2-1 to Ha2267/2-4), the Novartis Foundation, The Federal Ministry of Education and Research of Germany (01GS0413) and the Verein für Tissue Engineering (VTE) e.V.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CPB and TH performed patient recruitment, PCR, immunohistochemistry and data interpretation and drafted the manuscript. UU was involved in in situ hybridization and PCR. VK was involved in patient recruitment and performed the 'synovitis score'. AP and CK conducted part of the patient recruitment and data evaluation. FS, GAM and GRB provided substantial input into data evaluation. All authors read and approved the final manuscript.