Case of epidermolysis bullosa acquisita with concomitant anti-laminin-332 antibodies

Emi NISHIDA,1 Eiichi NISHIO,2 Hiroko MURASHIMA,3 Norito ISHII,4 Takashi HASHIMOTO,5 Akimichi MORITA1

ABSTRACT
Subepidermal autoimmune blistering disease including bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, anti-laminin-c1 pemphigoid, linear immunoglobulin A bullous disease and epidermolysis bullosa acquisita (EBA), are all characterized by direct immunofluorescence microscopy or immunoglobulin depo- sition on the basement membrane zone. Among them, EBA is a rare acquired subepidermal autoimmune blister- ing disease of the skin and mucous membranes reactive with type VII collagen, a major component of the epidermal basement membrane zone. Anti-laminin-332-type mucous membrane pemphigoid has pathogenic autoantibodies against laminin-332, which is a basement membrane heterotrimeric protein composed of a3, b3 and c2 laminin chains. We describe a 73-year-old Japanese man presenting with multiple, annular, tense blisters on the lower legs and oral lesions. Despite the severe clinical manifestations, the disease was successfully con- trolled by combination therapy of oral prednisolone and mizoribine. This case was confirmed to have autoantibod- ies to both type VII collagen and laminin-332 a3 chain by indirect immunofluorescence of 1 mol NaCl-split normal human skin, various immunoblot analyses and enzyme-linked immunosorbent assays. This case was a rare case of EBA with concomitant anti-laminin-332 antibodies.

INTRODUCTION
We report a patient who was diagnosed with inflammatory epi- dermolysis bullosa acquisita (EBA) with concomitant anti-lami- nin-332 antibodies. We consider that this patient initially developed anti-type VII collagen autoantibodies, followed by production of anti-laminin-332 autoantibodies via epitope spreading phenomenon.

CASE REPORT
A 73-year-old Japanese man, with past histories of type 2 dia- betes mellitus, myocardial infarction and cerebral infarction, presented with blisters on the lower legs more than 13 years prior to referral. A skin biopsy indicated a diagnosis of bullous pemphigoid (BP). He had been treated with 17.5–30 mg/day prednisolone for 10 years and had discontinued the therapy for 3 years. He then restarted the prednisolone, but the skin lesions gradually worsened. Then, he was referred to our hos- pital. He was admitted for reconsideration of the diagnosis and more precise treatment, as his cutaneous lesions differed from those typical of BP. Physical examination revealed bullae on the trunk (Fig. 1a), and ulcers and erosions in the oral cavity (Fig. 1b). He also showed scaly erythema with many milia on the fingers and feet (Fig. 1c). Milia was also seen on the forehead. Laboratory tests revealed elevated C-reactive protein (0.93 mg/dL) and white blood cells (10 200/lL), but no circulating antibodies to BP180 and BP230 based on enzyme-linked immunosorbent assay. Computed tomography revealed no specific findings, including malignant tumors. Histopathological examination of a biopsy obtained from a lesion on the abdomen revealed a subepithelial cleft and lymphocytic infiltration. Direct immunofluorescence showed a linear deposition of immunoglobulin (Ig)G and C3 at the epider- mal basement membrane zone (BMZ).

Indirect immunofluorescence of 1 mol NaCl-split normal human skin revealed circulating IgG and anti-BMZ autoanti- bodies bound to the dermal side of the split at a titer of 1:40. Immunoblot analyses of normal human epidermal and dermal extracts, recombinant proteins of BP180 NC16a and C-terminal domains, and concentrated culture supernatant of HaCaT cells showed no positive reactivity, while the 290-kDa full-length type VII collagen was detected in normal human dermal extracts (Fig. 2a), and the 165- and 145-kDa a3 subunits of laminin-332 were detected in purified human laminin-332 (Fig. 2b). The patient was diagnosed with EBA with concomi- tant anti-laminin-332 antibodies. After combination treatment with systemic prednisolone (35 mg/day) and mizoribine (150 mg/day), the inflammatory cutaneous lesions cleared, but the mucosal lesions remained. The prednisolone dose was gradually reduced weekly. He currently takes prednisolone (5 mg daily) and is in stable condi- tion (Fig. 1d) and there is no scarring of the oral mucosa.

