Antibody to bullous pemphigoid antigen 1 binds to the antigen at perilesional but not uninvolved skin, in localized bullous pemphigoid

ARTICLE

Localized bullous pemphigoid is a rare variant of bullous pemphigoid (BP), which shows tense, bullous lesions at limited sites. It is classified into two types [1]: 1) the Brunsting and Perry type characterized by scarring, plaque-like lesions on the head and neck, and 2) the Eberhartinger and Niebauer (or pretibial) type characterized by non-scarring, bullous lesions on the extremities, especially the lower legs [2, 3]. BP is an autoimmune blistering disease which is characterized by the presence of circulating autoantibodies to two major hemidesmosomal molecules, the 230 kDa BP antigen (BPAG1) and the 180 kDa BP antigen (BPAG2) [4-7]. Recent molecular genetic studies on these antigens revealed that BPAG1 is an intracellular plaque protein of hemidesmosome, which is a member of the plectin family [8-10], and that BPAG2 is a transmembrane collagenous protein [11, 12]. It is now widely accepted that antibody to BPAG2 is the most likely pathogenic agent for BP, because the blistering in the animal model that mimicks BP can been generated by the injection of antibody against the noncollagenous domain (NC16a) of BPAG2 to mouse [13]. However, antibodies to BPAG2 are present at lower levels to than those to BPAG1 in BP sera [15]. BPAG1 can be detected by the sera from 50-90% of typical BP patients, while BPAG2 can be detected by the sera from 35-50% of patients by immunoblotting [16] and the BPAG2 NC16a domain was positive for 80% of typical BP sera [16]. On the other hand, it has been reported that the sera in 7 (87%) out of 8 cases of localized BP reacted with BPAG1, whereas only 1 case (13%) reacted with BPAG2 [16], suggesting that localized BP sera apparently react less frequently with BPAG2 in epidermal extracts than typical BP sera. Furthermore, 7 (87%) out of 8 cases reacted with the human BPAG2 NC16a domain recombinant protein, but at a lower antibody titer to this protein [16]. Therefore, it is suggested that the atypical clinical features of localized BP may be related to a low titer of autoantibodies to BPAG2, particularly to the NC16a domain [16]. However, the precise pathogenic roles of BP antigens in localized BP are still unknown. We present here a case of localized bullous pemphigoid in which perilesional skin showed a linear deposition at the basement membrane zone (BMZ) on direct immunofluorescence study, but the intact skin distant from the lesion showed no deposition.

Case report

A 59-year-old woman with vesicles and erosion on both lower legs was admitted to our hospital on September 24, 1997. Vesicles and bullae with erythematous patches had appeared recurrently only on her lower legs over the previous 2 years (Fig. 1). No involvement was observed elsewhere or on any mucous membranes and Nikolsky's sign was absent. Since the age of 46 years, she had had gait disturbance following a cerebral thrombosis. Laboratory data were all within normal. Histological findings for the vesicle showed a subepidermal blister with infiltration by eosinophils and neutrophils in the dermis (Fig. 2).

Immunoblot analysis

Extracts from normal human epidermis were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 6% gel according to the standard method [17, 18]. Separated proteins were electrophoretically transferred on to a nitrocellulose membrane. After blocking with 5% skimmed milk in 0.2% Tween-20 in phosphate buffered saline (PBS), the membrane was incubated with a 1: 100 dilution of the patient's serum overnight at 4° C. Specific reactions by the serum were detected with enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham International, Buckinghamshire, U.K.). The patient's serum reacted with a polypeptide with a molecular weight of 230 kDa (Fig. 3), but not with the 180 kDa polypeptide even in 1:10 diluted patient serum, whereas the serum from another patient with typical bullous pemphigoid reacted with the 180 kDa polypeptide.

Immunofluorescence study

Biopsy samples were obtained from the perilesional skin on the lower leg and the thigh skin, 20 cm distant from the edge of the lesion, and normal skin samples were obtained from volunteers. Prior to the biopsy, the patient was informed about this study and gave written consent. Tissue specimens were embedded in OCT compound and stored at ­ 80° C. On indirect immunofluorescence study (IDIF), cryosections of 4 µm in thickness on slides were rinsed with PBS at room temperature and were reacted with various dilutions of the patient's serum or normal control serum for 45 min. After washing with PBS, the sections were incubated for 45 min at room temperature with fluorescence-conjugated goat anti-human IgG. On direct immunofluorescence (DIF), 4 µm cryostat sections were rinsed with PBS and then reacted for 45 min with fluorescence-conjugated goat antibodies against human IgG, IgA, IgM and C3. The sections for IDIF or DIF were washed with PBS and covered with glass coverslips. Observation was carried out using a fluorescence microscope, Nikon FX (Nikon, Tokyo, Japan). To study the localization of immunoreactants in detail, we performed an IDIF study using normal tissue specimens incubated in 1 M NaCl for 48 h at 4° C to separate the epidermis and dermis.

