ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression

Abstract

Introduction: Autoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients.

Methods: Twenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (iSPR) of biomolecular interactions on the sensor chip.

Results: Cluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations.

Conclusions: Compared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.

Figure 1

Schematic overview of autoantibody detection by microarray surface plasmon resonance imaging. (A) Biotinylated peptides are immobilized on a sensor chip which contains a gold surface with a streptavidin hydrogel on top. Each peptide (48 in total) is pumped back and forth along a specific position of the chip for one hour. This leads to a 48-spot microarray, in which each spot contains a different peptide. (B) Interactions of serum antibodies with peptides on the 48-spot microarray are detected by surface plasmon resonance imaging (iSPR). The incident light is reflected at the sensor chip surface and detected by a charge-coupled device (CCD) camera. (C) At a certain incidence angle, the amount of reflected light will be at a minimum. The binding of an antibody leads to a mass change at the surface and results in a shift in the resonance angle. The resonance angle is plotted against time to generate a sensorgram.

Figure 2

Detection of autoantibodies against citrullinated protein in anti-cyclic citrullinated peptide 2-positive early arthritis sera by microarray surface plasmon resonance imaging. Sensorgrams for the binding of 20 anti-CCP2-positive early arthritis sera to four peptide sets are shown. The different citrullinated peptides are depicted in different shades of red, and the arginine control peptides are shown in different shades of green. The different colors the four different citrullinated peptides illustrate the data listed in Table 1. Widely reactive autoantibodies against citrullinated protein (ACPA)-positive serum from a rheumatoid arthritis patient (X) and a mixture of two monoclonal ACPAs (Y) were used as controls.

Figure 3

Clustering of early arthritis patient sera based upon microarray surface plasmon resonance imaging data. Microarray surface plasmon resonance imaging data for 374 early arthritis patient sera were subjected to cluster analysis using Cluster 3.0 software. The results were used to generate a heat map, with the results for the individual peptides given in rows (peptide numbers indicated on the right) and those for the patients given in columns. (A) through (L) On the basis of the dendrograms, 12 autoantibodies against citrullinated protein profiles were defined. Below the heat map, the anti-cyclic citrullinated peptide 2 (CCP2) reactivity is shown: anti-CCP2-positive is shown in different shades of brown and orange, and anti-CCP2-negative is shown in blue. Fib: fibrinogen; FN: fibronectin; Pep. Lib.: peptide library; MNDA: myeloid cell nuclear differentiation antigen; Vim: vimentin.