Key Points
Question
What is the clinical and immunological phenotype in patients with bullous pemphigoid (BP) who are negative for BP180 noncollagenous 16A (NC16A) domain in enzyme-linked immunosorbent assay (ELISA) and/or chemiluminescent enzyme immunoassay (CLEIA)?
Findings
Our retrospective study revealed that patients who had negative results on these assays presented with milder phenotypes and less erythema and required less extensive treatments. The reactivity to NC16A domain rarely became positive during the later stages of the disease.
Meaning
Nonreactivity of IgG to the NC16A domain may predict a mild disease course.
This case series examines the association of nonreactivity of IgG to the noncollagenous 16A domain using the enzyme-linked immunosorbent assay and the chemiluminescent enzyme immunoassay and severity of disease course in 14 patients with bullous pemphigoid.
Abstract
Importance
Enzyme-linked immunosorbent assay (ELISA) and/or chemiluminescent enzyme immunoassay (CLEIA) for BP180 noncollagenous 16A (NC16A) extracellular domain is a sensitive diagnostic tool for bullous pemphigoid (BP). However, some patients with BP have negative results for these assays.
Objective
To elucidate the clinical and immunological features of patients with BP without antibodies that react to BP180 NC16A.
Design, Setting, and Participants
This retrospective case series study included 152 patients who were diagnosed with BP and followed up at the Kurume University Hospital in Japan from 2007 to 2016. The diagnosis was made using clinical, histological, and immunological findings.
Main Outcomes and Measures
Clinical and immunological features of patients with BP who had negative results for BP180 NC16A using ELISA and/or CLEIA.
Results
Of the 152 patients, 69 (45.4%) were men and 83 (54.6%) were women. The mean (SD) age of participants was 75.2 (14.4) years. Of the 152 patients with BP, 14 (9.2%) had negative results for BP180 NC16A on ELISA and/or CLEIA; most of these patients exhibited no erythema and had relatively mild phenotypes. Two (14%) of the 14 patients had positive results for intact BP180 in epidermal extracts, 10 (71%) had positive results for a 120-kD fragment of BP180 (LAD-1) and 3 (21%) had positive results for BP180 C-terminal domain. Seven (50%) patients tested positive in BP230 ELISA. Five (36%) patients did not require oral prednisolone treatment, whereas the others required a dose of prednisolone at less than 30 mg per day. Three (21%) patients were administered a dipeptidyl peptidase-4 inhibitor (DPP4i) before disease onset. This ratio was not significantly higher than that in patients with BP who tested positive for BP180 NC16A ELISA and/or CLEIA (19 [14%] of 138 patients). Our follow-up study (mean [SD], 31.9 [33.2] weeks; range, 0-108 weeks) revealed that patients with BP tested negative for BP180 NC16A ELISA and/or CLEIA during the later stages of the disease.
Conclusions and Relevance
This study indicates that patients with BP negative for BP180 NC16A ELISA and/or CLEIA had milder phenotypes, fewer erythemas, and required less extensive treatments.
Introduction
Among autoantibody-induced autoimmune diseases, bullous pemphigoid (BP) is characterized by tense blisters and edematous erythemas, with the highest prevalence and susceptibility in elderly individuals. Immunologically, BP is characterized by circulating antibasement membrane zone (BMZ) antibodies. Their targets are mainly the 230-kD-protein BP antigen 1 (BPAG1, BP230) and the 180-kD-protein BP antigen 2 (BPAG2, BP180, also known as type XVII collagen). An extracellular domain of BP180, the noncollagenous 16A (NC16A) domain contains the main epitope targeted in BP. Anti-BP180 NC16A are pathogenic; they reduce BP180 on the surface of keratinocytes via macropinocytosis and induce complement and neutrophil activation through the Fc gamma receptor, which results in blister formation. Enzyme-linked immunosorbent assay (ELISA) for anti-BP180 NC16A is available and is useful for diagnosis and disease monitoring. Recently, a chemiluminescent enzyme immunoassay (CLEIA) was developed, which is a highly effective autoantibody detection system that is as reliable as ELISA. However, a small number of patients with BP have negative results for BP180 NC16A in these assays (hereafter referred to as non-NC16A patients with BP). The characteristics of non-NC16A patients with BP have not been well described, although a few studies have been reported. In this study, we analyzed patients with BP in our department and summarized their immunological and clinical features.
