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 (100Â %) 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 A2 receptor (PLA2R) 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-PLA2R antibody has high affinity and is considered pathogenic in idiopathic membranous
nephropathy 20]. Why IgG4 anti-PLA2R 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-PLA2R, 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.
Table 3. Summary of predominant subclasses in autoantibodies in IgG4-RD and autoimmune diseases
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 pneumoniae33], 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,
PLA2R, 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.