Bullous pemphigoid and mucous membrane pemphigoid humoral responses differ in reactivity towards BP180 midportion and BP230

Abstract

Background: Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) are rare autoimmune blistering disorders characterized by autoantibodies (autoAbs) targeting dermo-epidermal junction components such as BP180 and BP230. The differential diagnosis, based on both the time of appearance and the extension of cutaneous and/or mucosal lesions, is crucial to distinguish these diseases for improving therapy outcomes and delineating the correct prognosis; however, in some cases, it can be challenging. In addition, negative results obtained by commercially available enzyme-linked immunosorbent assays (ELISAs) with BP and MMP sera, especially from patients with ocular involvement, often delay diagnosis and treatment, leading to a greater risk of poor outcomes.

Objectives: Our aim was to find potentially different reactivity profiles in BP and MMP and improve available approaches for diagnosis with focus on ocular MMP.

Methods: Two cohorts of 90 BP and 90 MMP, recruited from different Italian clinical centers, were characterized also employing a novel ELISA based on the BP180 extracellular domain (ECD-BP180).

Results: Immunoglobulin G (IgG) reactivity to BP180 and BP230 in MMP sera was significantly reduced in comparison with BP, mostly affecting BP230 and E-1080 (53% and 36% in BP vs. 11% and 3% in MMP, respectively, p < 0.0001). The combined sensitivity of BP180-NC16A and ECD-BP180 ELISAs was greater compared to BP180-NC16A and BP230 ELISAs both in BP (97% and 92%, respectively) and in MMP (42% and 31%, respectively). The present study shows that MMP patients with ocular involvement rarely reacted to BP180 by IgG in contrast with patients with oral and/or cutaneous involvement (p = 0.0245 and p = 0.0377, respectively), suggesting that an oral and/or cutaneous MMP positive to BP180 hardly evolves to ocular MMP. Of note, one-third of ocular MMP showed immunoglobulin A (IgA) reactivity to ECD-BP180 by immunoblotting.

Conclusions: The present study provides several hints to perform a correct and timely diagnosis in BP and MMP, which is crucial for improving therapy outcomes and delineating the correct prognosis.

Keywords: autoantibody; autoantigen; bullous pemphigoid; epitope; humoral response; mucous membrane pemphigoid.

Figure 1

Setting up of BP180-ectodomain ELISA. (A) Schematic representation of the extracellular portion of human BP180 (ECD-BP180), spanning amino acids 490–1,497. Leader sequence for secretion, 6-His tag, and NC16A region are indicated. Black and light blue boxes refer to non-collagenous and collagenous protein domains, respectively. (B) ECD-BP180 production by transient transfection of Expi293™ cells is depicted. (C) Area under the curve (AUC) and cutoff value were calculated with 50 bullous pemphigoid patients and 50 normal donors.

Figure 2

ELISA and immunoblotting reactivity to BP180 of bullous pemphigoid sera. (A) Scatter plot representation of IgG reactivity of bullous pemphigoid (BP) sera tested in BP180-NC16A and ECD-BP180 ELISAs. Red dots represent sera negative for both NC16A and BP230. The cutoff value is indicated by the dashed black line; PIV, pemphigoid index value, calculated as reported in Materials and Methods. (B) Immunoblotting filters showing IgA reactivity of BP and normal donor (ND) sera against recombinant ECD-BP180. Representative BP sera (1, 2, 3, and 4 positive and 5 negative), two ND (6 and 7 negative), and positive control serum (C+) are shown in the figure.

Figure 3

ELISA and immunoblotting reactivity to BP180 of mucous membrane pemphigoid sera. (A) Scatter plot representation of IgG reactivity of mucous membrane pemphigoid (MMP) sera tested in BP180-NC16A and ECD-BP180 ELISAs. Red dots represent NC16A and BP230 negative sera. The cutoff value is indicated by the dashed black line; PIV, pemphigoid index value, calculated as reported in Materials and Methods. (B) Immunoblotting filters showing IgA reactivity of MMP and normal donor (ND) sera against recombinant ECD-BP180. Five representative MMP sera (1, 2, 3, and 4 positive and 5 negative), two ND (6 and 7 negative), and positive control serum (C+) are shown in the figure.

Figure 4

MMP patients’ sera show a global reduced reactivity to BP180 and BP230 in comparison with BP. The graph displays the comparison of immunological reactivity between bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) sera with BP230 and BP180 antigens/epitopes. E-1080, midportion of the extracellular domain of BP180, spanning amino acids (AA) 1,080–1,107; E-1331, C-terminal region of the extracellular domain of BP180, spanning AA 1,331–1,404; ECD-BP180, ectodomain region of BP180 covering the 490–1,497 AA residues; p-value was evaluated with Fisher’s exact test.