A Practical Technique for Differentiation of Subepidermal Bullous Diseases Localization of in Vivo–Bound Igg by Laser Scanning Confocal Microscopy
Total Page:16
File Type:pdf, Size:1020Kb
STUDY A Practical Technique for Differentiation of Subepidermal Bullous Diseases Localization of In Vivo–Bound IgG by Laser Scanning Confocal Microscopy Katarzyna Woz´niak, MD; Takashi Kazama, MD; Cezary Kowalewski, MD Objective: To develop a practical technique to distin- whereas basement membrane zone markers were la- guish autoimmune subepidermal bullous diseases. beled with anti–mouse Cy5-conjugated antibodies. Design: A prospective study. Results: In patients with bullous pemphigoid, in vivo– bound IgG was localized on the epidermal side of lami-  Setting: Academic referral center—the Department of nin 5 and co-localized with 4 integrin. In patients with Dermatology, Medical University of Warsaw. mucous membrane pemphigoid, IgG was in vivo bound to the dermal-epidermal junction between localization Patients: Forty-two patients fulfilling clinical, immu- of laminin 5 and type IV collagen. In patients with epi- nological, and/or immunoelectron microscopic criteria dermolysis bullosa acquisita, in vivo–bound IgG was for bullous pemphigoid (n=31), mucous membrane pem- present on the dermal side of type IV collagen. phigoid (n=6), or epidermolysis bullosa acquisita (n=5), diagnosed as having disease and treated from January 1, Conclusions: Laser scanning confocal microscopy al- 1997, to December 31, 2002. lows precise localization of in vivo–bound IgG in pa- tients’ skin and, thus, it is a rapid method for the differ- Main Outcome Measures: We applied laser scan- entiation of mucous membrane pemphigoid from bullous ning confocal microscopy to determine the localization pemphigoid and epidermolysis bullosa acquisita. This tool of in vivo–bound IgG at the basement membrane zone is suitable for the routine diagnosis of individual pa- in biopsy specimens taken from patients’ skin to com- tients and for retrospective studies. This method is of spe- pare the localization of basement membrane zone cial value in those patients in whom circulating autoan-  markers: antibody against 4 integrin, antibody against tibodies are not detectable. laminin 5, and antibody against type IV collagen. In vivo– bound IgG was visualized by labeling with fluorescein isothiocyanate–conjugated anti–human IgG antibody, Arch Dermatol. 2003;139:1007-1011 ULLOUS PEMPHIGOID (BP), cult or even impossible.4,5 The character- mucous membrane pemphi- ization of BMZ antigens on a molecular goid (MMP), and epider- level6,7 and the introduction of immuno- molysis bullosa acquisita blotting, radioimmunoprecipitation, an en- (EBA) belong to the group zyme-linked immunosorbent assay, and fu- Bof autoimmune subepidermal bullous dis- sion protein techniques for diagnosing eases (ASBDs) characterized by the devel- ASBDs allow differentiation of these en- opment of tense blisters on apparently tities in patients in whom circulating an- healthy skin. These entities clinically can tibodies are detectable. In patients with mimic each other, especially at the onset BP and MMP, BP180 antigen is a target of the disease, but they differ in course, molecule that plays a crucial role in the prognosis, and response to treatment.1-3 pathogenesis of these dermatoses.8-10 BP180 From the Departments The ASBDs are defined by the presence of antigen is a transmembrane protein con- of Dermatology, Medical circulating antibodies directed against dif- sisting of an amino terminal domain, pres- University of Warsaw, ferent basement membrane zone (BMZ) ent in the cytoplasm of basal keratino- Warsaw, Poland (Drs Woz´niak and Kowalewski), and Niigata antigens and the presence of in vivo– cytes and in the extracellular portion, that University School of Medicine, bound IgG at the dermal-epidermal junc- started with the NC16A domain just be- Niigata, Japan (Dr Kazama). tion in patients’ skin; thus, differentia- low the keratinocyte plasma membrane; The authors have no relevant tion of these dermatoses by routine it extends across the whole lamina lucida financial interest in this article. immunofluorescence (IF) may be diffi- into the upper part of the lamina densa, (REPRINTED) ARCH DERMATOL / VOL 139, AUG 2003 WWW.ARCHDERMATOL.COM 1007 ©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 ending by the carboxyterminal domain on the border of microscopy using a peroxidase technique was performed on the the lamina lucida and the lamina densa.11 In patients with skin originating from 2 patients whose samples were negative BP, the target epitopes for BMZ antibodies are located on for circulating anti–BMZ antibodies, and showed the presence the NC16A domain of BP180 antigen, ultrastructurally of IgG deposits within the lamina lucida. localized in the upper part of the lamina lucida, whereas Patients With MMP the serum samples of most patients with MMP recog- nize the carboxyterminal domain of BP180 antigen at the 12 Six patients (2 men and 4 women) fulfilled the clinical and im- lamina lucida–lamina densa border. In some patients