Methods
Patients
A total of 152 patients with BP who visited the Kurume University Hospital between January 2007 and September 2016 were enrolled in this study. The study was approved by the ethics committee of the Kurume University and was performed in adherence with the Declaration of Helsinki Guidelines. All participants provided written informed consent and they were not compensated. Thirty-one NC16A-positive patients with BP and all the 14 non-NC16A patients with BP were evaluated including their disease severity with the BP Disease Area Index (BPDAI). Detailed information is provided in eMethods in the Supplement.
Results
Of the 152 patients with BP, 14 had negative results for BP180 NC16A on ELISA or CLEIA during their first visit to our hospital.
The clinical and immunological features of non-NC16A patients with BP are summarized in the eTable in the Supplement. We evaluated the disease severity using the BPDAI scores. Scores for cutaneous erosions/blister and urticaria/erythema were significantly lower in non-NC16A patients with BP: Non-NC16A patients with BP vs NC16A-positive patients with BP (mean [SD], 14.57 [12.41] vs 23.65 [13.46]; P = .01 for erosions/blister; 4.57 [9.66] vs 20.10 [13.98]; P < .001 for urticaria/erythema; 1.29 [2.67] vs 0.84 [2.04]; P = .98 for mucous erosions/blister). Notably the score for cutaneous urticaria/erythema was low in non-NC16A patients with BP. The representative clinical pictures of the 2 phenotypes and patients administered dipeptidyl peptidase-4 inhibitor (DPP4i) are shown in Figure 1.
Figure 1. Representative Clinical Manifestations of Non-NC16A Patients With BP.
A, Bulla dominant type (patient 5): this type presented with relatively mild skin involvement, including blisters and erosions without erythema. B, Bulla plus erythema type (patient 4): this type presented with erythema and blisters on the trunk and extremities, which correspond with typical BP. C, DPP-4i–related type (patient 12): this type presented with milder skin involvement and without erythema. The BPDAI scores are presented in eTable in the Supplement.
We then tested the patients’ sera for immunoreactivity to outside of NC16A and for other components at the epidermal BMZ using immunofluorescence, western blotting (WB) and BP230 ELISA, which are well established in our department. Western blotting analyses revealed that epidermal extracts were positive for the 180-kD protein in 2 patients, indicating the reactivity to BP180; for the 190-kD protein in 2 patients, indicating the reactivity to periplakin and for the 230-kD protein in 3 patients, indicating the reactivity to BP230. No patients exhibited reactivity to the recombinant BP180 NC16A protein. Ten and 3 patients exhibited reactivity to LAD-1 in HaCaT cell supernatant and the recombinant BP180 C-terminal domain protein, respectively. Results of ELISA revealed that 7 patients were positive for BP230 (Figure 2).
Figure 2. Summary of DPP4i-Administration in 2 Groups and Immunological Findings in Non-NC16A Patients With BP.
A, The left column shows a prevalence of DPP-4 inhibitors (DDP4i) administration in NC16A-positive patients with BP and non-NC16A patients with BP. B and C, reactivity to each protein or materials detected by WB or ELISA in 2 non-NC16A patients with BP.
Because non-NC16A patients with BP had milder phenotypes, we also analyzed their responses to treatment. Consistent with the low BPDAI scores, 5 (35.7%) patients did not require any systemic corticosteroids and the others required a dose of prednisolone at less than 30 mg per day.
Izumi et al reported that non-NC16A patients with BP exhibited less erythema and had a high prevalence of administration of DPP4i. In our non-NC16A patients with BP, 3 (21.4%) were administered DPP4i before disease onset; however, we could not confirm the duration and end of DPP4i administration.