IDIF findings with normal skin showed that there was a linear deposition of IgG at the BMZ (Fig. 4A), and an antibody titer of 1:160 on the epidermal side of skin split by 1 M NaCl (Fig. 4B). In addition, positive staining was observed at the BMZ in the thigh skin of the patient (Table I) at a titer of 1:160 by IDIF. On the other hand, in the DIF study, IgG and C3, but not IgA and IgM, were positive at the BMZ of perilesional skin (Fig. 4C), whereas IgG, C3, IgA and IgM were all negative for the thigh skin of the patient (Table I).

Treatment

Topical treatment with betamethasone valerate was effective. Brown pigmentation without scarring remained at the lesional sites after the vesicles had disappeared.

Discussion

Localized BP sera react with BPAG1 [19-21] in epidermal extracts more frequently as compared with BPAG2 [16]. Antibody to BPAG2 is detected less frequently in localized BP than generalized BP. In addition, most cases with localized BP reacted with the BPAG2 NC16a domain, even though there was a lower end point of antibody titer to this protein [16]. These results suggest that antibody to BPAG1 plays a more crucial role in the pathogenesis of localized BP than in generalized BP [21]. It is not known how antibody to BPAG1, which is an intracellular protein, relates to bulla formation. When basal keratinocytes are injured, the BPAG1 antigen may be uncovered and subsequent complement activation will be induced with the generation of C5 antigens [22]. This speculation is supported by the reports showing that localized BP is induced by X-rays [19], injuries, UV light [23] or PUVA therapy.

It is now accepted that the mechanism for the blister formation in BP involves BP-IgG binding to BP antigen in the lamina lucida inducing fixing and activation of complement. A variety of inflammatory cells recruited by these factors produce cytokines and chemical mediators, some of which digest or disrupt the dermal-epidermal junctions [24]. On the other hand, another mechanism for the blistering in BP has been recently proposed that involves the internalization of BPAG2 after the binding of the BP-IgG on the lateral-apical cell surface of basal cells which may play an important role in blistering [24-26]. Briefly, the binding of BP-IgG to the BPAG2 NC16a domain impairs the molecular function of BPAG2 to assemble with other hemidesmosomal components such as alpha integrin [27], so that the internalization of IgG-bound BPAG2 may be generated in order to clean off the BP-IgG-impaired molecule from the cell surface [24]. In any case, once the basal cells are injured, the BPAG1, which may possess a higher antigenicity because it is an intracellular protein, is exposed to the immune system in large quantities, so that sufficient immune complex may be generated to cause the inflammation and blisters.

Here we present a patient with localized bullous pemphigoid. The perilesional skin showed a linear deposition at the BMZ in the DIF study: the uninvolved thigh skin, distant from the lesion, did not show any deposition. Immunoblot analysis using epidermal extracts demonstrated the presence of IgG antibody directed to BPAG1 in the patient's serum. The immune complex of BP antigen (probably BPAG1) and BP-IgG were detected by DIF in the close-perilesional skin of the lower leg but not in the distant thigh skin. Therefore, these findings suggest that BPAG1 in the close-perilesional skin had been exposed to the immune system, possibly due to the injury by BP-IgG. The intact skin showed a linear deposition at the BMZ on IDIF because BP antigen (probably BPAG1) at the BMZ bound with the patient's serum. Although we could not check the reactivity of the serum to the BPAG2 NC 16a domain, there is a possibility that the patient's serum did not contain enough antibody to detect BPAG2 by immunoblot using epidermal extracts. Therefore, antibody to BPAG2, which may injure the basal cells even at a lower titer, might be a primary trigger for blister formation, antibody to BPAG1 exposed to the immune system by this injury may then play a role in the subsequent blister formation. However, the possibility cannot be ignored that the antigenicity of BPAG1 depends on the skin site.

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