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• W2024496190 abstract "Epidermolysis bullosa acquisita (EBA) is an acquired bullous disease of the skin characterized by IgG autoantibodies against type VII (anchoring fibril) collagen. We previously defined four immunodominant antigenic epitopes within the noncollagenous 1 (NC1) domain of type VII collagen. In this study, we produced an additional recombinant fusion protein from the NC1 domain corresponding to the N-terminal 227 amino acids (residues 1 to 227), which contains homology with cartilage matrix protein (CMP). Using enzyme-linked immunosorbent assay and immunoblot analysis, we tested sera from EBA patients (n = 32), bullous systemic lupus erythematosus patients (n = 3), bullous pemphigoid patients (n = 15), and normal humans (n = 12). Twenty-six of 32 EBA sera and two of three bullous systemic lupus erythematosus sera reacted with the CMP domain, whereas none of the control sera did. Affinity-purified anti-CMP EBA antibodies injected into hairless mice produced the clinical, histological, immunological, and ultrastructural features of EBA. F(ab′)2 fragments generated from anti-CMP EBA autoantibodies did not induce disease. Our studies provide the first evidence that EBA autoantibodies to the CMP subdomain of NC1 are pathogenic and induce blister formation. This is the first antigenic epitope on type VII collagen demonstrated to be a pathogenic target for EBA autoantibodies. Epidermolysis bullosa acquisita (EBA) is an acquired bullous disease of the skin characterized by IgG autoantibodies against type VII (anchoring fibril) collagen. We previously defined four immunodominant antigenic epitopes within the noncollagenous 1 (NC1) domain of type VII collagen. In this study, we produced an additional recombinant fusion protein from the NC1 domain corresponding to the N-terminal 227 amino acids (residues 1 to 227), which contains homology with cartilage matrix protein (CMP). Using enzyme-linked immunosorbent assay and immunoblot analysis, we tested sera from EBA patients (n = 32), bullous systemic lupus erythematosus patients (n = 3), bullous pemphigoid patients (n = 15), and normal humans (n = 12). Twenty-six of 32 EBA sera and two of three bullous systemic lupus erythematosus sera reacted with the CMP domain, whereas none of the control sera did. Affinity-purified anti-CMP EBA antibodies injected into hairless mice produced the clinical, histological, immunological, and ultrastructural features of EBA. F(ab′)2 fragments generated from anti-CMP EBA autoantibodies did not induce disease. Our studies provide the first evidence that EBA autoantibodies to the CMP subdomain of NC1 are pathogenic and induce blister formation. This is the first antigenic epitope on type VII collagen demonstrated to be a pathogenic target for EBA autoantibodies. Epidermolysis bullosa acquisita (EBA) is a severe, chronic, subepidermal bullous disease of the skin and mucosa characterized by skin fragility, blisters in trauma-prone sites, scarring with milia formation, and nail dystrophy.1Roenigk HH Ryan JG Bergfeld MA Epidermolysis bullosa acquisita: report of three cases and review of all published cases.Arch Dermatol. 1971; 103: 1-10Crossref PubMed Scopus (231) Google Scholar It is a prototypic autoimmune disease in which EBA patients have in vivo tissue-bound and circulating IgG autoantibodies directed against type VII collagen, a major component of anchoring fibrils, structures that anchor the epidermis onto the dermis.2Woodley DT Briggaman RA O'Keefe EJ Inman AQ Queen LL Gammon WR Identification of the skin basement membrane autoantigen in epidermolysis bullosa acquisita.New Engl J Med. 1984; 310: 1007-1013Crossref PubMed Scopus (434) Google Scholar, 3Woodley DT Burgeson RE Lunstrum G Bruckner-Tuderman L Reese MJ Briggaman RA The epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen.J Clin Invest. 