Prevalence of pemphigus and pemphigoid autoantibodies in the general population
- Wiebke Prüßmann†1, 2,
- Jasper Prüßmann†1, 2,
- Hiroshi Koga†1, 2Email author,
- Andreas Recke1, 2,
- Hiroaki Iwata1, 6,
- David Juhl3,
- Siegfried Görg3,
- Reinhard Henschler4,
- Takashi Hashimoto5,
- Enno Schmidt1,
- Detlef Zillikens1, 2,
- Saleh M. Ibrahim1, 2 and
- Ralf J. Ludwig1, 2
© Prüßmann et al.; licensee BioMed Central. 2015
Received: 20 February 2015
Accepted: 29 April 2015
Published: 15 May 2015
Mucocutaneous blistering is characteristic of autoimmune bullous dermatoses (AIBD). Blisters are caused by autoantibodies directed against structural components of the skin. Hence, detection of specific autoantibodies has become a hallmark for AIBD diagnosis. Studies on prevalence of AIBD autoantibodies in healthy individuals yielded contradictory results.
To clarify this, samples from 7063 blood donors were tested for presence of anti-BP180-NC16A, anti-BP230 and anti-Dsg1/3 IgG by indirect immunofluorescence (IF) microscopy using a biochip.
Cumulative prevalence of these autoantibodies was 0.9 % (CI: 0.7–1.1 %), with anti-BP180-NC16A IgG being most prevalent. Validation of IF findings using ELISA confirmed presence of autoantibodies in 7/15 (anti-Dsg1), 6/7 (anti-Dsg3), 35/37 (anti-BP180-NC16A) and 2/3 (anti-BP230) cases. Moreover, in 16 samples, anti-BP180-NC16A autoantibody concentrations exceeded the cut-off for the diagnosis of bullous pemphigoid. Interestingly, these anti-BP180-NC16A autoantibodies from healthy individuals formed immune complexes with recombinant antigen and dose-dependently activated neutrophils in vitro. However, fine-epitope mapping within NC16A showed a different binding pattern of anti-BP180-NC16A autoantibodies from healthy individuals compared to bullous pemphigoid patients, while IgG subclasses were identical.
Collectively, we here report a low prevalence of AIBD autoantibodies in a large cohort of healthy individuals. Furthermore, functional analysis shows differences between autoantibodies from healthy donors and AIBD patients.
KeywordsAutoimmunity Skin Type XVII collagen BP180 Desmoglein Pemphigoid Pemphigus
Autoimmune bullous dermatoses (AIBD) are clinically characterized by chronic mucocutaneous blistering, leading to severe morbidity and increased mortality [1–4]. Blister formation is directly or indirectly caused by autoantibodies binding to structural proteins of the skin [5, 6]. Depending on the location of the blister and the targeted autoantigens, AIBD can be classified as pemphigus and pemphigoid disease, epidermolysis bullosa acquisita (EBA) and dermatitis herpetiformis [7, 8]. Epidemiological studies have documented the incidence of AIBD in several geographic regions. In central Europe, bullous pemphigoid (BP) had the highest incidence, with 6.1 to 42.8 cases per million persons per year [1, 3, 4, 9–13]. For pemphigus disease, including pemphigus vulgaris (PV) and pemphigus foliaceus (PF), the reported incidence ranged from 0.6 to 6.8 cases per million persons per year [1, 14–16].
Previously reported prevalence rates of autoantibodies to structural proteins of the skin
Normal subjects (53)
Blood donors (401)
Normal subjects (53)
Blood donors (401)
Healthy volunteers (47)
Blood donors (494)
Normal subjects (336)
Healthy subjects (61)
Normal controls (109)
Healthy controls (56)
Blood donors (483)
This study included 7063 normal blood donors from the Institutes for Transfusion Medicine Lübeck, Kiel and Frankfurt between August 2010 and March 2011. All samples were anonymized immediately after blood drawing to comply with requirements by the ethics committees. To avoid duplicate testing of the same person, blood samples from all frequent donors were collected within eight weeks, which is the shortest possible donation interval for men. Further collection was restricted to first-time donors. All plasma aliquots were stored at −20 °C until further testing. All participants signed an informed consent. The study was performed according to the principles of the Declaration of Helsinki and was approved by the local ethics committees (10–094, the ethics committee of the University of Lübeck).
