Serum levels of IgE anti-BP180 and anti-BP230 autoantibodies in patients with bullous pemphigoid

Journal of Dermatological Science (2008) 49, 153—161

www.intl.elsevierhealth.com/journals/jods

Serum levels of IgE anti-BP180 and anti-BP230 autoantibodies in patients with bullous pemphigoid Nobuko Ishiura a, Manabu Fujimoto b,*, Rei Watanabe a, Hiroko Nakashima a, Yoshihiro Kuwano a, Norihito Yazawa a, Takeshi Echigo b, Hitoshi Okochi c, Kunihiko Tamaki a a

Department of Dermatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan b Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan c Department of Tissue Regeneration, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan Received 9 April 2007; received in revised form 20 August 2007; accepted 25 August 2007

KEYWORDS Bullous pemphigoid; Anti-BP180 antibody; Anti-BP230 antibody; IgE; Autoimmunity

Summary Background: Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins, BP180 and BP230. NC16A, a non-collagenous stretch of the BP180 ectodomain is the primary target of pathogenic IgG antibodies. Whereas IgG anti-BP180 autoantibodies play a primary role in the pathogenesis, there is a growing number of data regarding the potential pathogenic roles of IgE class autoantibodies in BP. Objectives: To examine the levels of IgG and IgE autoantibodies against BP180 and BP230, and to investigate mutual association and clinical relevance. Methods: Sera obtained from 67 BP patients and 36 healthy donors were subjected to ELISA assays to measure serum IgG and IgE levels of anti-BP180 and anti-BP230 antibodies. Results: IgG anti-BP180 antibodies were positive in 63 (94%) of 67 BP patients. IgG anti-BP230, IgE anti-BP180, and IgE anti-BP230 antibodies were found in 48 (72%), 20 (30%) and 45 (67%), respectively. IgG anti-BP180 levels were correlated with the affected areas. IgG anti-BP230 antibodies tended to increase in proportion to elongation of disease duration. IgE anti-BP230 levels showed a strong association

* Corresponding author. Tel.: +81 76 265 2341; fax: +81 76 234 4270. E-mail address: [email protected] (M. Fujimoto). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.08.008

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N. Ishiura et al. with local eosinophil accumulation, while the levels were reversely related with the affected areas in BP. Conclusions: IgE autoantibodies to BP180 and BP230 are detected at high frequencies in BP. IgE anti-BP230 antibodies may have a role in attracting eosinophils to the skin lesions. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies (autoAbs) against components of the epidermal basement membrane zone (BMZ) of the skin [1]. BP is characteristic from other autoimmune blistering diseases in that blood eosinophils and serum IgE are increased in many cases [2,3]. Eosinophils are predominant cells of the lesional infiltrates. BP autoAbs predominantly recognize two structural components of hemidesmosomes, BP180 (BPAG2, type XVII collagen) and/or BP230 (BPAG1) [4—8]. BP180 is a transmembrane protein of the collagen family [9,10], and has been commonly accepted as the primary target of pathogenic IgG autoAbs [11—14]. The major epitope of IgG anti-BP180 Abs is mapped to the NC16A domain, a non-collagenous stretch of its ectodomain [15]. By contrast, BP230 is a plakin family protein located in the cytoplasm [16—18]. Although several studies indicate that anti-BP230 Abs may also play a pathogenic role [19], it is unclear whether BP230 autoAb reactivity directly contributes to blister formation or whether these autoAbs represent an epiphenomenon. That autoAbs to BP180 are critical in subepidermal blister formation in BP patients has been strongly supported by a passive transfer model. AutoAbs to BP180 can reproduce many characteristic features of BP [11,20], dependently upon complement activation, neutrophil recruitment, and protease release [12,21—25]. Nonetheless, in this mouse model the infiltrate is composed almost exclusively of neutrophils, which may suggest that other components, such as IgE autoAbs, may also play an important role. A growing number of data have demonstrated the specificity and potential pathogenic role of IgE class autoAbs in BP [3,26—28]. Elevated circulating IgE levels were observed in 70% of BP patients [2,28], and IgE deposition at the BMZ is also found in 25% of BP patients [2,26,29,30]. Several studies have suggested that IgE autoAbs also target the NC 16A region of BP180 [3,28,31], whereas others reported that the IgE autoAbs primarily target BP230 or the intracellular domain of the BP180 [3,32]. In this study, we examined IgG and IgE Ab titers against

two hemidesmosomal proteins, BP180 and BP230 in the sera from BP patients by ELISA, and analyzed their relative contributions.

