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

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 67BP 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 67BP 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 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.     

Clinical and immunological profiles of anti-BP230-type bullous pemphigoid: Restriction of epitopes to the C-terminal domain of BP230, shown by novel ELISAs of BP230-domain specific recombinant proteins

Objectives

To confirm that sera from some BP patients reactive exclusively to the BP230 and to study the clinical and immunological characteristics of this condition.

Materials and methods

BP patients were divided into three groups: BP reactive only to BP230 (BP230- BP), BP reactive to both BP180 and BP230 (BP180-BP230-BP) and BP reactive only to BP180 (BP180-BP), based on the results of standard ELISAs for BP180 and BP230. Clinical features were statistically analyzed among the three groups. Then, targeted epitopes in each group were studied by immunoblotting and novel ELISAs using three domainspecific BP230 recombinant proteins.

Results

Forty-one, 65 and 47 of 153 BP patients were categorized as BP230-BP, BP180-BP230-BP and BP180-BP, respectively. Clinically, BP230-BP patients showed significantly lower severity, less need of systemic steroids and better responses to various treatments, suggesting that BP230-BP is a milder condition. Immunoblotting and ELISAs of domain-specific BP230 recombinant proteins indicated that, while BP180-BP230-BP sera reacted with all three domains of BP230, BP230-BP sera reacted more frequently with epitopes in the BP230 C-terminal domain.

Conclusion

We propose a new disease entity, named anti-BP230-type BP, in which anti-BP230 antibodies might be pathogenic and react specifically with the BP230 C-terminal domain. While anti-BP230 antibodies in BP180-BP230-BP seem to be produced via intermolecular epitope spreading, anti-BP230 antibodies in BP230-BP are considered to be produced by different mechanisms.

     

Role of BP230 autoantibodies in bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune disease associated with subepidermal blistering due to autoantibodies directed against BP180 and BP230. BP180 is currently considered as the major pathogenic autoantigen. However, previous clinical findings suggested that anti-BP230 autoantibodies alone can cause skin lesions in animal models and many BP patients. The characteristics of BP230 and the pathogenic roles of anti-BP230 antibodies have been proposed. First, at the molecular level, BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque and interacts with other constituents of hemidesmosomes. Second, the presence of BP230 autoantibodies may correlate with specific clinical features of BP. The immunoglobulin (Ig)G autoantibodies from BP patients react mainly against the C-terminus of BP230, while the IgE autoantibodies are still inconclusive. Third, in vivo, autoantibodies against BP230 involved in the disease may not only induce the inflammatory response but also impair the structural stability of hemidesmosomes. This article reviews recently published work about the role of BP230 and its antibodies, including IgG and IgE, aiming to find clues of its clinical association and lay the foundation for the research on the pathogenicity of antibodies against BP230.     

大疱性类天疱疮患者外用糖皮质激素治疗抵抗的相关因素分析

目的:依据临床和实验室数据,分析大疱性类天疱疮(BP)患者外用糖皮质激素(简称激素)治疗抵抗的相关因素。方法:收集BP患者64例,其中外用激素治疗有效组(即外用激素治疗4周内,连续3 d,每日新发水疱数<3个,以下简称有效组)22例,外用激素治疗无效组(即外用激素治疗4周内皮损未控制,连续3 d,每日新发水疱数≥3个,以下简称无效组)42例。对2组BP患者的皮损类型进行统计,比较2组患者的大疱性类天疱疮疾病面积指数(BPDAI)评分、外周血中白蛋白浓度和嗜酸性粒细胞(EOS)计数以及外周血中抗BP180和抗BP230 IgG抗体、总IgE、抗BP180和抗BP230 IgE抗体浓度。结果:有效组患者的皮损以单纯水疱为主(68%),无效组则以红斑水疱为主(63%)。无效组患者的BPDAI评分、EOS计数、总IgE、抗BP180 IgG抗体、抗BP230 IgG抗体浓度及抗BP230 IgE抗体浓度均较有效组患者明显增高,差异具有统计学意义(P<0.05);而2组患者白蛋白水平及抗BP180 Ig E抗体浓度无明显差异。结论:除BPDAI评分及特异性IgG水平之外,还可根据BP患者的皮损类型、外周血EOS计数、总IgE及抗BP230 IgE抗体的水平指导BP患者治疗方法的选择。     

大疱性类天疱疮患者血清总IgE与血清自身抗体的关系

目的 分析大疱性类天疱疮患者血清总IgE与抗BP180IgG抗体、抗BP230IgG抗体、抗表皮基底膜IgG抗体滴度(即间接免疫荧光滴度)的关系.方法 收集沈阳市第七人民医院2014年1月-2020年1月大疱性类天疱疮病例,进行回顾性分析,根据抗BP180IgG抗体、抗BP230IgG抗体阳性情况将患者分组,比较组间血...     

