Takeji Nishikawa

The clinical phenotype of pemphigus is defined by the anti-desmoglein autoantibody profile

BACKGROUND Some patients with pemphigus vulgaris (PV) have mucous membrane erosions with minimal skin involvement (mucosal dominant type), and others show extensive skin blisters and erosions in addition to mucous membrane involvement (mucocutaneous type). Patients with pemphigus foliaceus (PF) show only skin involvement. OBJECTIVE The purpose of this study is to determine whether there is a difference in autoantibody profile among mucosal dominant PV, mucocutaneous PV, and PF. METHODS Antibody titer against desmoglein 1 (Dsg 1) and desmoglein 3 (Dsg3) were measured with enzyme-linked immunosorbent assay using recombinant Dsg1 and Dsg3. Sera were obtained during clinically active disease from 24 patients with mucosal dominant PV, 20 with mucocutaneous PV, and 23 with PF. RESULTS All sera samples from patients with mucosal dominant PV sera were negative against Dsgl but positive against Dsg3. All sera samples from those with mucocutaneous PV were positive against both Dsg1 and Dsg3. All sera samples from patients with PF were positive against Dsg1, but negative against Dsg3. CONCLUSION Each subtype has its own anti-Dsg autoantibody profile, indicating that the clinical phenotype of pemphigus is defined by the autoantibody profile.

Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that occurs in association with underlying neoplasms. Patients with PNP develop characteristic IgG autoantibodies directed against multiple antigens, most of which have been identified as cytoplasmic proteins of the plakin family (desmoplakin I, II, BPAG1, envoplakin, and periplakin). This study identified cell surface target antigens of PNP. We focused on desmoglein (Dsg) 3 and Dsg1, the autoantigens of pemphigus vulgaris and pemphigus foliaceus. ELISA using baculovirus-expressed recombinant Dsgs (rDsg3, rDsg1) has revealed that 25 out of 25 PNP sera tested were positive against Dsg3 and 16 of 25 were positive against Dsg1. All of 12 PNP sera tested immunoprecipitated Dsg3. Removal of anti-Dsg3 autoantibodies by immunoadsorption was sufficient to eliminate the ability of PNP sera to induce cutaneous blisters in neonatal mice in vivo. Furthermore, anti-Dsg3-specific antibodies that were affinity purified from PNP sera were proven to be pathogenic and caused blisters in neonatal mice. These findings indicate that Dsg3 and Dsg1 are the cell surface target antigens in PNP and that IgG autoantibodies against Dsg3 in PNP sera play a pathogenic role in inducing loss of cell adhesion of keratinocytes and causing blister formation.

BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid

Bullous pemphigoid (BP) is an acquired autoimmune subepidermal blistering disease against hemidesmosomal cytoplasmic BP230 and transmembrane BP180 proteins. Epitope mapping studies have shown that the membrane-proximal noncollagenous (NC) 16a domain of BP180 harbors clusters of antigenic sites recognized by the vast majority of BP sera. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) using bacterial recombinant NC16a protein and evaluated its clinical benefit for diagnosis and monitoring disease activity. Fifty four (84.4%) of 64 sera from BP patients were positive, while only one (1.1%) of 91 sera from collagen disease patients and five (1.5%) of 336 sera from normal control barely exceeded the cut-off value. None of 69 pemphigus vulgaris sera and none of 42 pemphigus foliaceus sera exceeded the cut-off value. Thus, the sensitivity and specificity of NC16a ELISA were 84.4 and 98.9%, respectively. The correlation between ELISA scores and disease activity along the time course was examined using seven BP patients. NC16a ELISA scores tended to fluctuate in parallel with the disease activity along the time course and reflected the disease activity much better than indirect immunofluorescence. These findings indicate that NC16a ELISA will be a valuable tool not only for the diagnosis of patients with BP but also for the monitoring of the disease activity.

Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins

Desmoglein (Dsg) is a cadherin-type adhesion molecule found in desmosomes. Dsg1 and Dsg3 are the target Ags in the autoimmune blistering diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. To map conformational epitopes of Dsg1 and Dsg3 in PF and PV, we generated Dsg1- and Dsg3-domain-swapped molecules and point-mutated Dsg3 molecules with Dsg1-specific residues by baculovirus expression. The swapped domains were portions of the N-terminal extracellular domains of Dsg1 (1–496) and Dsg3 (1–566), which have similar structures but distinct epitopes. The binding of autoantibodies to the mutant molecules was assessed by competition ELISAs. Domain-swapped molecules containing the N-terminal 161 residues of Dsg1 and Dsg3 yielded >50% competition in 30/43 (69.8%) PF sera and 31/40 (77.5%) PV sera, respectively. Furthermore, removal of Abs against the 161 N-terminal residues of Dsg1 by immunoadsorption eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. Within these N-terminal regions, most of the epitopes were mapped to residues 26–87 of Dsg1 and 25–88 of Dsg3. Furthermore, a point-mutated Dsg3 molecule containing Dsg1-specific amino acid substitutions (His25, Cys28, Ala29) reacted with anti-Dsg1 IgG, thus defining one of the epitopes of Dsg1. Using the predicted three-dimensional structure of classic cadherins as a model, these findings suggest that the dominant autoimmune epitopes in both PF and PV are found in the N-terminal adhesive surfaces of Dsgs.

Monitoring disease activity in pemphigus with enzyme-linked immunosorbent assay using recombinant desmogleins 1 and 3.

Summary Background Pemphigus is an antidesmoglein (Dsg) autoimmune disease that is divided into two major subtypes: pemphigus foliaceus (PF) and pemphigus vulgaris (PV). We previously developed enzyme‐linked immunosorbent assays (ELISAs) using recombinant Dsg1 and Dsg3 to detect IgG autoantibodies in patients with pemphigus. The protocol for the ELISAs was optimized for serological diagnosis, but under the conditions used, these assays were not particularly useful for monitoring disease activity in certain patients. That is, the sera from some patients with high‐titre antibodies continued to show high index values in the ELISA after clinical improvement.

Immunoblotting studies of linear IgA disease

Patients with linear IgA deposits at the basement membrane zone (BMZ) detected by direct immunofluorescence (IF) may show diverse clinical and laboratory findings. The aim of this study was to investigate the issue of target antigens for linear IgA disease (LAD) antibodies. We examined sera from 46 adults and children with exclusive IgA deposits at the BMZ, by both indirect IF on 1 M NaCl split human skin and immunoblotting. IgA anti-BMZ antibodies binding to the epidermal side of the split were found in 31 LAD sera. IgA anti-BMZ antibodies binding to the dermal side of the split were detected only in 4 LAD sera. No sera contained IgA anti-BMZ antibodies binding to both sides of the split. Immunoblotting revealed that 12 epidermal side-positive LAD sera reacted with the 97 kDa protein in the human epidermal extracts. Moreover, we found that 2 dermal side-positive LAD sera reacted with a protein of approximately 255 kDa on immunoblotting of the dermal extract. We conclude that there are at least two types of LAD. However, the nature of target antigens for LAD antibodies remains to be determined.

A case of linear IgA bullous dermatosis with IgA anti-type VII collagen autoantibodies

Summary In this study we present a patient with the sublamina densa type of linear IgA bullous dermatosis (LABD). with IgA autoantibodies reactive with the 290‐kDa type VII collagen (the epidermolysis bullosa acquisita (EBA) antigen) and with immunoblotting of normal human dermal extracts. The clinical and histological features of the present case were compatible with those of LABI) but quite different from those of RBA. Although EBA sera reacted with the bacterial fusion protein of the N‐terminal globular (NC1) domain of type VII collagen, this patients serum did not show reactivity. Furthermore, ultrastructural localization of target epitopes on the anchoring fibrils in this patient was considerably different from EBA. These results indicate that, whereas EBA antibodies react with the NC1 domain of type VII collagen, the epitope in this case is different from that of EBA (and is most likely on the central triple helical domain). This difference may be responsible for the clinical presentation in this patient being distinct from that of EBA.

