The use of skin substrates deficient in basement membrane molecules for the diagnosis of subepidermal autoimmune bullous disease

ARTICLE

In some cases it is impossible to differentiate between epidermolysis bullosa acquisita (EBA) and bullous pemphigoid (BP) by mere clinical presentation. In such cases laboratory investigations, such as immunofluorescence microscopy and Western blot, could be helpful to differentiate between these two diseases. But what if repetitive tests suggest an unexpected diagnosis contradicting the clinical presentation?

Case report

A 40-year old Hungarian male presented with complaints of blisters, oozing erosions and excoriation on the scalp, trunk, hands and feet which had lasted for more than a year [1]. These lesions had developed spontaneously or had evolved following minor trauma. The family history was negative for blistering disorders.

On physical examination, 3-5 mm, tense blisters with crusts and milia were present on the distal portion of the limbs, feet and toes. On the hands and feet, crusted lesions and milia were found. The tongue mucosa was also affected. The toe nails were dystrophic.

The initial laboratory tests were performed in Hungary. Direct immunofluorescence revealed linear depositions of IgG and C3 along the epidermal basement membrane zone (EBMZ). Direct IF on the salt-split skin of the patient showed a "combined" staining for IgG on both sides of the split as can be found in a minority (5%-10%) of patients with BP [2, 3]. The serum of the patient was shown to be positive by the ELISA technique using a substrate of synthetic oligopeptide of the 230 kDa bullous pemphigoid antigen (BPAG1) as described elsewhere [4]. The BPAG1 oligopeptide used comprised 21 amino acids located at position 1914-1934 according to the peptide sequence of Sawamura et al. (Genbank accession number M69225) [5].

Diagnostic challenge

So the question arose as to whether this patient had EBA as suggested by the clinical presentation [6], or BP mimicking EBA as was suggested from direct IF with salt-split skin and ELISA with BP230 oligopeptide, or was this a combination of both. Clinically, BP may mimic EBA [7] and vice versa, EBA may mimic BP [8]. Although the simultaneous presence of BP and EBA in one person would be extremely unusual, the coexistence of two autoimmune subepidermal blistering disorders has been known to occur [9]. Direct or indirect IF using salt-split skin cannot exclude this possibility. Western blot with the patient's serum may fail to detect conformational epitopes as a result of the denaturing conditions [10].

To solve the problem, we used frozen skin samples lacking the EBA antigen/type VII collagen from a person with severe, mutilating dystrophic epidermolysis bullosa, type Hallopeau-Siemens, as the substrate for indirect IF to test for the presence of circulating antibodies against non-EBA antigens in the patient. This skin substrate is deficient in the protein because of a genetic mutation, while the other proteins of the EBMZ are naturally present. Monoclonal antibody clone II, 32 (Gibco BRL, Gaithersburg, MD) and LH7:2 against respectively, the helical and globular aminoterminal domains [11] did not bind to the type VII collagen-deficient skin substrate. All other antigens of the EBMZ were normally present, including 120, 180 and 230 kDa BP-antigens, as investigated with monoclonal antibodies.

The indirect IF was performed with serum from the patient and with positive and negative control sera from patients with proven EBA or BP (BP180 and BP230 positive) or from normal human controls. In addition, we performed fluorescence overlay antigen mapping (FOAM) on the patient's skin [12].

Indirect IF on skin lacking the EBA antigen showed no binding of the patient's serum IgG or IgA (Fig. 1A). Reference sera from EBA patients did not bind to the type VII collagen-deficient substrate. BP control sera bound in a normal linear pattern along the EBMZ (Fig. 1B). As was expected, normal human sera showed no binding. FOAM of the patient's skin showed overlap of type VII collagen as detected by monoclonal LH7:2 and IgG depositions along the EBMZ (Fig. 2A). The patient thus had only EBA and not BP.

Direct IF of lesional skin showed duplication of the epidermal basement membrane. Antigen mapping of a blister revealed a spontaneous split below the lamina densa: staining with monoclonal antibody GB3 against laminin 5 was seen exclusively in the blister roof, consistent with the diagnosis of EBA (Fig. 2B). Indirect IF on normal salt-split skin now showed exclusive binding of IgG and IgA to the dermal site. The blister level in salt-split skin was always consistently through the lamina lucida as checked by electron microscopy and antigen mapping. Western blot with the patient's serum using a dermal extract showed a band of 290 kD, corresponding to the EBA-antigen [13]. Western blot using cultured keratinocyte and epidermal extracts was always negative. We concluded that this patient was suffering from EBA and not BP. Treatment with prednisolone 100 mg daily and azathioprine 150 mg daily gave a good clinical response. The patient's history, the presence of circulating autoantibodies and the response to treatment suggest that he had an inflammatory form of EBA.

Comment

In this case, it was possible using type VII collagen-deficient skin from a patient with mutilating dystrophic EB to exclude BP in a case of clinical EBA, but with initial laboratory tests which suggested the exclusive or concomitant presence of BP.

The combined linear staining pattern of IgG and C3 initially found by direct IF on salt-split skin of the patient was apparently due to the duplicate epidermal basement membrane. The positive ELISA on BP230 substrate was obviously false or a result of some immunological cross-reaction.

Another method which could have been used to arrive at the correct diagnosis in this patient, would have been the use of the more expensive and time-consuming technique of direct immunoelectron microscopy. The advantage of an EBMZ molecule-deficient skin in indirect IF for the detection of antigen-specific autoantibodies over Western blot or ELISA, is that in the former all other antigens of the EBMZ are naturally present in the substrate. Moreover, the technique is more simple and can be performed in any laboratory with equipment for immunofluorescence microscopy. Obtaining skin substrates that are deficient in an EBMZ molecule may be difficult if no epidermolysis bullosa patients are available.

The concept of using an EBMZ molecule-deficient skin substrate for indirect IF can also be applied to differentiate between other autoimmune subepidermal bullous disorders of which an analogous hereditary EB form exists. For instance, to differentiate between anti-epiligrin cicatricial BP and EBA ­ two disorders with a dermal staining pattern on indirect IF of salt-split skin ­ laminin 5-deficient skin substrate from patients with Herlitz junctional EB (H-JEB) would be the substrate of choice. However, skin of H-JEB patients that lack laminin 5 has reduced binding to monoclonal antibody BM165 directed to the alpha3 chain, even if the H-JEB is caused by LAMA3 truncation mutations [14]. The reduced alpha3 staining in H-JEB skin can be explained by the presence of alpha3 in laminin 6 and laminin 7, laminin isoforms that are present in the EBMZ [15]. Since autoantibodies in anti-epiligrin cicatricial BP bind to the alpha3 chain in laminin isoforms [16], one may expect reduced staining with anti-epiligrin cicatricial BP serum on H-JEB skin, whereas the staining intensity is not reduced when type VII collagen-deficient skin from a patient with mutilating dystrophic EB is used.

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