131例神经系统疾病患者血清中抗BP180和抗BP230抗体的检测

目的：检测神经系统疾病患者血清中抗BP180、抗BP230和抗基底膜带抗体的阳性率。方法：收集神经系统疾病患者和正常对照血清,采用酶联免疫吸附试验（ELISA）和间接免疫荧光（IIF）检测两组血清中抗BPl80NC16A抗体和BP230抗体水平。结果：共收集到131例神经系统疾病患者血清(脑卒中109例,脑肿瘤17例,其他神经系统疾病19例)和131例正常对照血清。病例组中抗BP180NC16A1阳性率为1.45%,低于对照组的3.05%,差异具有统计学差异（P = 0.009）,病例组中抗BP230抗体阳性率5.34%与对照组（2.29%）比较,差异无统计学意义。IIF检测抗基底膜带抗体结果均为阴性。结论：抗BP180NC16A抗体在神经系统疾病患者中有较高的阳性率,可能与神经系统疾病患者合并大疱性类天疱疮相关。     

Usefulness of Enzyme-linked Immunosorbent Assay Using Recombinant BP180 and BP230 for Serodiagnosis and Monitoring Disease Activity of Bullous Pemphigoid

Background

Bullous pemphigoid (BP) is an autoimmune subepidermal bullous disease associated with autoantibodies against BP180 and BP230. Enzyme-linked immunosorbent assay (ELISA) is a sensitive tool for the detection of immunoglobulin G (IgG) anti-BP180 and anti-BP230 autoantibodies.

Objective

The aim of this study was to evaluate the usefulness of ELISA for diagnosing and monitoring the disease activity of BP.

Methods

We evaluated serum IgG levels of anti-BP180 and anti-BP230 autoantibodies in 47 BP patients, 16 epidermolysis bullosa aquisita patients, and 15 healthy volunteers using ELISA. Through retrospective review of the medical records, the clinical characteristics of BP including disease activity, duration, pruritus severity and peripheral blood eosinophil counts were assessed.

Results

The sensitivity of BP180 ELISA was 97.9%, BP230 ELISA 72.3%, and a combination of the two was 100%. The specificity of BP180 ELISA was 90.3%, BP230 ELISA 100%, and a combination of the two was 90.3%. BP180 ELISA scores showed strong associations with disease activity, pruritus severity, peripheral blood eosinophil counts, and disease duration, whereas BP230 ELISA scores did not.

Conclusion

BP180 and BP230 ELISAs are highly sensitive methods for the diagnosis of BP, and BP180 ELISA, in particular, is a sensitive tool for monitoring the disease activity of BP.     

Absence of anti-BP180 antibodies in mothers of infants with bullous pemphigoid

BACKGROUND: It is not clear whether bullous pemphigoid (BP) of infancy is linked to maternal transmission of pathogenic autoantibodies. Objectives To search for anti-BP180 antibodies in the sera of infants with BP and their mothers, using sensitive and specific methods. METHODS: Four infants (<6 months) with BP and their mothers were tested for anti-BP180 antibodies by indirect immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found anti-BP180 antibodies in the sera of the four infants with all methods. These antibodies reacted with the extracellular domain NC16A. In the serum of their mothers we found 180 and 160 kDa proteins, each in one case, but indirect immunofluorescence and ELISA were negative, suggesting the absence of anti-BP180 autoantibodies reacting with the extracellular domain NC16A. CONCLUSIONS: BP of infants is not due to maternofetal transmission of pathogenic autoantibodies. Other hypotheses for the pathophysiology of BP are discussed.     

