Osteoarthritis (OA) is generally accepted as a slowly progressive degenerative disease, mainly caused by physical stresses and aging factors. However, involvement of immunologic pathways has also been recently implicated in the pathophysiology of OA. Specifically, autoimmunity to cartilage-related components, such as anti–chondrocyte surface antibodies (1), as well as cellular immune responses to chondrocyte membranes (2), the cartilage link protein, and cartilage aggrecans (3) have been described. In this context, we have also recently reported the existence of autoimmunity against cartilage-related proteins such as YKL-39 (4), cartilage intermediate layer protein (5), and osteopontin (6). However, most of the autoimmune responses to such antigens are detected both in patients with OA and in patients with rheumatoid arthritis (RA).
Considering that chronic synovitis is observed in both OA and RA (7–9), the autoimmune responses could be a part of epiphenomena that are associated with destruction/degradation of the cartilage. In this respect, if OA-specific autoimmunity is demonstrated, the pathologic role of this autoimmunity in OA would be strongly implicated. To investigate the OA-specific autoimmunity, it would be of great help to compare the overall profiles of autoimmunity between OA and RA patients.
To this end, we carried out this study to detect the proteins comprehensively recognized by autoantibodies in patients with OA as compared with those in patients with RA, using a proteomic approach. Specifically, with serum samples from patients with OA and RA, we separated the human chondrocyte proteins by 2-dimensional (2-D) electrophoresis, detected antigenic protein spots by Western blotting, and identified the antigenic proteins by mass fingerprinting. We next prepared some of the identified proteins as recombinant proteins, by which we confirmed their antigenicity and determined the prevalence of the autoantibodies in OA, RA, and other disease categories. Furthermore, we mapped the autoepitopes.
As a result, we found that the overall profiles of the chondrocyte-related autoantigens overlapped only partially between OA and RA. Interestingly, some proteins were recognized as an autoantigen much more frequently by the OA samples than by the RA samples. One such protein was identified as triosephosphate isomerase (TPI). In fact, TPI was recognized by 24.7% of the tested serum samples from the OA patients, but by only 5.6% of the tested serum samples from the RA patients, in an enzyme-linked immunosorbent assay (ELISA) using recombinant TPI.
TPI is a glycolytic enzyme that interconverts D-glyceraldehyde-3-phosphate (GDP) and dihydroxyacetone phosphate (DHAP). Since another glycolytic enzyme of glucose-6-phosphate isomerase (GPI) was reported to be a disease-causing autoantigen in RA-like murine arthritis, we hypothesized that the autoimmunity to TPI may be involved in the pathology of OA. Taken together, our results could promote the understanding of the role of autoimmunity in OA.
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- PATIENTS AND METHODS
In the present study, we sought to detect the autoantibodies produced by chondrocytes in patients with OA and RA, using a proteomic approach to compare autoimmune profiles between the two diseases. In the first experiment involving 2-D electrophoresis/Western blotting of 20 serum samples each from the OA, RA, and healthy control groups, we detected 62 immunogenic protein spots, of which 52 were recognized by the serum samples from patients with OA or RA. It is of importance that 41 of the 52 protein spots were recognized by the OA serum samples, whereas 33 of the 52 protein spots were recognized by the RA serum samples. Thus, the range of autoimmunity in OA is at least comparable with that in RA.
Since inflammation is much more severe, generally, in RA than in OA, the wide range of autoimmunity would not simply reflect the severity of the joint inflammation. Furthermore, 19 protein spots reacted with the OA serum samples only, and 11 protein spots reacted with the RA serum samples only. This demonstrated that RA and OA possess unique autoimmune profiles. The difference in the profiles may reflect different involvement of the autoimmunity in the pathology between OA and RA.
We concentrated on protein spot 4, one of the apparently OA-specific autoantigens in the 2-D electrophoresis/Western blot screening. Mass fingerprinting revealed that protein spot 4 was most likely TPI, which was confirmed by ELISA and Western blotting. We confirmed a higher frequency of detection (24.7%) of the anti-TPI autoantibodies in the patients with OA, but these were infrequent in RA patients as well as SLE patients and healthy donors. The frequency of 24.7% determined by ELISA corresponded well with the result of the first screening by 2-D electrophoresis/Western blotting (autoantibody frequency of 20%).
Predominant detection of the anti-TPI autoantibodies in OA was more distinct in the measurement of synovial fluid. Furthermore, in the paired samples of sera and synovial fluid from individuals, the anti-TPI antibody titers were higher in the synovial fluid samples than in the sera (Figure 4c). This finding may reflect the pathologic importance of the anti-TPI autoantibodies in the joints.
