Screening for IgG4-type anti-nuclear antibodies in IgG4-related disease

In IgG4-RD patients, we found no IgG4 ⁺ ANA, but did detect IgG1 ⁺ and IgG2 ⁺ ANA (Fig.  1 and 2). We also found IgG4 ⁺ ANA was very rare, whereas IgG1/2/3 ⁺ ANA were detected in systemic autoimmune diseases (Fig.  2 and 3). Autoantibodies with cytoplasmic patterns in the Fluoro-HepANA™ test are not exact ANA; “anti-cytoplasmic” antibodies—e.g., anti-SS-A/Ro, anti-aminoacyl-tRNA synthetase, and anti-signal recognition particle antibodies—are known in SS and PM. Subclass-based ANA tests found IgG1/2/3 ⁺ anti-cytoplasmic antibodies, but not IgG4 (Fig.  2, 3).

IgG4 ⁺ ANA is very rare in systemic autoimmune diseases, possibly because serum IgG4/IgG ratios are low, less than 5 %, in these diseases (Table  2). However, IgG4 ⁺ ANA was not detected despite high serum IgG4/IgG ratios (43 %) in IgG4-RD. This implies that IgG4 itself is not used to make ANA.

Several studies have investigated ANA subclasses in systemic autoimmune diseases. Zouali et al. reported that in SLE and mixed connective tissue disease, anti-double-stranded DNA (dsDNA) antibody was IgG1/3-dominant, and anti-RNP was IgG2-dominant [ 11]. Anti-Sm, anti-RNP, and anti-dsDNA in SLE [ 12], anti-SS-A/Ro and anti-SS-B/La in SS [ 13], and anti-Scl-70 in SSc [ 14] are reportedly IgG1-dominant. However, IgG4-type ANA was hardly detected in all the above reports. Rigopoulou et al. examined primary biliary cirrhosis cases, and found that ANA was IgG1/3-dominant but IgG4 was not detected by subclass-based IIF [ 15]. The reason IgG2-type ANA was remarkably frequent in our study whereas IgG1 and IgG3 were predominant in previous studies might be that second antibody affinities differed between studies. In the subclass-based ANA test, titers cannot be accurately compared between subclasses, as the second antibodies are different. In past studies, IgG2-type ANA was also detected at moderate levels, whereas IgG4-type ANA was constantly negative or at low levels. In our study, IgG4-type ANA was also hardly detected.

Autoimmune pancreatitis (AIP) is an organ-specific disorder seen in IgG4-RD. Various autoantibodies, such as anti-lactoferrin [ 5] and anti-carbonic anhydrase II [ 6] antibodies, are seen in AIP. Asada et al. found anti-pancreatic secretory trypsin inhibitor (PSTI) antibody in AIP, and showed that the titers of anti-PSTI antibody moved in parallel with serum IgG4 levels [ 16]. IgG4 levels change in parallel with IgG4-RD disease activity, as reported in many studies, including our previous study [ 17]. Asada et al. thought that anti-PSTI might be an important factor in the pathophysiology. However, immunoblotting of subclasses with anti-IgG1 or anti-IgG4 as second antibodies showed the subclass was not IgG4 but IgG1. Possibly, IgG4-type autoantibodies are difficult to produce in IgG4-RD patients.

