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Rheumatology 2001; 40: 721-723
© 2001 British Society for Rheumatology

Editorial

Extra domain-positive fibronectins in arthritis: wolf in sheep's clothing?

S. Carsons

Division of Rheumatology, Allergy and Clinical Immunology, Winthrop University Hospital, Mineola, New York 11501 and Department of Medicine, School of Medicine, State University of New York at NY Stony Brook, Stony Brook, New York, USA

Twenty years ago, elevated quantities of fibronectin (Fn) were detected in inflammatory synovial fluid and synovial tissue [1–3]. Levels were found to be consistently higher in rheumatoid arthritis (RA), but elevations in synovial fluid Fn were also noted in other inflammatory arthropathies [4]. In contrast, Fn levels in RA plasma were similar to those found in normal human plasma [2, 4, 5].

It is reasonable to hypothesize that the increased amounts of Fn found in inflamed joints serve a reparative and/or anti-inflammatory function as Fn has been demonstrated to play a significant role in wound-healing [6] and the opsonic clearance of inflammatory debris such as collagen and fibrin [7, 8]. Deciphering the function of Fn in arthritis has been complicated by its molecular heterogeneity. For example, the forms of Fn found in plasma and tissue have different primary structures [9]. In addition, local, tissue-specific post-translational modifications, such as glycosylation and phosphorylation, may contribute to functional heterogeneity [10, 11]. In synovial fluid, immunoreactive Fn may consist of molecules which have diffused from plasma in addition to those produced locally by synoviocytes and by inflammatory cells infiltrating the synovium. Finally, it has been amply demonstrated that Fn is susceptible to proteolytic cleavage at the flexible regions connecting its globular functional domains [12]. Proteolytic fragments may lose the binding activities that are present in the native molecule or may acquire new functions [13]. Fn present in the inflamed joint has been shown to undergo limited proteolytic fragmentation [14].

What has traditionally been measured in synovium and cartilage as ‘immunoreactive Fn’ is now seen to actually consist of several isoforms and peptide fragments. This has led to a re-evaluation of the concept that synovial Fn is a single protein acting in a purely anti-inflammatory or reparative manner. Fn fragments may acquire chemotactic properties [15], chondrolytic activity [16] and de novo proteolytic activity such as that localized to a region in Fn containing a hexapeptide (LTSRPA) homologous to a streptokinase sequence [17]. When intact Fn is cleaved by plasmin, a fragment containing LTSRPA acquires streptokinase enzymatic activity. Fn isoforms may also possess alternative adhesion sequences that have the ability to modulate cell attachment. Facilitating adhesion of inflammatory cells to high endothelial venules and adhesion of fibroblast-like synoviocytes (FLS) to the surface of cartilage is another potential proinflammatory function of Fn in the inflamed joint.

An important mechanism underlying the functional diversity of Fn is alternative splicing of the primary transcript [9]. Three main Fn alternative splice sites have been identified. Two alternative splice sites include extra domains. Extra domains are similar to the 15 minidomains (type III repeats) that constitute approximately two-thirds of the Fn monomer. Extra domain A (ED-A) is found in between the 11th and 12th type III repeats, while extra domain B (ED-B) is found between the 7th and 8th type III repeats. ED-A and ED-B are never included in the hepatocyte Fn transcript; thus plasma Fn does not include these alternative splice domains. The presence of ED-A, therefore, is indicative of ‘cellular’ (tissue) Fn and the expression of ED-A and ED-B is increased during tissue injury. An additional alternative splice site is found in the flexible region near the heparin domain connecting the 14th and 15th type III repeats and is called III-CS (type III repeat connecting segment). Five sets of synthetic peptides, labelled CS-1 to C5-5, span the entire III-CS sequence. A peptide (LDV) in CS-1 mediates cell adhesion via the {alpha}4ß1 integrin (VLA-4) [18]. III-CS/{alpha}5ß1 adhesion is modulated in part by inclusion or exclusion of the variably spliced LDV sequence. In contrast, the RGD sequence, which is always present in the 10th type III repeat mediates cell adhesion via the {alpha}5ß1 integrin (the classical fibroblast Fn receptor). Thus, alternative splice sites may regulate cell adhesion in inflammatory environments. CS-1 Fn has been localized to the microvasculature and lining layer of the synovium. It appears to be expressed to a greater degree in RA synovium, where it may play a role in the adhesion of lymphocytes to high endothelial venules and the adhesion of type B synoviocytes to articular cartilage [19]. Boyle et al. [20] recently showed that inflammatory cytokines such as interleukin1 (IL-1) enhance CS-1 expression on cultured endothelial cells, resulting in increased adhesion of Jurkat cells.