人源性抗大疱性类天疱疮抗原BP180单链抗体的体外功能鉴定

目的 探讨人源性抗大疱性类天疱疮抗原BP180单链抗体的生物学功能。方法 亲和层析方法纯化大疱性类天疱疮（BP）患者血清自身抗体,通过竞争性ELISA、竞争性免疫荧光和补体活化的竞争性抑制实验来观察所制备的抗BP180单链抗体对BP-IgG自身抗体结合人BP180抗原的竞争性抑制作用。结果 竞争性ELISA结果表明,在0 ~ 60 μg/ml范围内单链抗体对BP-IgG自身抗体的竞争性抑制作用呈剂量依赖关系,最大抑制率可达69.50%（与对照组相比,均有统计学意义,P < 0.01）。竞争性免疫荧光实验中,单链抗体浓度增加至40 μg/ml时,BP-IgG在基底膜带的沉积及其介导的补体C3活化作用均被抑制,呈阴性。结论 人源性抗BP180单链抗体在体外对BP致病性自身抗体与抗原结合及后续的补体活化具有一定的抑制作用。     

大疱性类天疱疮患者噬菌体抗体库的构建及抗BP180-NC16A抗体筛选

目的构建噬菌体抗体库并筛选抗BP180-NC16A抗体。方法以大疱性类天疱疮(BP)患者外周血淋巴细胞为基因来源,扩增多样性的轻链和重链Fd段基因,构建Fab段表面展示的噬菌体抗体库,用BP180分子的NC16A片段为抗原对抗体库进行筛选并对筛选得到的抗体用ELISA,W estern b lot和免疫荧光等方法进行鉴定。结果经过轻、重链基因的重组和转化,获得了库容为5×107的噬菌体抗体库。以BP180-NC16A抗原进行"亲和吸附—洗脱—扩增"淘筛,获得2株特异性抗体。结论利用噬菌体抗体库技术成功制备了抗BP180抗体,为深入研究BP自身抗体、探讨新的治疗策略奠定了基础。     

IgE autoantibodies against the intracellular domain of BP180

Background  Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230. BP not only involves IgG-mediated neutrophil activation, leading to blistering, but also IgE-dependent activation of mast cells and basophils. While IgG and IgE autoantibodies target the extracellular noncollagenous (NC) 16A domain of BP180, little is known whether other BP180 regions are targeted by these antibody classes.
Objectives  To characterize IgE and IgG autoantibody binding to antigenic sites on the intracellular domain (ICD) of BP180 compared with BP180 NC16A.
Methods  IgE/IgG autoreactivity against recombinant BP180 ICD and NC16A was determined by immunoblotting of sera from 18 patients with BP and 10 controls.
Results  Total serum IgE was elevated in 16 of 18 BP sera. Most BP sera tested positive (15 of 18) to NC16A with both immunoglobulin classes. Additionally, 14 of 18 sera showed IgE reactivity with an epitope mapped to the ICD of BP180 (amino acid residues 103–266). Mapping of ICD antigenic sites revealed similar IgE and IgG reactivities for most regions except for greater IgE reactivity to amino acid residues 234–398 (11 of 18 BP sera) than IgG (five of 18). Control sera failed to display IgE reactivity to these antigens.
Conclusions  The results indicate that BP180 NC16A is not the only antigenic determinant of IgE autoantibodies in BP and that additional, novel epitopes exist on different regions of the ICD of BP180. The heterogeneous autoimmune response against BP180 suggests intramolecular epitope spreading during disease progression.     

Coexistence of psoriasis and linear IgA disease in a patient with recent herpes zoster infection

We report the case of a 29-year-old man with chronic plaque psoriasis who developed linear IgA disease following herpes zoster infection. There has only been one previous report describing the coexistence of psoriasis and linear IgA disease, which was confirmed by immunopathological studies. In our patient, immunoblotting studies identified IgA antibodies binding to BP180 and BP230 antigens, and IgG autoantibodies binding weakly to the BP180 antigen. This is an interesting case that we believe is an example of epitope spreading in the development of autoimmune subepidermal bullous diseases.     

大疱性类天疱疮自身反应性IgG抗体亚型研究进展

大疱性类天疱疮（BP）是一种自身免疫性表皮下大疱性皮肤病,以c3和（或）IgG在基底膜带线状沉积为特征。研究提示,抗BP180IgG抗体通过激活补体,可能是诱发大疱形成的主要原因,而自身抗体结合的抗原表位多数包括BP180NC16A结构域。由于不同IgG抗体亚型含量以及激活补体的能力不同,其致病性也不同。免疫荧光和酶联免疫吸附试验提示,IgG1和IgG4为主要的自身反应抗体亚型。体外实验和动物模型均证实,IgG1为主要的致病抗体,其含量与BP的严重程度平行;IgG4具有较弱的活化炎症细胞的致病作用和封闭抗原表位的保护作用。最近的研究提示,抗BP180IgG抗体可以不依赖激活补体和炎症细胞这两种方式诱导表皮真皮分离。因而关于各IgG亚型的致病能力,特别是IgG4的作用现在仍然不清楚。     