Predominant IgG4 subclass in autoantibodies of pemphigus vulgaris and foliaceus

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1. We have previously developed ELISAs using recombinant Dsg3 and Dsg1 expressed by baculovirus as a diagnostic tool for pemphigus. In this study, we determined the frequency of coexistence of IgA class as well as distribution of IgG subclass. Two out of 49 PV and PF sera tested had anti-Dsg1 IgA in addition to anti-Dsg1 IgG. Interestingly, one of them showed prominent pustular formation. Among IgG subclass, IgG4 was predominant and found in all of the 30 PV and 19 PF sera tested, followed by IgG1, detected in 25 out of 30 PV and 12 out of 19 PF sera. Even though IgG2 and IgG3 were detected in 13 and one PV and 6 and 4 PF sera, respectively, the ELISA titers had barely exceeded the cutoff value in most of the cases. There was no IgG subclass shift during the course of the disease in seven cases examined. These findings indicate that IgG4 subclass is the predominant autoantibodies in both PV and PF, while IgG1 is also frequently found.

Pathogenic autoantibody production requires loss of tolerance against desmoglein 3 in both T and B cells in experimental pemphigus vulgaris

Mechanisms of tolerance break against desmoglein 3 (Dsg3) in patients with pemphigus vulgaris (PV) producing pathogenic anti‐Dsg3 IgG autoantibodies are unclear. In this study, using a novel PV mouse model involving Dsg3 knockout mice, we investigated the mechanisms leading to production of autoantibodies against Dsg3. Adoptive transfer of Dsg3–/– splenocytes immunized with recombinant mouse Dsg3 to Rag2–/– recipient mice expressing Dsg3 resulted in the stable production of anti‐Dsg3 IgG and development of PV phenotypes including oral erosions with suprabasilar acantholysis. When purified T and B cells from Dsg3–/–, Dsg3+/– or Dsg3+/+ mice were mixed with various combinations and transferred to Rag2–/– mice, pathogenic anti‐Dsg3 IgG production was observed only with a combination of Dsg3–/– T and Dsg3–/– B cells but not with the other combinations. These results suggest that loss of tolerance against Dsg3 in both B and T cells is important for the development of autoimmune state of PV.

Dissociation of intra- and extracellular domains of desmosomal cadherins and E-cadherin in Hailey-Hailey disease and Darier's disease

In order to clarify the pathomechanism of acantholysis in Hailey–Hailey disease (HHD) and Darier’s disease (DD), the distribution of desmosomal and adherens junction‐associated proteins was studied in the skin of patients with HHD (n = 4) and DD (n = 3). Domain‐specific antibodies were used to determine the cellular localization of the desmosomal transmembrane glycoproteins (desmogleins 1 and 3 and desmocollin), desmosomal plaque proteins (desmoplakin, plakophilin and plakoglobin) and adherens junction‐associated proteins (E‐cadherin, α‐catenin, β‐catenin and actin). A significant difference in staining patterns between intra‐ and extracellular domains of desmosomal cadherins and E‐cadherin was demonstrated in acantholytic cells in both HHD and DD, but not in those in pemphigus vulgaris and pemphigus foliaceus samples used as controls. In acantholytic cells in HHD and DD, antibodies against attachment plaque proteins and intracellular epitopes of desmosomal cadherins exhibited diffuse cytoplasmic staining, whereas markedly reduced staining was observed with antibodies against extracellular epitopes of the desmogleins. Similarly, membrane staining of an intracellular epitope of E‐cadherin was preserved, while immunoreactivity of an extracellular epitope of E‐cadherin was destroyed. While the DD gene has been identified as ATP2A2, the gene for HHD has not been clarified. The dissociation of intra‐ and extracellular domains of desmosomal cadherin and E‐cadherin is characteristic of the acantholytic cells in HHD and DD, and not of pemphigus. This common phenomenon in HHD and DD might be closely related to the pathophysiological mechanisms in both conditions.