1988; 81: 683-687Crossref PubMed Scopus (269) Google Scholar, 4Woodley DT Briggaman RA Falk RJ Reese MJ Tomsick RS Gammon WR O'Keefe EJ Epidermolysis bullosa acquisita antigen, a major cutaneous basement membrane component, is synthesized by human dermal fibroblasts and other cutaneous tissues.J Invest Dermatol. 1986; 87: 227-231Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 5Briggaman RA Wheeler Jr, CE The epidermal-dermal junction.J Invest Dermatol. 1975; 65: 71-84Crossref PubMed Scopus (260) Google Scholar, 6Burgeson RE Type VII collagen, anchoring fibrils, and epidermolysis bullosa.J Invest Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar, 7Sakai LY Keene DR Morris NP Burgeson RE Type VII collagen is a major structural component of anchoring fibrils.J Cell Biol. 1986; 103: 1577-1586Crossref PubMed Scopus (433) Google Scholar, 8Keene DR Sakai LY Lundstru GP Morris NP Burgeson RE Type VII collagen forms an extended network of anchoring fibrils.J Cell Biol. 1987; 104: 611-621Crossref PubMed Scopus (288) Google Scholar EBA autoantibodies bind to type VII collagen within anchoring fibrils. EBA patients have a diminution of normal anchoring fibrils and subsequent epidermal-dermal disadherence. The clinical appearance of EBA patients and the histology of their cutaneous lesions are often very reminiscent of hereditary dystrophic epidermolysis bullosa. These two diseases are etiologically unrelated but share the common feature of decreased anchoring fibrils. In the case of inherited dystrophic epidermolysis bullosa, the cause of decreased or absent anchoring fibrils is a genetic defect in the gene that encodes for type VII collagen.9Uitto J Christiano AM Molecular basis for the dystrophic forms of epidermolysis bullosa: mutations in the type VII collagen gene.Arch Dermatol Res. 1994; 287: 16-22Crossref PubMed Scopus (96) Google Scholar, 10Uitto J Christiano AM Molecular genetics of the cutaneous basement membrane zone: perspectives on epidermolysis bullosa and other blistering skin diseases.J Clin Invest. 1992; 90: 687-692Crossref PubMed Scopus (138) Google Scholar Type VII collagen is composed of three identical α chains, each consisting of a 145-kd central collagenous triple-helical segment characterized by repeating Gly-X-Y amino acid sequences, flanked by a large 145-kd amino-terminal noncollagenous domain (NC1), and a small 34-kd carboxyl-terminal noncollagenous domain (NC2).6Burgeson RE Type VII collagen, anchoring fibrils, and epidermolysis bullosa.J Invest Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar, 7Sakai LY Keene DR Morris NP Burgeson RE Type VII collagen is a major structural component of anchoring fibrils.J Cell Biol. 1986; 103: 1577-1586Crossref PubMed Scopus (433) Google Scholar, 8Keene DR Sakai LY Lundstru GP Morris NP Burgeson RE Type VII collagen forms an extended network of anchoring fibrils.J Cell Biol. 1987; 104: 611-621Crossref PubMed Scopus (288) Google Scholar, 11Lunstrum GP Kuo HJ Rosenbaum LM Keene DR Glanville RW Sakai LY Burgeson RE Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain.J Biol Chem. 1987; 262: 13706-13712Abstract Full Text PDF PubMed Google Scholar, 12Lunstrum GP Sakai LY Keene DR Morris NP Burgeson RE Large complex globular domains of type VII procollagen contribute to the structure of the anchoring fibrils.J Biol Chem. 1986; 261: 9042-9048Abstract Full Text PDF PubMed Google Scholar Within the extracellular space, type VII collagen molecules form anti-parallel, tail-to-tail dimers stabilized by disulfide bonding through a small carboxyl-terminal NC2 overlap between two type VII collagen molecules. The anti-parallel dimers then aggregate laterally to form anchoring fibrils with large globular NC1 domains at both ends of the structure. Sequence analysis of the NC1 domain revealed multiple submodules with homology to adhesive proteins.13Christiano AM Greenspan DS Lee S Uitto J Cloning of human type VII collagen: complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms.J Biol Chem. 1994; 269: 20256-20262Abstract Full Text PDF PubMed Google Scholar These include a segment with homology to