IgG anti-hinge antibodies against IgG4 F(ab’)(2) fragments generated using pepsin are useful diagnostic markers for rheumatoid arthritis: implications of the possible roles of metalloproteinases and IgG subclasses in generating immunogenic hinge epitopes

BACKGROUND: Pepsin agglutinators, discovered over 50 years ago, have been recently referred to as anti-hinge antibodies (AHAs) because of their reaction with the IgG hinge epitope. AHAs have different reactivity for each hinge epitope generated by each protease that cleaves the hinge region at different sites. Moreover, AHAs have different reactivity against different hinge epitopes derived from each IgG subclass even when the same protease is used. Since the expression of matrix metalloproteinase-3 (MMP-3) is enhanced in rheumatoid arthritis (RA), AHA production could also be increased. The purpose of this study was to determine whether the levels of AHAs against IgG hinge epitopes produced by MMP-3 are elevated in RA. METHODS: The serum levels of IgG or IgA AHAs against the IgG1/IgG4 F(ab’)(2) fragments, generated by either MMP-3 or pepsin, were measured using ELISA in 111 patients with RA and 81 healthy controls (HC). Receiver operating characteristic (ROC) analysis was used for obtaining optimal cutoff values and cutoff values indicating high specificity (> 95%) of the AHA. The targeted epitope of a specific AHA was investigated through inhibition ELISA. RESULTS: Seven AHAs were statistically higher in RA patients than in HC, except IgG AHA against IgG1 F(ab’)(2), which was generated by MMP-3 proteolytic cleavage. The areas under the ROC curve were 0.66–0.80, although the sensitivities at high specificity were low (5.4–24.3%). The cumulative number of positive AHAs in each individual was statistically higher in RA patients than in HC, suggesting the extreme extent of AHA repertoires in RA. Inhibition studies revealed that IgG AHAs against IgG4 F(ab’)(2) fragments generated by pepsin cross-reacted with IgG1 F(ab’)(2) fragments generated by pepsin. Multivariate logistic regression analysis identified the IgG AHA against IgG4 F(ab’)(2) fragments generated by pepsin as an independent variable for RA diagnosis, even in RA patients who were negative for both RF and ACPA (odds ratio 1.18, 95% CI 1.06–1.32; P = 0.003). Additional experiments using non-RA patients finally strengthened the diagnostic utility. CONCLUSION: In RA patients, we observed diversification and amplification of AHA repertoires and diagnostic utility of the specific AHA against IgG4 F(ab’)(2) fragments generated by pepsin but not MMP-3.