munopathological criteria for MMP.8 All patients with MMP with MMP, autoantibodies are directed against the ␣ chain had mucous membrane and skin involvement. They had tense of laminin 5, localized in the upper part of the lamina bullae on the skin, which healed and left atrophic scars and densa. The production of autoantibodies directed against milia. Mucous membrane involvement in all patients referred type VII collagen, localized in the lower lamina densa and to scarring conjunctivitis and chronic painful erosions of oral the sub–lamina densa, leads to the development of mucosa. Of 6 patients’ serum samples, 2 were positive for cir- EBA.13,14 culating IgG anti–BMZ antibodies and reacted with the epider- In patients with ASBDs in whom circulating anti– mal and dermal side of sodium chloride salt–split skin by in- BMZ antibodies are not detectable, a final diagnosis can direct IF. The results of immunoblot studies were negative (in our studies and in the studies performed at another laboratory). be established based on direct immunoelectron micros- Direct immunoelectron microscopy using the peroxidase copy, which is time-consuming. In patients with BP, im- technique, performed in 4 patients (who were negative for 15 munoreactants are localized to the lamina lucida ;inpa- circulating anti–BMZ antibodies), showed the presence of IgG tients with MMP, immunoreactants are localized to the deposits within the lamina lucida and the lamina densa. lamina lucida and the lamina densa16; and in patients with EBA, immunodeposits are bound below the lamina Patients With EBA densa.17 In a previous study, Kazama et al18 proved that it is possible to distinguish BP from EBA based on the Three men (aged 20, 38, and 42 years at the onset of disease) comparison of the localization of target antigens for cir- and 2 children (a 3-year-old boy and a 13-year-old girl) were culating anti–BMZ antibodies and/or in vivo–bound IgG included in this study. Three patients (2 adults and 1 child) fulfilled the criteria of Roenigk et al3 for mechanobullous EBA. in patients’ skin with the localization of well-defined BMZ They had tense bullae on the skin, localized on traumatized areas markers using laser scanning confocal microscopy that were healing, and scars and milia. Two other patients had (LSCM). In the present study, we applied LSCM to in- an inflammatory type of EBA, clinically resembling BP.2 Of 5 vestigate whether it is possible to differentiate MMP from patients with EBA, 3 had oral mucous involvement. Of 5 pa- BP and EBA based on the localization of in vivo–bound tients’ serum samples, 3 were positive for circulating IgG anti– IgG within the BMZ. BMZ antibodies and revealed reactivity with the exclusively der- Therefore, this study investigates whether it is pos- mal side of sodium chloride salt–split skin by indirect IF. sible to differentiate subepidermal bullous diseases based Immunoblot studies of these serum samples showed reactivity on the localization of in vivo–bound IgG at the BMZ with with a 290-kDa molecule on the dermal extract. Serum stud- regard to the localization of lamina lucida and lamina ies using postembedding immunogold electron microscopy on Lowicryl-embedded healthy human skin demonstrated the re- densa markers using LSCM. activity of IgG anti–BMZ antibodies with the lamina densa and anchoring fibrils. Direct immunoelectron microscopy using the METHODS peroxidase technique, performed on the skin specimens of 2 patients who were negative for circulating anti–BMZ antibod- PATIENTS ies, revealed the presence of IgG deposits below the lamina densa. Data are summarized in the Table. Forty-two patients positive for in vivo–bound IgG and comple- ment C3 at the BMZ by direct IF were selected from a group of LASER SCANNING CONFOCAL MICROSCOPY 102 patients who were diagnosed as having an ASBD and treated at the Department of Dermatology, Medical University of War- Punch biopsy specimens taken from perilesional patients’ skin saw, from January 1, 1997, to December 31, 2002. Patients posi- were mounted in tissue freezing medium (Leica Instruments, tive for in vivo–bound IgA at the BMZ by direct IF as a predomi- GmbH, Nussloch, Germany) and cut into 10-µm cryosec- nant or concomitant component were not included in this study. tions. These sections were incubated with monoclonal anti–  BMZ antibodies directed against 4 integrin, a marker of the Patients With BP upper part of the lamina lucida (clone 3E1; Chemicon Inter- national, Temecula, Calif); laminin 5, also called epiligrin, a Thirty-one patients (14 men and 17 women) fulfilled the clini- marker of the upper part of the lamina densa (clone P3E4; cal and immunopathological criteria for BP. Patients were aged Chemicon International); and type IV collagen, a marker of between 73 and 90 years. They developed tense blisters on the the lamina densa (clone COL-94; Sigma, Steinheim, Ger- trunk and extremities, which healed without scars or milia. Pa- many), for 30 minutes, followed by 5-minute washings with tients with BP who had bullous lesions localized on trauma- phosphate-buffered saline, performed 3 times.