Plasma samples from all 7063 donors were analyzed for the presence of pemphigus- and pemphigoid-related antibodies with a commercial indirect immunofluorescence (IF) assay (dermatology-mosaic 7, EUROIMMUN AG, Lübeck, Germany) at a 1:10 dilution. The assay included the following substrates: primate esophagus, primate salt split skin, recombinant tetrameric BP180-NC16A and transfected HEK293 cells that express recombinant BP230, Dsg1 or Dsg3. Specific fluorescence (Additional file 1) at a dilution of 1:10 was considered positive, as advised in the instruction manual. Indirect IF microscopy-positive samples were subsequently evaluated for specific antibodies (IgG) with enzyme-linked immunosorbent assays (ELISA, Anti-BP180-NC16A-ELISA, Anti-BP230-CF-ELISA, Anti-Dsg-1-ELISA, Anti-Dsg3-ELISA, all EUROIMMUN AG) [19, 20]. These different ELISAs share a detection threshold of 0.5 to 0.6 RU/ml and linearity from at least 10 to 199 RU/ml. Calibrators with 2, 20 and 200 RU/ml were used to quantify the measured extinction rates with computer software. For values below 1 RU/ml, the software is technically unable to calculate exact values, so that values for these samples are set “<1 RU/ml” and were considered negative. To consolidate the read out from indirect IF microscopy, a comparable number of indirect IF microscopy-negative samples were also tested for each of the relevant antigens by ELISA.
ROS release from human PMN
ROS release from immune complex-activated human PMN was performed as described in  with some modifications. Briefly, 500 ng of tetrameric form of the NC16A domain of BP180 recombinant protein  was coated each wells followed by addition of 1:10 diluted plasma samples and incubation for 1 h. After washing well, PMN was added to each well. Each sample was tested with PMNs isolated from 3 healthy donors in duplicate.
Determination of IgG subclasses
For determination of BP180-NC16A IgG1-4 reactivity, a commercial BP180-NC16A ELISA was used  and adapted to detect IgG1-4 antibodies. After incubation with human plasma, ELISA plates were incubated with biotin-conjugated mouse anti-human IgG1, IgG2, IgG3 and IgG4 antibodies (Invitrogen, Camarillo, CA), followed by incubation with peroxidase-conjugated donkey anti-mouse IgG antibody (minimal cross-reaction to serum proteins of other species, Jackson ImmunoResearch Laboratories, West Grove, PA). As we found low BP180-NC16A-IgG4 reactivity, the sensitivity of the protocol was evaluated using sera from pemphigus patients. Here, we frequently detected anti-Dsg3 IgG4 autoantibodies (not shown). The cut-off values were determined as mean value +3SD from 10 healthy blood donors.
Fine epitope mapping of anti-BP180-NC16A reactivity
For determination of the reactivity of BP180-NC16A+ sera to BP180-NC16A2-3 (Biotin-RSILPYGDSMDRIEKDRLQGMAPAAGADL, Peptides and elephants GmbH, Potsdam, Germany), we followed previously described protocols . Briefly, streptavidin-coupled microtiter plates were coated for 1 h on a shaker with 200 ng of the biotinylated synthetic peptide. After washing the plate, sera diluted 1:200 in PBST were added to the plate and incubated for 1 h, followed by HRP-conjugated anti-human IgG incubation for 1 h. Bound IgG were visualized by 1-Step Turbo TMB-ELISA (Pierce, Rockford, IL) and measured at 450 nm. The cut-off value was determined as mean value +3SD from 10 healthy blood donors. Each sample was tested in duplicate.
Dsg3 internalization assay
HaCaT cells were cultured with keratinocyte growth medium 2 (PromoCell GmbH, Heidelberg, Germany). At confluence, the calcium concentration of the medium was adjusted to 1.5 mM and incubated for 24 h. Then, 1 mg/ml of IgG purified from healthy donors (Intratect®, Biotest Pharma GmbH, Dreieich, Germany), PV patients or healthy donors with or without anti-Dsg3 antibodies at dilution indicated at the Figure legends was added to HaCaT cells and incubated at 37 °C in a CO2 incubator for 24 h. After incubation, the cells were fixed and permeabilized followed by blocking with 1 % BSA and 10 % normal goat serum. Dsg3 was detected by mouse anti-Dsg3 antibody (Acris antibodies, Herford, Germany) and Cy3-goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Fluorescence images were obtained using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). To determine the anti-Dsg3 antibody index value, all samples were adjusted 15 mg/ml and measured by anti-Dsg3-ELISA (EUROIMMUN AG).
Statistical analysis was performed with Gnu R (version 2.14.1, http://www.r-project.org) together with package “epitools” (version 0.5–6) for calculation of Wilson confidence intervals for binomial counts. ELISA values of IIF-positive and IIF-negative samples were compared by the Wilcoxon rank-sum test with continuity correction using SigmaPlot (Version 12).
