Rheumatoid arthritis (RA) is a disabling disease with both a large impact on the quality of life for the patient and a high economic impact on society. It is known that early intervention minimizes tissue damage and disease progression. Therefore specific and sensitive diagnostics are essential for early discovery of disease. Even though rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs) are widely used as diagnostics for RA, improvements are needed to enhance the specificity and sensitivity of current molecular markers in RA. Therapy should also improve when new diagnostic assays can differentiate RA patients into groups with different underlying pathologic mechanisms. Several approaches to develop novel serologic markers for RA have been attempted, one of them being measurement of cartilage oligomeric matrix protein (COMP) in serum or synovial fluid. COMP is a structural component of cartilage, and it has been shown to be released during erosive joint diseases such as RA and osteoarthritis (OA) [1]. Excessive production of COMP in the skin in systemic sclerosis (SSc) is also reflected by an increase in serum COMP [2]. COMP is a homopentamer of 435 kDa in which each individual monomer is composed of four epidermal growth factor (EGF) domains, eight thrombospondin type 3 (TSP3) domains, and a globular C-terminus. The chains polymerize via their N-terminal coiled coil domains, and this is stabilized by interchain disulfide bonds [3]. One function of COMP in tissue is to catalyze collagen fibrillogenesis [4, 5] and, in the adult, to stabilize tissue structure by interacting with other collagen-bound matrix proteins [6].

In a previous study, we showed that COMP released from the joints during RA is able to activate complement both in vitro and in vivo [7]. Complexes between COMP and the complement-activation product C3b were found both in the serum and synovial fluid of RA patients, whereas no COMP-C3b was found in the serum of healthy controls or in patients with OA; therefore, it was concluded that COMP-C3b might be diagnostic in RA. The complement-activating site was shown to reside within the C-terminal proportion of COMP, by interaction with both properdin and C3.

Complement is an important part of innate immunity, and its uncontrolled activation has been strongly implicated in many autoimmune diseases, among them, RA. Several studies have shown that complement deficiency or inhibition ameliorates disease activity in rodent models of RA [8, 9], and complement-activation products have been found in the joints of patients with RA [10–12]. Several proteins that are found in affected joints have been shown to activate complement, among them RF [13, 14], ACPA [15], cartilage molecules of the small leucine-rich-repeat protein (SLRP) family [16, 17], as well as apoptotic cells [18] and thereby most likely contribute to disease progression by feeding the inflammatory response. Measurement of complexes between complement-activation products and joint-specific molecules might provide improved specificity to the diagnostics of RA and may give better distinction between patients with complement-mediated joint inflammation and healthy individuals. This might also provide valuable information on the patients' need of complement-inhibition therapy that is currently undergoing clinical trials.

Our previous pilot study on the presence of COMP-C3b in the circulation of RA and OA patients, as well as in healthy controls, was conducted on a small number of patients, and therefore, in the present study, we set out to validate our results in larger patient cohorts. To investigate whether such complexes are a specific feature of RA or whether they can be found in patients with other rheumatologic disorders or joint diseases, we have now included patients with SSc, reactive arthritis (ReA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), and systemic lupus erythematosus (SLE), as well as a larger group of healthy individuals. Furthermore, we have also related serum concentrations of COMP-C3b complexes to clinical and biochemical markers commonly used to monitor disease activity, as well as studied changes of COMP-C3b levels over a 3-month period in relation to treatment of RA, PsA, or AS with TNF inhibitors to elucidate how changes relate to modulation of disease activity.

In the present study, we showed that elevated COMP-C3b can be found in the circulation of RA patients, extending previous observations in a smaller patient cohort. COMP-C3b correlates with several inflammatory parameters describing mainly synovitis in RA, suggesting that joint inflammation causes the release of COMP fragments that are able to activate complement. Interestingly, with TNF-α inhibition, serum COMP-C3b decreased significantly, which indicates that the molecular process of cartilage turnover has been altered. Because serum COMP did not significantly decrease during TNF-α inhibition, it appears that the intervention causes a reduction in the release of complement-activating COMP, perhaps in the form of unique fragments, as opposed to a reduction in total COMP. This might be a process of modified cartilage degradation or turnover due to an alteration of protease activity. A previous study reported that serum COMP levels decreased on TNF-α inhibition [26]. The fact that this decrease was not seen in our patient cohort might be a reflection of the lower baseline COMP values in our cohort (9.1 U/L) compared with the other cohort (11.3 U/L and 12.2 U/L in the different treatment groups). Furthermore, in the other study, the major reduction was seen in the patients with the highest baseline COMP values.

The reduction in CRP levels and COMP-C3b were observed to follow different kinetics, with a decrease in COMP-C3b occurring at a slower rate. This indicates that formation of COMP-C3b is not an immediate response to inflammation-induced joint damage but rather a complicated process regulated by cartilage metabolic and catabolic activity and by the surrounding microenvironment.