However, some autoimmune diseases reportedly show IgG4-type autoantibodies. Rock et al. reported that IgG4 was the most common (100 %) of anti-desmoglein (Dsg)-1 antibodies detected in sera of patients with pemphigus foliaceus, and showed the pathogenicity of IgG4-type anti-Dsg-1 antibody using Balb/c mice [ 18]. Anti-Dsg-3 antibody in pemphigus vulgaris was also IgG4-predominant [ 19]. Beck et al. showed by immunoblotting that anti-phospholipase A ₂ receptor (PLA ₂R) antibody in idiopathic membranous nephropathy mainly consisted of IgG4 [ 20]. IgG4 is reportedly predominant in anti-neutrophil cytoplasmic antibody (ANCA). C-ANCA (IIF), proteinase-3 (PR3)-ANCA (ELISA), and myeloperoxidase (MPO)-ANCA (ELISA) in granulomatosis with polyangiitis (GPA) [ 21], and MPO-ANCA (ELISA) in propylthiouracil-induced vasculitis [ 22] were IgG1/4-dominant. Others similarly reported that IgG4 made up most C-ANCA (IIF) and PR3-ANCA (ELISA) in vasculitides [ 23, 24]. Engelmann et al. reported that anti-cyclic citrullinated peptide (CCP) antibody was IgG1/4-dominant in RA [ 25]. However, IgG4 in vasculitides and RA might not be pathophysiologically important. In functional analyses of ANCA, IgG1 and IgG3 PR3-ANCA can stimulate neutrophils [ 26], whereas IgG4 PR3-ANCA was only weakly stimulatory to neutrophils [ 27]. In RA patients who had HLA-DR4-shared epitope, Engelmann et al. found IgG3 anti-CCP antibody to be predominant, and considered that IgG3-type antibody might be more important in the pathophysiology of RA [ 28]. As IgG4 has poor ability to activate complements and antibody-dependent cellular cytotoxicity [ 29– 32], IgG4 is unlikely to take part in mechanisms of tissue damage in autoimmune diseases.

Interestingly, there seem to be pathogenic and non-functional IgG4-type autoantibodies. IgG4-type ANCA is considered less pathogenic, compared to other subclass ANCA in ANCA-associated vasculitis [ 26, 27]. The affinities between IgG4-type and other subclass ANCA should be equal, but the abilities of complement activation are different, so that the role of IgG4-type ANCA can be less significant than that of other subclass ANCA. On the other hand, IgG4 anti-PLA ₂R antibody has high affinity and is considered pathogenic in idiopathic membranous nephropathy [ 20]. Why IgG4 anti-PLA ₂R antibody can exert pathogenicity without ability of complement activation may be because the pathogenicity is brought by the destruction of electrical barriers of glomerular basement membrane.

Taken together, IgG4 usage rates differ among autoantibodies and among diseases. IgG4 is associated with anti-Dsg-1/3, anti-PLA ₂R, anti-CCP antibodies, and ANCA, but not with anti-PTSI antibody in AIP or ANA in IgG4-RD and systemic autoimmune diseases (Table  3). This asymmetry implies that IgG4 has unknown but certain physiological or pathological functions. Further analyses are needed to know its role.

In the present study, we observed ANA patterns differed among IgG subclasses in some cases (Fig.  3, 4). When a case has several autoantibodies, the utilized subclasses differ by autoantigens. This can be explained by the hypothesis that each IgG subclass prefers to cover its own spectrum of antigens. The reason we hardly found IgG4 in ANA might be that IgG4 does not cover antigens that can be detected by the ANA test—i.e., nuclear antigens or related microbial antigens. Selective IgG2 subclass deficiency is often associated with bacterial infection by Neisseria meningitidis and Streptococcus pneumoniae [ 33, 34], so that IgG2 is considered to have a role in protection from these bacteria. The role of IgG4 has not been sufficiently understood. If IgG4 is related to some microorganism type, and if the microorganism antigens and autoantigens are similar, as with Dsg-1/3, PLA ₂R, PR3, and citrullinated proteins, it would explain why IgG4-type antibody against those proteins was dominantly generated.

Our results imply that IgG4-RD is not an autoimmune disease, and that high levels of serum IgG4 in IgG4-RD are only nonspecific. Subclass-based ANA tests in this study covered both nuclear and cytoplasmic antigens in HEp-2 cells, and can screen a wide range of unmodified ubiquitous antigens. However, this analysis has limitations: modified antigens like citrullinated proteins and organ-specific antigens are not screened. The number of cases is limited in this study. There remains a possibility that unknown IgG4-type autoantibodies might be found in IgG4-RD. A further analysis is needed.