2. Materials and methods 2.1. Patients Sera were obtained from 67 patients (35 males and 32 females) with BP who presented to University of Tokyo Hospital and Kanazawa University Hospital from 1993 to 2005. Patients with BP fulfill the four criteria: characteristic clinical findings of, typically, pruritic plaques and tense blisters of the trunk and extremities, without forming scars or milia; histological evidence of subepidermal blisterings with many infiltrating polymorphonuclear cells, especially eosinophils, along the basal membrane and within the blister cavity; evidence of in-vivo bound C3 and/or IgG autoAbs along the basement membrane by direct immunofluorescence; and evidence of circulating anti-hemidesmosomal Abs by indirect immunofluorescence, especially evidence of the deposit on the epidermal side of bulla by use of 1 M NaCl-split skin. Ages of BP patients were 72.4  16.1 (range; 17—93) years. The sera were obtained when the patients first presented and were diagnosed with BP and were taken prior to treatment. No patients had been treated with corticosteroids or with any immunosuppressive drugs before. Control sera were collected from 36 healthy donors (18 males and 18 females). Ages of controls were 70.1  9.8 (range; 51—85) years. There was no statistical significant difference in sex and age between control and the patient’s group. No patients had asthma, atopic dermatitis, allergic rhinitis, lymphomas or any other diseases which could affect serum IgE or eosinophils. In the correlation analysis between each autoAb and the other disease markers, 28 BP patients (17 males and 11 females) who satisfied all serological, pathological and clinical data were examined. As the disease markers, we examined five items: disease severity (the percentage of the area covered with the skin lesions), disease duration, serum IgE levels, serum eosinophil counts, and local eosinophil counts

Serum levels of IgE anti-BP 180 and anti-BP230 autoantibodies infiltrating around edematous erythemas and blisterings of skin biopsy specimens. Other complications in the studied patients were hypertension (7 patients), diabetes mellitus (5), cerebral infarction (5), gastric ulcer (2), gallstone (2), benign prostate hyperplasia (2), hysteromyoma (1), Parkinson’s disease (1), Marfan’s syndrome (1), lung cancer (2), esophageal carcinoma (1), hepatocellular carcinoma (1), and prostate cancer (1). Fresh venous blood samples were centrifuged shortly after clot formation. All samples were stored at 70 8C prior to use as archival sera. Clinical manifestations and laboratory findings of each patient at the time of serum sampling were obtained from the medical records. Institutional review board approval and informed consent from all patients were obtained.

2.2. Pathological assessment The number of infiltrating eosinophils around edematous erythemas (infiltrated plaques or papules) and blisterings was counted in H&E-stained sections of skin biopsy specimens. The number of eosinophils in a microscopic field (200) beneath the center of the fresh bulla cavity and those in two microscopic fields (200) on the border between the bulla and edematous erythemas were summed up. Eosinophils in the bulla were not included. Each specimen was examined independently by three investigators in a blind fashion. In 28 BP patients, the local eosinophil counts were 296  349 (range; 26—1846) per power field at 200. There was no statistically significant difference of local eosinophil counts between sex and age.

2.3. Clinical assessment As an index of disease severity, we estimated the percentage of the area covered with the skin lesions (primary edematous erythemas and blisters) on the whole body surface, with rule of nine, at the time when serum samples were obtained. In 28 BP patients, the percentage of the skin lesions was 58  31 (range, 9—90)%. The time interval between the onset of symptoms and the time when the diagnosis was made and serum samples were obtained was defined as the disease duration. In 28 BP patients, the disease duration was 38  47 (range, 2—180) days. There was no significant difference of percentage of the skin lesions or the disease duration between sex and age, respectively.

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patients, serum IgE levels were 2071  3807 (range, 16—14,000) IU/ml, and blood eosinophil counts were 1626  1677 (range, 122—5809)/mm3. There was no significant difference of serum IgE levels or blood eosinophil counts between sex and age, respectively.