抗BP180NC16A IgG亚型检测方法的建立以及在大疱性类天疱疮中的意义

【摘要】 目的 建立抗BP180NC16A IgG亚型的检测方法,并探讨其在大疱性类天疱疮（BP）中的意义。方法 原核表达GST-NC16A融合蛋白,并采用亲和层析法纯化。优化ELISA关键环节,建立抗BP180NC16A IgG各亚型的ELISA检测方法,并对10例未经治疗的BP、5例妊娠疱疹、1例成人线状IgA大疱性皮病、2例天疱疮患者血清分别进行检测。结果 通过方阵测定法确定GST-NC16A融合蛋白的包被浓度为500 μg/L,包被条件为4 ℃ 12 h,血清稀释倍数为1 ∶ 100,酶标二抗为1 ∶ 2000,孵育条件为37 ℃ 1 h,底物反应条件37 ℃ 20 min。10例大疱性类天疱疮患者10例IgG1阳性,9例IgG2阳性,5例IgG3阳性,9例IgG4阳性。2例寻常型天疱疮、1例成人线状IgA大疱性皮病均阴性。5例妊娠疱疹所有亚型均阳性,以IgG1和IgG3亚型为主。结论 抗BP180NC16A ELISA检测法特异性强、重复性好,是检测BP和妊娠疱疹患者抗BP180NC16A抗体亚型的半定量方法。     

Localisation of bullous pemphigoid antigen 180 (BP180) in cultured human keratinocytes: functionally relevant modification by calcium

The expression of BP180 has previously been demonstrated to be influenced by both calcium (Ca²⁺) concentration and binding of anti-BP180-antibodies in cultured keratinocytes of the skin squamous cell carcinoma line DJM-1. Here, BP180 expression was studied in cultured normal human epidermal keratinocytes by confocal laser scanning microscopy. We exploited an experimental system, in which BP180 was previously shown to mediate, upon incubation with anti-BP180 antibodies, a specific signal-transducing event that leads to the release of inflammatory mediators, such as IL-8 from cultured normal human epidermal keratinocytes (NHEK). We found that without addition of BP180-specific IgG, BP180 is predominantly expressed on the cell surface irrespective of the Ca²⁺ concentration. In contrast, cell surface BP180 was greatly reduced in NHEK kept in high Ca²⁺ medium after incubation with BP180-specific IgG for 12 h compared to low Ca²⁺ medium. This effect was seen with antibodies to both N- and C-terminal fragments of the BP180 ectodomain, respectively. In addition, a slightly higher BP180 expression was found in NHEK cultured in low compared to high Ca²⁺ medium by Western blotting. Interestingly, in contrast to NHEK kept under low Ca²⁺ conditions, in NHEK grown in high Ca²⁺ medium, no elevated levels of IL-8 were released after treatment of cells with anti-BP180 IgG compared to normal IgG. Our data indicate that the Ca²⁺-modulated expression of BP180 is functionally relevant. This finding sheds further light on the complex pathomechanism in blister formation of BP180-related autoimmune blistering skin diseases.     

Clinical manifestations in 100 Japanese bullous pemphigoid cases in relation to autoantigen profiles

The relationship between clinical findings and antigen profiles in 100 bullous pemphigoid (BP) patients has been investigated. The patients were divided into four groups based upon the results of immunoblot analysis, namely patients whose sera detected the 230-kPa BP antigen (BP230) and the 180-kDa BP antigen (BP180), those recognizing either BP23Q or BP180 alone, and those recognizing neither antigen, analysis by the chi-squared test showed predominant occurrence of oral (P < 11(15) and facial lesions (P < 0.005) in patients whose sera detected BP180, and these patients also tended to have more extensive lesions (P < 0.005). Patients that were positive for UP 180 alone needed treatment with higher doses of steroids than the patients positive for BP230 alone (P < 0.05). Furthermore, all rue recalcitrant cases, which did not respond well to steroid treatment, were shown to possess auto antibodies against UP I NO in their sera. Patients with antibodies to BP230 had a tendency to have a high titre of anti-HMZ antibodies (P < 0 (105). These results suggest that anti-BPI80 antibodies nun be more related to the disease severity than anti-BP230 antibodies.     

