IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin

Abstract

In the rheumatoid synovium, deiminated ('citrullinated') forms of fibrin are the major targets of IgG autoantibodies to citrullinated proteins (ACPA), the most specific serological markers of rheumatoid arthritis (RA). To further the characterization of ACPA, we determined their subclass distribution. From a previously validated highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) onto in vitro deiminated human fibrinogen - antihuman fibrin(ogen) autoantibodies (AhFibA)-ELISA - we derived and calibrated four ELISAs, using monoclonal antibodies to each of the four IgG subclasses, to determine the proportions of AhFibA subclasses in the sera. A series of 186 serum samples from RA patients was analysed. All AhFibA-positive sera contained IgG1-AhFibA, which reached the highest titres and accounted for more than 80% of AhFibA in three-quarters of the sera. One or two other subclasses were associated with IgG1 in 39% of the sera, IgG4-AhFibA being observed much more frequently and at higher titres than IgG3- or IgG2-AhFibA. IgG1 alone or IgG(1 + 4)-AhFibA were the AhFibA subclass profiles found in more than 80% of patients. AhFibA are mainly IgG1 and, to a lesser extent, IgG4. Such IgG subclass profiles may influence the effector phases of the immunological conflict between ACPA and deiminated fibrin that takes place specifically in the rheumatoid synovium and therefore may play a critical role in the self-maintenance of rheumatoid inflammation.

Fig. 1

Titration of the MoAbs to IgG subclasses. The curves correspond to the optimal dilution (see Methods) of the anti-IgG1, -IgG2, -IgG3 or -IgG4 MoAb, i.e. the dilution giving the same OD for the same concentration of the corresponding detected IgG subclass. Curves are the means of at least five duplicate determinations, performed in independent experiments. Bars indicate standard error. Titration curves obtained with the unlabelled NL16 anti-IgG1 MoAb (1 : 9000) followed by HRP-labelled antimouse IgG F(ab)′₂ (1 : 2500), with the HRP-labelled HP6014 anti-IgG2 MoAb (1 : 2500), the HRP-labelled HP6050 anti-IgG3 MoAb (1 : 1500) and the HRP-labelled HP6025 anti-IgG4 MoAb (1 : 6000) are indicated by arrows. The thick full line represents the reference curve obtained with the HRP-labelled JDC10 anti-IgG MoAb (1 : 3000).

Fig. 2

Distributions of titres obtained with the reference AhFibA-ELISA and those obtained with the IgG-, IgG1-, IgG2-, IgG3- and IgG4-ELISAs on the whole population of patients. For each serum, titres obtained in the (a) IgG-, (b) IgG1-, (c) IgG2-, (d) IgG3- and (e) IgG4-ELISAs were plotted against the titre obtained in the original AhFibA-ELISA.

Fig. 3

Box-plot of the titre distributions in the IgG-, IgG1-, IgG2-, IgG3- and IgG4-ELISAs. Boxes contain 90% of the OD values obtained, bars extend to the extreme values. The median is indicated by a square.

Fig. 4

IgG subclass profiles of AhFibA. For each of the 141 AhFibA-ELISA positive sera, subclass proportions were calculated using titration curves established as described in the Patients and methods section. The histogram bars represent the proportion and number of patients with the subclass profiles described below. (+): subclass accounting for more than 10% of total AhFibA; (–): subclass accounting for less than 10% of total AhFibA.