2.5. ELISA assays for detection of IgG and IgE class Abs against BP180 and BP230 Serum titers of anti-BP180 and anti-BP230 Abs were measured using ELISA kits from MBL (Nagoya, Japan). In BP180 kit, BP180 NC16A recombinant proteins were coated in the microplate wells (Fig. 1A). Serum samples were diluted to 1:100 in assay diluent. For detecting anti-BP180 IgG Abs, standards and diluted serum samples were added to the wells. After rinsing any unbound substances, peroxidase-conjugated goat polyclonal anti-human IgG Abs were added to the wells. Color developed using a microplate reader set to 450 nm, in proportion to the amount of anti-BP180 Abs bound in the initial step in each sample. IgG anti-BP230 Abs were also measured similarly using BP230 ELISA kit, in which C- and N-terminal recombinant proteins of BP230 were coated (Fig. 1B). Serum samples were diluted to 1:100. Standards and diluted serum samples were added to the wells, and after rinsing any unbound substances, peroxidase-conjugated goat polyclonal anti-human IgG Abs were added to the wells. Color developed using a microplate reader set to 450 nm, in proportion to the amount of anti-BP230 Abs bound in the initial step in each sample. Cut-off levels for IgG anti-BP180 and anti-BP230 Abs have been already determined as 99 percentile of normal controls by the manufacturer. For detecting IgE class Abs against BP180 and BP230, we first incubated serum samples twice in the microplate wells coated with protein A (Pierce, Rockford) in order to preabsorb IgG in the serum

2.4. Laboratorial assessment For laboratorial assessment, we checked serum IgE levels and blood eosinophil counts. In the 28 BP

Fig. 1 Schematic diagrams depicting the structure and the regions of the recombinant proteins of human BP180 (A) and BP230 (B), respectively.

156 samples. Preabsorption using protein G was also tested, and essentially the same results were obtained. These precleared samples were next added to the microplate wells coated with BP180 or BP230 proteins as described above. After rinsing,

N. Ishiura et al. peroxidase-conjugated goat polyclonal anti-human IgE Abs were added, followed by color development. We set the cut-off value as average +5S.D. of normal controls for IgE anti-BP180 Abs and anti-BP230 Abs to exclude the false positive severely.

Fig. 2 (A and B) IgG reactivity against (A) NC16A domain of BP180 and (B) C- and N-terminal recombinant proteins of BP230 in the sera from BP patients and normal controls. The dotted line shows the cut-off value (BP180, 5 index; BP230, 9 index). (C and D) IgE reactivity against (C) NC16A domain of BP180 and (D) C- and N-terminal recombinant proteins of BP230 in the sera from BP patients and normal controls. The dotted line shows the cut-off value as evaluated by mean +5S.D. value of absorption levels (BP180, 0.398; BP230, 0.204).

Serum levels of IgE anti-BP 180 and anti-BP230 autoantibodies

2.6. Statistical analysis Correlations of the serum titers of anti-BP180 or anti-BP230 Abs with disease markers in BP patients were analyzed by Pearson’s product-moment correlation coefficient. Statistical analysis on disease markers between positive and negative group of each Ab levels in BP patients were tested by Mann—Whitney’s U-test. A p-value less than 0.05 was considered statistically significant. All data are shown as means  S.D.

3. Results 3.1. IgG and IgE reactivity of BP sera against BP180 and BP230 Using ELISA, IgG and IgE reactivity against hemidesmosomal proteins, BP180 and BP230 (Fig. 1), were measured in the sera from 67 patients with BP (35 males and 32 females), and 36 normal controls (18 males and 18 females). No association was observed between age or sex and serum Ab titers in patients or controls (data not shown). Fig. 2 shows levels of IgG Abs against BP180 (Fig. 2A), BP230 (Fig. 2B), and IgE Abs against BP180 (Fig. 2C), and BP230 (Fig. 2D).

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As for levels of anti-BP180 IgG Abs and anti-BP230 IgG Abs, the cut-off values have been already determined in the kits: 5 index and 9 index, respectively. As for IgE Abs to BP180 and BP230, the mean + 5S.D. absorption levels of sera from normal controls were determined as cut-off values. These values were of 0.398 for IgE anti-BP180 Abs and 0.204 for IgE antiBP230 Abs, respectively. When sera from 20 patients with atopic dermatitis were tested for IgE autoantibodies, none of them were above the cut-off values in either BP180 or BP230 ELISA (data not shown). IgG Abs against BP180 were positive in 63 (94%) out of 67 patients with BP. By contrast, 48 (72%) of 67 BP patients had elevated levels of IgG anti-BP230 Abs. IgE Abs to BP180 and BP230 were positive in 20 patients (30%) and 45 patients (67%) of BP patients, respectively. There was no correlation between each Ab titer and other complications including internal malignancy (data not shown).