The AS patients in our cohort, who were chosen for having only axial joint involvement, had slightly higher concentrations of circulating COMP-C3b than did the controls. Interestingly, in this group, CRP did not correlate with COMP-C3b. Similar observations were made for patients with peripheral joint PsA, in which a lack of correlation between CRP and COMP-C3b was also observed. The fact that both groups had similar levels of circulating complexes indicates that the origin of released COMP fragments (axial/peripheral joints) does not directly relate to the ability of COMP to activate C3. Furthermore, lack of correlation between CRP and these complexes shows that the level of COMP-C3b in these patient groups does not reflect disease activity. Surprisingly, an increased COMP-C3b was observed in both AS and PsA patients on TNF-α inhibition, even though systemic inflammation was reduced, as shown by a decrease in CRP. Therefore, it is possible that this intervention either stimulates tissue regeneration or changes the character of the COMP fragments released from the tissue. It also is possible that TNF-α inhibition is effective downstream of the process of tissue destruction and complement activation and therefore will primarily have an effect on the inflammation, its symptoms, and effects.

In the current study, we observed elevated levels of COMP-C3b in the sera of OA patients, in contrast to our previous report, in which this was observed only in the synovial fluid and not in serum. With further investigations of this divergence, we found that, for unexplained reasons, in the serum of OA samples previously used, all C3 was degraded to fragments smaller than 40 kDa, as shown by SDS-PAGE followed by Western blotting. Thus, the fraction of C3b that, in the patient, was deposited onto COMP was most likely similarly degraded and therefore unrecognizable in our assay. We found that this was not the case for the synovial fluid samples. We are not sure what caused the degradation of C3 in the archival OA samples during storage. We have found experimentally that serum can be thawed and frozen several times or stored for many hours at room temperature before freezing without C3 becoming degraded, and thus no extreme precautions must be taken for the sample to be suitable for analysis with our assay. However, the OA serum samples used in the previous study were obviously exposed to some harsh conditions that were not apparent to any co-authors but could have occurred during the many years during which these samples were stored. We confirmed that C3 was not degraded in any other patient group or in OA samples used in the current study. The new data clearly show that at least a proportion of the COMP released in OA contains the complement-activating region in the C-terminal part. It is important to stress that, even though the inflammation is much more pronounced in RA, an inflammatory component also exists in OA [27, 28].

Patients with SSc displayed in previous studies elevated COMP levels in their circulation as a result of increased COMP synthesis and turnover by their dermal fibroblasts [2]. We could now observe elevated levels of COMP-C3b in their circulation as well, which shows that the released COMP fragments have the potential to activate complement. Furthermore, patients with ReA displayed high levels of COMP-C3b in their circulation. The median COMP-C3b values in ReA were markedly higher than those in other studied disease groups, which most likely reflects the pronounced cartilage engagement and strong inflammatory stimulation of the disease. Similarly, SLE patients were found to have elevated levels of COMP-C3b in their circulation, independent of being in disease flare or remission. The serum COMP-C3b concentration was, however, significantly higher in patients with arthritis than in patients without arthritis, suggesting the importance of joint inflammation and possibly cartilage involvement for the release of complement-activating COMP. The fact that C1q and C4 levels were positively correlated with COMP-C3b shows that classic pathway components are not consumed during the disease process leading to COMP-C3b formation. Even though a negative correlation could be expected between COMP-C3b and C3, it might be that the overwhelming excess of C3 in serum compared with released COMP masks such changes. However, we cannot exclude that C3 has been consumed within the synovial fluid as a more-local response.

We found elevated levels of COMP-C3b complexes in the circulation of patients with several rheumatologic diseases, showing that the release of complement-activating COMP is not a specific feature of RA. Because levels of COMP-C3b correlate to the inflammatory variables in RA but not in the other diseases examined, it seems that formation and release of complexes can reflect somewhat different processes in different conditions. This may relate to downstream regulation of the inflammatory process and merits further investigation. Notably, TNF-α inhibition in RA reduced COMP-C3b serum levels in RA, but not in AS and PsA, which points to different pathophysiologic mechanisms regulating complex formation. It should be stressed that although a correlation was found to inflammation in RA, this was rather weak and, taken together with the different kinetics of response for CRP and COMP-C3b complexes after TNF-α inhibition, may suggest that the release of complexes is not merely due to inflammation but reflects other, yet unknown components of the disease process in RA (for example, being upstream of major cytokine release). Further studies, including longitudinal monitoring of early RA patients, are needed to elucidate the pathophysiologic role of COMP-C3b complexes and to establish their value as disease markers in the clinic.

The authors KEH, AMB, TS, and DH have filed a patent application on a method to detect tissue degradation leading to inflammation. Authors TS and DH own stocks in AnaMar Medical, who provided the COMP-kits for the study.