RA patients, healthy controls (HC), and non-RA patients
In this cross-sectional and case-control study, serum samples were collected from 111 patients with RA who met the ACR classification criteria . As HC, we obtained sera from 81 healthy staffs in our hospital. All sera were leftovers, and there was a shortage of five sera for testing IgA AHAs. All the samples were stored at -80°C until use.
Characterizations of the RA patients and the HC are shown in Table 1. The RA patient cohort was significantly older than the HC group, although there were no gender differences between the two groups. Many RA patients had long disease duration and were treated with biologics. Positive for RF and anti-CCP2 antibodies due to routine laboratory examination were 67/111 (60.4%) and 77/111 (69.4%), respectively.
Furthermore, sera from 61 patients (50 female, 17-83 years old; 11 male, 48—84 years old) with non-RA rheumatic diseases were used for measurement of AHAs. The non-RA was composed of SLE (n=22), polymyositis/dermatomyositis (n=9), Behcet disease (n=5), polymyalgia rheumatica (n=4), primary Sjögren’s syndrome (n=3), limited cutaneous systemic sclerosis (n=3), ANCA-associated vasculitis (n=3), MCTD (n=2), adult-onset Still’s disease (n=2), Takayasu’s arteritis (n=1), IgG4 related disease (n=1), polyarteritis nodosa (n=1), psoriatic arthritis (n=1), RS3PE (n=1), SAPHO syndrome (n=1), sarcoidosis (n=1), and acute transient arthritis (n=1)
Generation of F(ab’)2 fragments by proteolytic cleavage
The following biologics were used: tocilizumab (TCZ;IgG1), infliximab (IFX;IgG1), panitumumab (PAN;IgG2) and natalizumab (NTZ;IgG4). All biologics, except IFX, were humanized monoclonal IgG. Although IFX is chimeric, the hinge region and CH2 domain were derived from human IgG1 according to the database from the National Center for Biotechnology Information (NCBI, USA). We used pepsin (Sigma-Aldrich, St. Louis, USA) and human MMP-3 as proteases to cleave the biologics. The pro-MMP-3 was a kind gift from Daiichi-Fine Chemical, Toyama, Japan.
The biologics were dialyzed with 0.1 M sodium citrate buffer (SCB, pH3.5), and then 100 μg of pepsin in SCB was incubated at 37°C with 10 mg of each biologic. Incubation time was overnight except 2 h for PAN. Tris 1M was added to the IgG solution until the pH increased to 7.4 to stop the digestion. Proteolysis by MMP-3 was performed after the activation of pro-MMP-3 by incubation at 55°C for 25 min . The activated MMP-3 (50 μg) in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 10 mM CaCl2 was mixed with 5mg of each biologic at 37°C. After 2 h of incubation, each a small amount of the reaction mixture (20 μL) was removed, and the reaction was stopped by rapid freezing. The remaining reaction mixture was continued for 24 h and stopped by adjustment to 20 mM ethylenediaminetetraacetic acid (EDTA).
Detection of human IgG fragments
Cleaved human IgG fragments were analyzed by SDS-PAGE in Tris-glycine buffer using 10% gels under non-reducing conditions. Samples were heated at 100°C for 2 min in 50 mM Tris-HCl buffer (pH 6.8) with a final concentration of 20% glycerin and 1% (w/v) SDS. Protein bands were visualized by staining with 0.25% (w/v) Coomassie Brilliant Blue R-250 (Nacalai Tesque, Kyoto, Japan) in 50% (v/v) methanol and 10% (v/v) acetic acid.
Purification of IgG F(ab’)2 fragments
At the outset, human IgG digested by pepsin or MMP-3 was separated by gel filtration on a Sephadex G-150 column (Pharmacia Fine Chemicals, Uppsala, Sweden). The estimated IgG F(ab’)2 fractions were concentrated by Vivaspin 20 (Sartorius Stedium, Goettingen, Germany). Finally, to remove IgG possessing Fc, the concentrated crude IgG F(ab’)2 were applied to a Protein G Mag Sepharose column (GE Healthcare, Uppsala, Sweden) using 50 mM Tris buffer with 150 mM NaCl at pH 7.5. Purification of the IgG F(ab’)2 was confirmed by the SDS-PAGE described above. Each purified F(ab’)2 fragment was denoted by the addition of an italic subscript to specify the protease responsible for its cleavage, e.g., IgG1 F(ab’)2MMP-3.
Measurement of AHAs by enzyme linked immunosorbent assay (ELISA)
ELISA plates (Sumitomo Bakelite, Tokyo, Japan) were coated overnight at 4°C with 100 μL/well of a solution of 0.5 μg/ml IgG F(ab’)2 in 0.1 M carbonate/bicarbonate buffer at pH 9.6. After washing with 10mM Tris buffer containing 0.9% NaCl with 0.05% Tween-20 (TBST) at pH 7.4, serum samples diluted 1:200 with TBST were added to the plate (100 μL/well). These were incubated for 2 h at room temperature (RT). After washing, 100 μL/well of alkaline phosphatase (ALP)-conjugated anti-human IgG Fc (Sigma-Aldrich) diluted 1:10,000 with TBST, or ALP-conjugated anti-human IgA (Sigma-Aldrich) diluted 1:10,000 with TBST was added and incubated for 1 h at RT. After washing, the AHAs were visualized with 1mg/mL of p-nitrophenyl phosphate tablets (Sigma-Aldrich) in diethanolamine buffer at pH 9.8 for 30 minutes or for 2 h for IgA AHA measurement. Absorbance was measured at 405 nm using a microplate reader.
Levels of IgG AHA were calculated by a calibration curve using pooled human IgG purified using 40% ammonium sulfate and DEAE sephadex (Pharmacia Fine Chemicals, Uppsala, Sweden). We arbitrarily defined 1 mg/mL of the pooled IgG as containing 800 arbitrary units (AU)/mL of IgG AHA against IgG F(ab’)2pepsin. We also used this calibration curve for measuring IgG or IgA AHA to other IgG F(ab’)2 fragments.
Inhibition study for specificities of IgG AHAs against IgG1 /IgG4 F(ab’)2pepsin
An equal volume of inhibitors with various concentrations (5, 000, 1, 000, 200, 40, 8, 1.6, 0.32, 0 μg/mL) and 1:200 diluted IgG AHA positive serum from RA patients were thoroughly mixed, followed by incubation for 2 h at RT. The mixtures were added to the ELISA plate (100 μL/well) coated with IgG1/IgG4 F(ab’)2pepsin, and then allowed to react for 2 h at RT. The subsequent procedure was the same as the measurement of AHA described above. The extents of inhibition was expressed as percent inhibition of the AHA responses, calculated as follows: (See Formula 1 in the Supplementary Files)
A: absorbance in the presence of inhibitors, B: absorbance in the absence of inhibitors.
We used the Mann-Whitney U test and the Kruskal-Wallis test to compare the differences between two groups and among multiple groups, respectively. We also used Fisher’s exact and χ2 test for nominal characteristic. To elucidate the independent variables associated with RA diagnosis, univariate logistic regression followed by multivariate logistic regression analysis was performed. Age and gender added to the model. A two-tailed P<0.05 was considered statistically significant. Data were analyzed on a personal computer using SPSS version 19 (IBM Japan, Tokyo, Japan) and Statflex version 6 (Artech, Osaka, Japan).