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The diagnosis of rheumatoid arthritis (RA) remains imprecise, particularly early in the course of the disease. Up to now, rheumatoid factor (RF) has been used as a typical marker for RA; however, RF has a low specificity because it also occurs in many inflammatory diseases as well as in healthy people. It has recently been shown that autoantibodies directed toward cyclic citrullinated peptides (anti-CCP) are potentially important serological markers for diagnosis and prognosis in RA.1
The proteolytic pathways associated with cellular interactions also seem to have an important role in joint cartilage destruction of RA. Matrix metalloproteinase-3 (MMP-3) is secreted by fibroblasts and synovial cells,2 and it has been shown to be increased in RA serum, not only in the late stage but also in the early stage of the disease. Therefore, MMP-3 is a useful marker for predicting bone damage in early RA.3
In many early cases of RA, patients will not always fulfil the American College of Rheumatology criteria. Therefore, it is important to detect the point at which disease-specific autoantibodies and proteinases appear in RA serum. In this study we directly investigated the production of anti-CCP and MMP-3 using a severe combined immunodeficiency (SCID) mutant mouse, into which human RA synovial tissue was transferred.
SCID mice are a well known model for analysing the developmental mechanism of T and B cells. We previously developed SCID mice (CB.17/Icr; Charles River Japan, Tokyo, Japan) in which human RA synovial tissue was grafted (SCID-HuRAg), and we evaluated them as models for RA.4,5 The histological features of RA were observed in all SCID-HuRAg mice and these mice induced the production of human antibodies. Proliferative synovial fibroblasts and infiltration of inflammatory cells were also seen in the pannus.
The serum levels of anti-CCP and human MMP-3 of SCID-HuRAg mice were examined weekly after implantation. Non-implanted SCID mice sera were used as controls. Figure 1 shows representative results of independent experiments from two patients. The mean (SD) anti-CCP and MMP-3 levels in the serum of the patients were 702 (26.9) U/ml and 321 (12.0) ng/ml, respectively. Human, not mouse, MMP-3 appeared in mouse serum shortly after implantation and decreased immediately. On the other hand, anti-CCP increased gradually and reached a plateau from the fifth week. It is reported that anti-CCP are produced locally in the inflamed synovial tissue from RA.6,7 Therefore we think it is reasonable that the surviving, activated human B cells can produce autoantibodies, and the proliferative synovial fibroblasts around the engrafted tissue can produce MMP-3, and that producing autoantibodies spontaneously takes much longer than inducing enzyme. In our previous report, both human RF and interleukin 6 were also detected in these mice sera4 and not in mice with tissue implants from osteoarthritis synovia (data not shown).
MRL-lpr/lpr mice develop an autoimmune syndrome resembling human systemic lupus erythematosus.8 Anti-CCP were present in these mice and (NZW × B6)F1-hbcl-2 transgenic mice, which have defects in the regulation of B cell apoptosis.9 However, these animals showed not only antibody reactivity against the citrullinated peptide cfc1-cyc peptide, but also against the non-citrullinated control peptide; therefore, Vossenaar et al reported that the antibodies in these mice are not citrulline specific, and citrulline-specific autoantibodies are present only in human patients with RA and not in animal models of autoimmune disease.10
We think that the main problem is that citrulline-specific autoantibodies detected by both groups were of mouse origin. On the contrary, all the target cells within SCID-HuRAg mice which we used were of human origin, having migrated from the implanted tissue, and anti-CCP and MMP-3 were of human origin, too.
In conclusion, we showed the point at which disease-specific autoantibodies and proteinases appear in RA. SCID-HuRAg mice will be worth using in the study and development of new drugs for RA.
Dr Iwaki-Egawa’s work was supported, in part, by a grant from the Akiyama Foundation (Japan).
This study was approved by the ethics committee of Hokkaido College of Pharmacy.