Experimental models for the autoimmune and inflammatory blistering disease,Bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal skin blistering disease characterized immunohistologically by dermal-epidermal junction (DEJ) separation, an inflammatory cell infiltrate in the upper dermis, and autoantibodies targeted toward the hemidesmosomal proteins BP230 and BP180. Development of an IgG passive transfer mouse model of BP that reproduces these key features of human BP has demonstrated that subepidermal blistering is initiated by anti-BP180 antibodies and mediated by complement activation, mast cell degranulation, neutrophil infiltration, and proteinase secretion. This model is not compatible with study of human pathogenic antibodies, as the human and murine antigenic epitopes are not cross-reactive. The development of two novel humanized mouse models for the first time has enabled study of disease mechanisms caused by BP autoantibodies, and presents an ideal in vivo system to test novel therapeutic strategies for disease management.     

大疱性类天疱疮和妊娠疱疹患者血清抗BP180 NC16A抗体的纯化和鉴定

目的 探讨大疱性类天疱疮（BP）和妊娠疱疹（HG）患者血清抗BPl80 NC16A 抗体的纯化和鉴定方法。方法 原核表达系统pGEX-2TBP180NC16A表达GST/NC16A融合蛋白,将融合蛋白与谷胱甘肽琼脂糖凝聚微珠进行共价偶联。微珠亲和层析法纯化BP和HG患者血清中抗BP180 NC16A抗体,并用ELISA、免疫荧光、及Western印迹进行鉴定。结果 原核表达系统pGEX-2TBP180NC16A表达37 000 GST/NC16A融合蛋白,微珠亲和层析法纯化后得单一抗BP180 NC16A抗体。经ELISA方法定量后确定其含量为2.4 mg/ml;该抗体能与人皮肤基底膜带结合,证明抗体活性;免疫印迹可见单一片段,显示抗体纯度。结论 微珠亲和层析法纯化的BP和HG患者血清中抗BP180 NC16A自身抗体活性高、特异性强。     

Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid

The NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and, probably, pathogenic region in bullous pemphigoid (BP). In the present study, in order to determine whether serum level of circulating anti-BP180 autoantibodies is a valuable serum marker in BP, the immunoreactivity of sera against the NC16A domain of BP180 was measured using enzyme-linked immunosorbent assay (ELISA) in ten patients with BP. Serum levels of anti-BP180 autoantibodies correlated with the clinical course in BP patients, who received various therapeutic agents. The result suggests that this NC16A-ELISA is a useful method for evaluating the clinical course and efficacy of the therapy in patients with BP.     

Evaluation of a BP180-NC16a enzyme-linked immunosorbent assay in the initial diagnosis of bullous pemphigoid

BACKGROUND: Bullous pemphigoid (BP) is the most common subepidermal immunobullous disease, characterized by circulating IgG autoantibodies targeting BP180 and BP230 hemidesmosomal proteins. Several immunological studies have demonstrated that the membrane proximal noncollagenous domain NC16a of BP180 is the immunodominant region targeted by BP autoantibodies. Recently, a commercial BP180 NC16a-specific enzyme-linked immunosorbent assay (ELISA) has become available for detecting pathogenic anti-BP180 autoantibodies in BP sera. However, it remains unclear whether the diagnostic potential of the ELISA is equivalent to that of the 'gold-standard' diagnostic technique of immunofluorescence (IF). OBJECTIVES: To examine the usefulness of a commercially available BP180-NC16a ELISA in the initial serodiagnosis of BP. METHODS: Sera from a large cohort of patients with BP (n = 102) and control subjects (age- and sex-matched normal volunteers, n = 60; pemphigus foliaceus, n = 18; pemphigus vulgaris, n = 16) were assayed by BP180-NC16a ELISA. All BP sera were obtained at presentation before initiation of systemic immunosuppressive therapy. The values of IgG antibody levels measured by ELISA were compared with those measured by indirect IF on salt-split skin. Results Receiver operating characteristic analysis was used to calculate the cut-off value for the ELISA in the diagnosis of BP which maximizes both sensitivity and specificity, and to estimate the diagnostic accuracy of the ELISA as represented by the area under the curve (AUC = 0.965). A cut-off value of 9 was associated with a sensitivity of 89% (91 of 102 BP sera showed a positive result) and a specificity of 98%. Fifty-eight of 60 normal controls and all the pemphigus sera showed a negative result. There was a correlation between the mean ELISA values and indirect IF titres (Spearman rank correlation 0.286; P = 0.004). CONCLUSIONS: Our results suggest that the BP180-NC16a ELISA is a useful tool for the detection of pathogenic anti-BP180 IgG autoantibodies at the initial disease stage of BP. Because it is not only highly sensitive and specific, but is also easy to perform, is objective, and semiquantitative, the ELISA may provide valuable information for the accurate and reliable serodiagnosis of BP.