Low prevalence of pemphigus and pemphigoid autoantibodies in the general population
Prevalence of pemphigus and pemphigoid autoantibodies in healthy blood donors
IIFM positive* [n]
IIFM positive* [%]
Below diagnostic cut-off [n]
Above diagnostic cut-off (≥20 RU/ml) [n]
Validation of detected autoantibody prevalence
BP180-NC16A autoantibodies from healthy blood donors form immune complexes and dose-dependently activate neutrophils
BP180-NC16A autoantibodies detected in healthy blood donors are predominantly of the IgG3 subclass
Subclasses of anti-BP180-NC16A IgG in healthy blood donors and bullous pemphigoid patients
Healthy, < 20 RU/ml*
Healthy, ≥ 20 RU/ml*
Healthy blood donors
Epitopes targeted by BP180-NC16A autoantibodies detected in healthy blood donors do not overlap with those predominantly recognized by BP patients
IgG from healthy individuals with antibody reactivity to Dsg3 do not induce Dsg3 internalization in vitro
Our data document an overall low prevalence of pemphigus and bullous pemphigoid IgG autoantibodies in a collection of 7063 blood donors. To date, this is the largest population studied for pemphigus and pemphigoid autoantibodies. In line with BP being the most common AIBD in central European countries, we found BP-associated autoantibodies more frequently than pemphigus-associated autoantibodies. Compared to the reported prevalence of pemphigus (0.004 %) and pemphigoid (0.016 %) in Denmark , the autoantibody prevalence reported here is 25 or 37-fold higher. Hence, one may assume that in BP and pemphigus, as reported for other autoimmune diseases [17, 18], autoantibodies predate the onset of clinical disease. Based on the prevalence of pemphigus and pemphigoid antibodies in healthy individuals and the prevalence of AIBD, every 25th individual with autoantibodies against pemphigus antigens is likely to develop pemphigus; similarly, every 37th healthy individual with anti-BP180-NC16A IgG reactivity would be expected to develop BP. This process would be initiated by the break of tolerance and the generation of autoantibodies to structural proteins of the skin. Subsequently, an AIBD will become clinically manifest in only a minority of affected autoantibody-positive healthy individuals. In the future, additional biomarkers will hopefully allow clinicians to distinguish between “resistant” antibody-positive individuals and those who are prone to developing overt clinical disease.
In contrast to the detection of other autoantibodies in healthy individuals, e.g., ANA , the potential pathogenic activity of anti-BP180-NC16A IgG and Dsg1/3 can be tested . As shown here, anti-BP180-NC16A IgG from healthy blood donors induces neutrophil activation in vitro. Because neutrophil activation is a key requirement for BP pathogenesis , the blister-inducing ability of these autoantibodies can be assumed. Furthermore, anti-BP180-NC16A IgG subclasses were identical between healthy blood donors and BP patients (Table 3). Our result that no IgG4 anti-BP180-NC16A antibodies were detected in BP patients seemingly contradicts a previous report , although it might be due to the small number of BP serum samples included in our current study. In addition, the fine mapping of the autoantibody response in our samples revealed differences in the cohort of healthy blood donors compared to BP patients (Fig. 3). This is similar to observations made for endemic pemphigus with autoantibodies directed against Dsg1. Epidemiologic studies demonstrated a shift from IgG1 to IgG4 autoantibodies  and intramolecular epitope spreading towards certain epitopes  upon development of skin blisters in endemic pemphigus foliaceus. The anti-Dsg3-positive sera from our collection of healthy blood donors demonstrated a lack of pathogenic potential of these autoantibodies, which is most likely due to the low concentration of autoantibodies in the samples (Fig. 4).
Our results on the prevalence of autoantibodies to structural proteins of the skin also resolve the discrepancies among the reports on this topic to this point. Regarding Dsg1 antibodies, the reported prevalences in healthy individuals ranged from 0 % to 0.7 %. A study by Ishii et al. included 53 healthy control sera, and none reacted to baculovirus-expressed Dsg1 in an ELISA . In a study by Schmidt et al., 401 healthy blood donors were included, and circulating Dsg1 antibodies were detected using ELISA with the Dsg1 ectodomain, expressed in a human cell line, as a substrate. There, 3 of the 401 healthy controls had ELISA values above the diagnostic cut-off for pemphigus . We showed here that 0.1 % of healthy individuals have detectable Dsg1 autoantibodies, which were detected by indirect IF microscopy using Dsg1-expressing cells as a substrate. Validation of the 15 indirect IF-positive samples by ELISA showed reactivity in 7 of the samples. However, with the exception of one sample, the values were below the diagnostic cut-off value. Compared with the study of Schmidt et al. we found far fewer Dsg1-reactive samples. This may be explained by the fact that Schmidt et al. defined Dsg1 positivity sorely by ELISA. In contrast, in our study, only those sera were considered positive that reacted with Dsg1 by both indirect IF microscopy and ELISA. Similar considerations also hold true for the other autoantigens in AIBD tested within our study (Table 1).
In summary, we here document the prevalence of pemphigus and pemphigoid autoantibodies in a large cohort of healthy blood donors. The comparison of the autoantibody prevalence and the prevalence of the corresponding AIBD may suggest that these diseases develop over a prolonged period of time.
Grant support: DFG Excellence Cluster “Inflammation at Interfaces” (DFG EXC 306/1 and 2), GRK1727/1 “Modulation of Autoimmunity” and the Uehara Memorial Foundation.
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