Similarly, enhanced expression of alternatively spliced extra domains has been documented in various forms of arthritis. Hino et al. demonstrated the presence of ED-A-positive Fn in the lining layer of active RA synovium but not OA synovium. ED-A Fn was also detected at the cartilage–pannus junction, suggesting a role in pannus invasion, and it was concentrated in RA synovial fluid [21]. These authors also demonstrated that the inclusion of ED-A enhanced synoviocyte binding to Fn, perhaps via steric effects augmenting interactions of the carboxy-terminal heparin (Hep-2) binding domain with cell-surface heparan sulphate proteoglycan [22].

Recently, Saito et al. [23] suggested a mechanism whereby ED-A-containing Fns may be directly proinflammatory. They demonstrated that recombinant ED-A increased proteoglycan release from cartilage explant cultures as well as enhancing matrix metalloproteinase (MMP) production in chondrocytes. Examination of cytokine message expression revealed that IL-1 was induced by ED-A before MMP expression. The ability of rIL-1 receptor antagonist to block ED-A-mediated MMP expression further demonstrates that ED-A promotes MMP production via IL-1 secretion in an autocrine fashion [23]. Okamura et al. [24] subsequently demonstrated that recombinant ED-A was able to stimulate MMP-9 production directly, via interaction with toll-like receptors (TRL4) and NF{kappa}B activation. Saito et al. [23] further showed that a 160-kDa ED-A-positive placental Fn fragment possessed the ability to augment MMP-1 synthesis. This fragment is devoid of the 12th type III repeat flanking ED-A on the carboxy-terminal end, leaving ED-A at least partially exposed, a feature of all biologically active ED-A-containing Fn peptides or fragments. Recently, Peters et al. identified an ED-A-positive 170-kDa synovial fluid Fn fragment [25]. Fragments such as these have the potential to mediate proinflammatory functions of ED-A-positive Fn.

In this issue, Shiozawa et al. [26] report a strong association between synovial fluid levels of ED-A-positive Fn and radiographic evidence of joint erosion in RA. They also found higher levels of ED-A-positive Fn in those patients who eventually required joint replacement surgery compared with those requiring synovectomy or no surgery at all. These findings, suggesting a link between ED-A-positive Fn and disease progression, support a proinflammatory role for ED-A-positive Fn in arthritis. Thus, in inflammatory arthritis, ED-A-positive Fn may truly be a wolf in sheep's clothing.

The report of Shiozawa et al. [26] also suggests that ED-A-positive Fn may have a role as a marker of disease progression in RA. Fns have attracted interest for some time as potential markers for RA disease activity, as it is conceivable that a form produced locally in the joint can be detected specifically and that Fn species can, in general, be measured easily in plasma. Over the years, it has been demonstrated nearly universally that RA synovial fluid has the highest Fn concentration when compared with normal plasma and synovial fluid from patients with other rheumatic diseases [1–5]. In the present study, total synovial fluid Fn did reveal a positive correlation with radiographic progression, although ED-A-positive Fn was superior [26]. In order to be useful as a marker of RA progression, a molecule should possess certain characteristics, including: (i) relative disease specificity, so that intercurrent conditions (such as secondary OA) will not confound the analysis; (ii) a high degree of sensitivity for radiographic change, as therapeutic decisions regarding potentially toxic medications will be made using these data, and it is generally felt that erosion is a relatively late event in terms of the reversibility of the RA lesion; and (iii) ease of measurement, as multiple specimens will need to be obtained in a longitudinal fashion. While it is encouraging that ED-A-positive Fn is superior to total Fn in predicting radiographic change, at the moment it appears to fall somewhat short of an ideal marker. ED-A-positive Fn is not entirely specific for RA, being detected in osteoarthritic synovial fluid, where it may be derived from osteoarthritic cartilage [25, 27]. ED-A-positive Fn appears not to be highly sensitive for radiographic change, as 11 of 37 patients with progression (about 30%) in the present study had levels <=10 µg/ml, the range of values for the 11 patients showing no apparent progression. Finally, there was no change in plasma ED-A-positive Fn with radiographic progression; plasma Fn concentration would be the most useful measurement in a clinical setting. Interestingly, Voskyul et al. [28] did find an elevation of ED-A-positive Fn in the plasma of RA patients, although this elevation was confined to a subset with rheumatoid vasculitis, where it could have represented the contribution of the inflamed vascular endothelium [29]. In contrast, Claudepierre et al. [30] noted elevations in ED-A-positive Fn and ED-B-positive Fn in RA plasma in patients without vasculitis. However, ED-B-positive Fn levels were actually highest in patients with spondyloarthropathy. The variability of these results may be due to the use of different antibodies to detect ED-positive Fns.

The data presented by Shiozawa et al. [26] support the literature suggesting that ED-A-positive Fn plays a proinflammatory role in RA, and take another step towards defining a connective tissue matrix macromolecule as a marker of disease progression in RA.

Notes

Correspondence to: S. Carsons, Division of Rheumatology, Winthrop University Hospital, 222 Station Plaza North, Mineola, NY 11501, USA. Back

References

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