Our experiments also sought to detect the IgG types of the anti-TPI autoantibodies. Although we also checked the IgA, IgM, and IgD types, they were rarely detected (data not shown). In the epitope mapping, each of the 8 regions (TPI-1 to TPI-5 and TPI-1a to TPI-1c) were recognized by a part of the OA serum samples. This indicates that there are at least 7 epitopes on TPI. Since the combination of the reactive regions was different among the serum samples, the antigenicity of the 7 epitopes would be different from each other. Furthermore, 1 serum sample recognized an average of 3.5 regions. The recognition of the multiple epitopes would not be produced by cross-reaction, but rather, would be more likely to be produced by the antigen-driven mechanism; that is, B cells specific for each of the epitopes were activated by the help of the antigen-specific T cell. The dominance of the IgG-type anti-TPI autoantibodies, which indicated class-switching, would also support the antigen-driven mechanism. In this respect, the generating mechanisms and pathologic roles of the anti-TPI autoantibodies in OA would be different from those of the IgM-type anti-TPI autoantibodies previously reported in patients with acute hepatitis A virus infection, infectious mononucleosis, or malaria (23–25).
Clinically, the mean radiographic grade of the OA joints was significantly lower in the anti-TPI–positive patients than in the anti-TPI–negative patients. This suggests that production of the anti-TPI autoantibodies would be related to the early phases of OA.
It remains unclear whether the anti-TPI autoantibodies play a substantial role in the development of OA. Informatively, TPI is a housekeeping glycolytic enzyme that interconverts GDP and DHAP. The autosomal recessive disease of TPI deficiency is characterized by chronic hemolytic anemia, neurologic disturbances, susceptibility to bacterial infection, and cardiomyopathy (26, 27). However, decreased production of ATP and accumulation of DHAP failed to explain the occurrence of hemolytic anemia (28).
In the case of chondrocytes, energy production in the chondrocytes has been demonstrated to be dependent mainly on the anaerobic metabolism (29). Since the anaerobic condition needs more amounts of glucoses than does the aerobic condition to produce the same level of energy, interconversion from DHAP to GDP by TPI would be of critical importance in chondrocytes. In this context, conditions involving stress on the joints, such as OA, may increase the need for TPI. Accordingly, we observed that the mRNA of TPI in cultured chondrocytes was increased by stimulation with interleukin-1β, which has a pathologic role in OA (data not shown). Considering these data, if the anti-TPI autoimmunity inhibits the enzymatic activity of TPI, it can directly lead to damage of the chondrocytes. Further studies are needed to elucidate whether such a pathway is possible.
TPI can be important as an antigen to generate chronic inflammation in OA, even though it is milder than in RA. Recently, GPI, a ubiquitously expressed protein and also one of the glycolytic enzymes, was demonstrated to be a pathogenic autoantigen in a K/B×N murine arthritis model, and the anti-GPI autoantibodies were detected in a subset of patients with RA (30–32). The ways in which the antibodies to a ubiquitous cytoplasmic enzyme provoke joint-specific autoimmune disease are further explained in some investigations.
Maccioni et al generated anti-GPI monoclonal antibodies (mAb) from spontaneously activated B cells in lymphoid organs of arthritic K/B×N mice and injected these mAb into healthy recipients individually (33). They found that the pathogenicity of the mAb depended on the ability to form mAb/GPI multimers by simultaneous recognition of different epitopes. Matsumoto and colleagues observed the deposits of GPI/anti-GPI and C3 on the lining of the articular cavity and cartilage surface in both arthritic mice and human arthritic joints (34). In contrast with the findings in the joints, C3 was not located with GPI–anti-GPI IgG complexes in kidney glomeruli. Based on these phenomena, the authors speculated that GPI could be passively transferred from the serum into synovial fluid and deposited on the surface of cartilage, which started inflammation (34). The deposition of immune complexes and complement components on the surface of articular cartilage was found in OA as well as in RA (35). In the present study, anti-TPI IgG from OA was demonstrated to recognize multiple epitopes on TPI, with high titers in both the serum and synovial fluid from the OA patients. Taking these facts together, the potential of the alternative complement pathway to play a pathophysiologic role in OA may be related to TPI and anti-TPI IgG. However, further studies are needed to elucidate this concept.
In summary, we demonstrated different autoimmune profiles in patients with OA as compared with patients with RA, by a proteomic approach. One of the OA-specific autoantibodies was the IgG-type anti-TPI autoantibody, which we have now described for the first time, to our knowledge. The anti-TPI autoantibody would be a potent diagnostic marker for OA and may play pathogenic roles in the development of OA.