DISCUSSION
Our case was initially diagnosed as BP, because direct immunofluorescence studies of the biopsy sections revealed the deposition of IgG and C3 at the dermoepidermal junction in a linear pattern. We could not confirm serration pattern1 by direct immunofluorescence microscopy. Vodegel et al.2 reported that IgG/IgA linear deposit at the BMZ show remark- able “u” or “n” serration patterns. The former pattern is found in EBA and bullous systemic lupus erythematosus, and the latter is in all other pemphigoid diseases. Epidermolysis bullosa acquisita is a blistering disease caused by autoantibody production against type VII collagen. Patients with EBA exhibit two major clinical subtypes: inflam- matory and non-inflammatory EBA, also called classic EBA. The inflammatory type of EBA can be further classified as: (i) clinically mimicking BP-like; (ii) mucous membrane pemphigoid (MMP)-like; (iii) Brunsting–Perry pemphigoid-like; and (iv) linear IgA bullous dermatosis-like.3–7 The classic form of EBA is a mechanobullous disease primarily involving trauma-prone areas of skin, such as the hands, elbows, knees and feet, and in some cases the oral mucosa. Reactivity with different epitopes leads to the different clinical phenotypes.

Figure 1. Clinical features of the patient on admission. (a) Erosions on the elbows, left hip and on the middle of the back. (b) Aph- tha on the border of the tongue and ulcers in the oral cavity. (c) Erosions and milia on the dorsum of the hands. (d) Two years after discharge. No symptoms were seen on the dorsal hand.

Figure 2. (a) Immunoglobulin G immunoblot analysis of normal human dermal extracts. Lane 1, serum of a patient with epider- molysis bullosa acquisita (EBA) reacts with the 290-kDa full-length type VII collagen. Lane 2, serum of a patient with anti-p200 pemphigoid recognizes a 200-kDa protein. Lane 3, serum of a normal control. Lane 4, serum of our patient reacted with the type VII collagen. (b) Immunoblot analysis of purified human laminin-332. Lane 1, serum of an anti-laminin 332-type mucous membrane pemphigoid (MMP) serum reacted with all a3, b3 and c2 subunits. Lane 2, serum of a normal control. Lane 3, serum of our patient reacted with the 165-kDa a3 subunit and the 145-kDa a3 subunit. On the other hand, MMP is a heterogeneous group of autoimmune subepithelial blistering diseases that mainly affect mucous membranes, such as the oral, conjunctival, nasal, pha- ryngeal, laryngeal, esophageal and anogenital mucosae, lead- ing to scarring of the affected tissue. Laminin-332, located in the epidermal BMZ, is a heterotrimeric glycoprotein comprising a3, b3 and c2 subunits that are covalently linked by disulfide bonds. It has been reported that patients with one form of MMP have pathogenic autoantibodies against laminin-332. Egan et al.8 reported an analysis of 35 patients with anti-lami- nin-332-type MMP, and demonstrated that IgG immunoblot analysis detected the a3 subunit alone in 24 cases, the b3 sub- unit alone in two cases, the a3 and b3 subunits in one case, the a3 and c2 subunits in three cases, and the b3 and c2 subunits in one case, indicating that most cases reacted with a subunit. Kato et al.9 reported a case of a patient with MMP with concomitant tracheal and bronchial lesions, in whom autoantibodies to the b3 subunit of laminin-332 were detected by immunoblot analysis. Thus, some phenotypes react with each of the subunits.

In our case, we made the diagnosis of inflammatory EBA with concomitant anti-laminin-332 antibodies, because it is dif- ficult to demonstrate the pathogenicity of the antibody against the a3 subunit of laminin-332. We considered that this patient initially developed anti-type VII collagen autoantibodies, fol- lowed by production of anti-laminin-332 autoantibodies via epi- tope spreading phenomenon.10 In addition, Jonkman et al. have reported the same case before, and they finally diag- nosed EBA based on the Mizoribine sublamina densa level of blister for- mation. Given the reality of this disease and apparent heterogeneity in anti-laminin-332-type MMP, it is important to register such patients with high-quality information in a data- base to determine the significance of association and path- omechanisms in patients with concurrence of the two diseases.

CONFLICT OF INTEREST: None declared.