It remains unclear how autoimmunity causes the recognition of BP180 NC16A during BP course. Di Zenzo et al analyzed the time course of reactivity to the epitopes of BP180 and BP230. In this study, we analyzed the time course of reactivity to NC16A as detected by BP180 ELISA and/or CLEIA. The mean (SD) duration of follow-up to this reactivity was 31.9 (33.2) (range, 0 to 108) weeks. In all the 14 patients, BP180 ELISA and/or CLEIA results remained negative.
Discussion
We report 14 patients who were negative for BP180 NC16A ELISA and/or CLEIA during their first visit to our hospital. These patients had relatively milder disease and fewer erythemas than those with the usual BP type and required lower systemic corticosteroid treatments along with a good response to the treatment. Recently, Izumi et al reported the features of non-NC16A patients with BP using a newly developed full-length eukaryotic BP180 ELISA, and these patients had relatively mild inflammation (erythema) compared with NC16A-positive patients with BP. We also observed similar features in non-NC16A patients with BP. The BP180 NC16A ELISA correlates with disease severity, and pathogenicity of anti-NC16A IgG has been demonstrated in vivo. Based on these reports, we suggest that anti-NC16A IgG is associated with the major pathogenicity and non-NC16A BP is a milder form of the disease. Imafuku et al reported that autoantibodies targeting the regions outside of NC16A depleted BP180 on keratinocytes, although their capacity was lower than that of NC16A autoantibodies in vitro, which may be related to the mild phenotype of non-NC16A BP. Recently the pathogenicity of anti-BP180 IgE was characterized. In non-NC16A patients with BP, anti-BP180 IgE may be pathogenic. Our results suggest that recognition of outside of NC16A rarely spreads to NC16A in patients with BP. Di Zenzo et al reported that no patients developed reactivity to NC16A (P490) later during the disease course. Studies on mice conducted by the same group revealed similar tendencies. Our results suggest that recognition of NC16A by the humoral immune system occurs during the early stage of the disease in most patients, whereas epitope spreading to NC16A rarely occurs in patients without anti-NC16A IgG.
In the study by Izumi et al DDP4i was administered to 7 of 14 non-NC16A patients with BP, and they suggested that DPP4i is involved in the development of non-NC16A BP. They used full-length BP180 and focused on patients who had reactions only with BP180 other than with NC16A, whereas our analysis contained anti-BP230 antibodies alone-positive cases. If we exclude patients with anti-BP230 solely from non-NC16A patients with BP, 3 (30.0%) of 10 non-NC16A patients with BP and 19 (13.8%) of 138 NC16A-positive patients with BP were administrated DPP4i before the disease onset; this was still not significant (P = .17). This discrepancy may be owing to different enrolment periods. The frequency of DPP4i use in treatment for type 2 diabetes dramatically increased in recent years worldwide, including in Japan. In our study, no patients were administrated DPP4i before 2012. When these cases were collected from 2012, 3 of 12 (25.0%) non-NC16A patients with BP and 19 of 75 (25.3%) NC16A-positive patients with BP were administrated DPP4i.
Limitations
This study included a small number of non-NC16A patients with BP, particularly non-NC16A-DPP4i-related BP patients. This was a single-center case series.
Conclusions
We report 14 non-NC16A patients with BP and characterized their unique features, including few erythemas, mild phenotypes, and no epitope spreading to NC16A during the later stages of the disease. The BP180 NC16A ELISA and/or CLEIA is useful for the diagnosis of BP. However, clinicians should keep in mind that some patients with BP exhibit no reactivity in these assays and have unique clinical and immunological features. The complete characterization of non-NC16A BP including with DPP4i administration requires the accumulation of more cases and further investigations.
eMethods
eTable. Clinical and immunological features of Non-NC16A BP in this study
eReferences
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Associated Data
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Supplementary Materials
eMethods
eTable. Clinical and immunological features of Non-NC16A BP in this study
eReferences