3.2. Mutual correlations of IgG/IgE antiBP180 and antiBP230 Ab levels Of 67 patients with BP, 46 (69%) were positive for IgG Abs against both BP180 and BP230 (Fig. 3A). Two patients had neither IgG anti-BP180 Abs nor IgG

Fig. 3 Correlations between (A) IgG anti-BP180 levels and IgG anti-BP230 levels; (B) IgG anti-BP180 levels and IgE antiBP180 levels; (C) IgE anti-BP180 levels and IgE anti-BP230 levels; and (D) IgG anti-BP230 levels and IgE anti-BP230 levels in BP patients. Cut-off values were shown by dotted lines. The numbers in quadrants indicate the numbers of patients positive or negative for the corresponding Abs.

158 anti-BP230 Abs by ELISA. There was no significant correlation between IgG anti-BP180 levels and IgG anti-BP230 levels (R = 0.233). Those who simultaneously possessed IgG and IgE anti-BP180 Abs were 20 (30%) of 67 patients (Fig. 3B). All patients positive for IgE anti-BP180 Abs showed IgG reactivity to BP180 as well, although correlation between IgG and IgE anti-BP180 levels was not significant

N. Ishiura et al. (R = 0.182). 15 (22%) of 67 BP patients were positive for both of IgE Abs against BP180 and BP230 (Fig. 3C). There was no significant correlation between IgE and IgG anti-BP180 levels (R = 0.196). Furthermore, 41 of 67 patients (61%) had elevated levels of both IgG and IgE anti-BP230 Abs (Fig. 3D), while those levels did not have correlation (R = 0.282). Thus, there were no significant mutual

Fig. 4 Correlations between (A) IgG anti-BP180 levels and the affected areas; (B) IgE anti-BP230 levels and the affected areas; (C) IgG anti-BP230 levels and the disease duration; (D) IgE anti-BP230 levels and serum IgE levels; (E) IgG antiBP180 levels and serum eosinophil counts; (F) IgG anti-BP230 levels and serum eosinophil counts; and (G) IgE anti-BP230 levels and local eosinophil counts in BP patients.

Serum levels of IgE anti-BP 180 and anti-BP230 autoantibodies correlations titers.

among

IgG/IgE

anti-BP180/BP230

3.3. Clinical correlations of antihemidesmosomal autoAb levels To determine clinical relevance, correlation of serum anti-hemidesmosomal autoAb levels with the areas of the skin lesions (edematous erythemas and blisters) was assessed. The affected areas were positively correlated with IgG ani-BP180 Ab levels (R = 0.468, p < 0.05, Fig. 4A) and negatively with IgE anti-BP230 Ab levels (R = 0.564, p < 0.05, Fig. 4B). Correlation of anti-hemidesmosomal autoAb levels with the disease duration without any systemic treatment was also analyzed. IgG anti-BP230 titers exhibited significant correlation with disease duration (R = 0.474, p < 0.05, Fig. 4C), while the others did not. Therefore, IgG anti-BP230 titers tended to increase in proportion to elongation of disease.

3.4. Laboratorial correlations of antihemidesmosomal autoAb levels Laboratorial correlations were also assessed. Serum IgE levels were elevated in 71% of BP patients. Serum IgE levels were significantly associated with IgE anti-BP230 levels (R = 0.464, p < 0.05, Fig. 4D) but not with IgE anti-BP180 levels (R = 0.115) or with IgG anti-hemidesmosomal Abs. Blood eosinophils often increase during the progression of BP. Significant correlations with blood eosinophil counts were observed in IgG anti-BP180 (R = 0.429, p < 0.05, Fig. 4E) and IgG anti-BP230 (R = 0.426, p < 0.05, Fig. 4F), but not in IgE autoAb levels. Therefore, IgE autoAbs to BP230 were related with serum IgE levels, while IgG autoAb levels against BP180 and BP230 were fairly related with blood eosinophil levels.

3.5. Correlation of anti-hemidesmosomal autoAb levels with local eosinophil counts of skin biopsy around fresh bullas in BP The major pathological characteristic of BP is increase of eosinophils beneath the bulla cavities and around the bullas. Local eosinophils of skin samples from edematous erythemas and blisters were counted and summed up of three points, as described in Section 2. IgE anti-BP230 titers showed a significant correlation with local eosinophil counts (R = 0.527, p < 0.05, Fig. 4G), while the others did not. Moreover, local eosinophil numbers in the specimen from BP patients positive for IgE anti-BP230 Abs were significantly higher than those negative (364  379 versus 91  61 per power field at 200,

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respectively, p < 0.05), while the other three autoAbs had no significant difference. IgE anti-BP230 levels seemed to be strongly related with local eosinophil accumulation.

4. Discussion The current study has investigated the profiles of IgG and IgE autoAbs against BP180 and BP230 in BP sera. Since the antigen proteins in these ELISA systems are not in full length, there was a possibility that autoAbs against other domains could not be detected. Nonetheless, as was expected, the highest prevalence among the four autoAbs was IgG anti-BP180 Ab. In concordant with the previous reports [33], more than 90% of BP sera had IgG Abs directed against the NC16A domain of BP180, confirming that IgG anti-BP180 Abs are strongly related with the pathogenesis of BP. IgG anti-BP180 Ab levels demonstrated a significant correlation with the affected area, which is consistent with its central role in pathogenesis [34]. The prevalence of IgG anti-BP230 Abs was 72%. This was similar to the study by Hamada et al. [35], who reported that 80% of BP sera possessed IgG Abs reactive with C-terminal domain of anti-BP230. Our study showed that IgG anti-BP230 titers tended to increase in proportion to elongation of disease. Therefore, once IgG anti-BP180 Abs initiate the disease, the exposure of the cytoplasmic proteins from damaged basal keratinocytes may boost IgG antiBP230 Ab production during the disease progress. Among four BP patients negative for anti-BP180 IgG Abs by ELISA, two had IgG anti-BP230 Abs. Interestingly, a 73-year-old female patient had both IgG and IgE anti-BP230 Abs but not IgG anti-BP180 Ab at the onset of the disease, and later showed the increase of IgG anti-BP180 levels as skin lesions spread. This suggests that the appearance of IgG anti-BP180 Ab may not be the initial event in some exceptional cases. Several previous studies have indicated the hypothesis that BP230-specific autoAbs are relevant for the pathogenesis of BP: (1) Immunization of rabbits with BP230-derived peptides appeared to increase the local inflammatory response following epidermal damage [36]; (2) BP230-specific autoAbs were readily detectable in the majority of patients even after a relatively short disease duration (1.5 months); (3) High-titer BP230specific polyclonal rabbit IgG, after subcutaneous injection into neonatal mice, induced an inflammatory response and skin fragility. The frequencies of IgE anti-BP180 and anti-BP230 Abs were 30% and 67%, respectively. The tendency of coexistence of IgG and IgE Abs for each antigen was observed. This might suggest that these two isotypes

160 have arisen mostly via common clonal selection. Nonetheless, four patients showed high titers of IgE anti-BP230 Abs with negative IgG anti-BP230 Abs. Moreover, a patient with severe BP showed an extremely high titer of IgE anti-BP180 Ab (O.D value, 2.06) with borderline IgG anti-BP180 Ab (7.1 index). Therefore, in several cases, IgE Abs may precede IgG Ab induction. While IgE has been classically considered to be important in allergic diseases and in parasitic infections [3,37,38], possible roles for IgE in autoimmune diseases have been described [39]. IgE antinuclear Abs as well as elevated serum IgE levels have been demonstrated in autoimmune diseases including systemic lupus erythematosus, autoimmune thyroid diseases, and rheumatoid arthritis (RA) [40—44]. IgE rheumatoid factor is positive especially in cases with extra-articular lesions, such as vasculitis, suggesting that IgE rheumatoid factor may be particularly relevant in the pathogenesis of systemic manifestations of RA [41]. It remains unclear whether IgE antihemidesmosome autoAbs play a role in the pathology of BP. One possible senario is that IgE antihemidesmosome Abs bind to Fce receptor on the surface of mast cells, eosinophils, and basophils. A recent study has clarified that IgE immune complex binds with macrophages through FcgRIV in mice [45]. IgE deposition may result in activation and degranulation of these cells, setting off an inflammatory response in BP [46]. This hypothesis is supported by the previous data of antigen-specific release of histamine from basophils of BP patients whose sera were positive for IgE immunoblot using recombinant NC16A [31]. It has been also shown that mast cells in the dermis of BP patients have BP180specific IgE on their surface by the immunofluorescent technique [28]. Another report has shown that an IgE-dependent activation of eosinophils can induce a selective release of eosinophil peroxidase and major basic protein [47,48]. In the current study, IgE anti-BP230 titers were strongly correlated with local eosinophil accumulation. In summary, the current study has demonstrated the positivity of IgE anti-BP180 and anti-BP230 Abs by ELISA and has suggested their possible roles. Future directions would include a passive transfer mouse model using IgE Abs against BP180 and/or BP230, which may possibly elucidate the pathogenesis of BP more precisely.

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