CMP, nine consecutive fibronectin type III-like repeats (FNIII), and a segment with homology to the A domain of von Willebrand factor (VWF-A) (Figure 1A). We and others have shown that NC1 interacts with various extracellular matrix components including fibronectin, laminin-5, type I collagen, and type IV collagen.14Chen M Marinkovich MP Veis A O'Toole EA Rao CN Cai XY Woodley DT Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components: a potential role in epidermal-dermal adherence in human skin.J Biol Chem. 1997; 272: 14516-14522Crossref PubMed Scopus (159) Google Scholar, 15Chen M Marinkovich MP Jones JC O'Toole EA Li YY Woodley DT NC1 domain of type VII collagen binds to the beta3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats.J Invest Dermatol. 1999; 112: 177-183Crossref PubMed Scopus (95) Google Scholar, 16Lapiere JC Chen JD Iwasaki T Hu L Uitto J Woodley DT Type VII collagen specifically binds fibronectin via a unique subdomain within the collagenous triple helix.J Invest Dermatol. 1994; 103: 637-641Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 17Rousselle P Keene DR Ruggiero F Champliaud MF Rest M Burgeson RE Laminin 5 binds the NC-1 domain of type VII collagen.J Cell Biol. 1997; 11: 719-728Crossref Scopus (222) Google Scholar Therefore, the NC1 domain may facilitate binding of type VII collagen to other basement membrane zone (BMZ) and matrix components. These matrix interactions are thought to stabilize the adhesion of the BMZ to the underlying dermis. Using a panel of recombinant fusion proteins or fragments of type VII collagen, we and others have shown previously that EBA autoantibodies recognize four major antigenic epitopes confined to the FNIII and VWF-A subdomains of NC1.18Lapiere JC Woodley DT Parente MG Iwasaki T Wynn KC Christiano AM Uitto J Epitope mapping of type VII collagen: identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.J Clin Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar, 19Jones DA Hunt III, SW Prisayanh PS Briggaman RA Gammon WR Immunodominant autoepitopes of type VII collagen are short, paired peptide sequences within the fibronectin type III homology region of the noncollagenous (NC1) domain.J Invest Dermatol. 1995; 104: 231-235Crossref PubMed Scopus (60) Google Scholar, 20Gammon WR Murrell DF Jenison MW Padilla KM Prisayanh PS Jones DA Briggaman RA Hunt III, SW Autoantibodies to type VII collagen recognize epitopes in a fibronectin-like region of the noncollagenous (NC1) domain.J Invest Dermatol. 1993; 100: 618-622Crossref PubMed Scopus (81) Google Scholar At that time, the amino terminus of NC1 had not been cloned or characterized. Moreover, none of the EBA autoantibodies to the identified antigenic epitopes was shown to be pathogenic. The pathogenicity of rabbit anti-type VII collagen antibodies in the induction of EBA has been established in animal models by passively transferring immune rabbit antibodies against type VII collagen into hairless mice.21Woodley DT Chang C Saadat P Ram R Liu Z Chen M Evidence that anti-type VII collagen antibodies are pathogenic and responsible for the clinical, histological, and immunological features of epidermolysis bullosa acquisita.J Invest Dermatol. 2005; 124: 958-964Crossref PubMed Scopus (98) Google Scholar, 22Sitaru C Mihai S Otto C Chiriac MT Hausser I Dotterweich B Saito H Rose C Ishiko A Zillikens D Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen.J Clin Invest. 2005; 115: 870-878Crossref PubMed Scopus (208) Google Scholar Recently, we immunized rabbits and raised a high titer antiserum to the NC1 domain of human type VII collagen. We injected the antibody into hairless immunocompetent mice, and the mice developed a bullous eruption that had many of the features of EBA patients.21Woodley DT Chang C Saadat P Ram R Liu Z Chen M Evidence that anti-type VII collagen antibodies are pathogenic and responsible for the clinical, histological, and immunological features of epidermolysis bullosa acquisita.J Invest Dermatol. 2005; 124: 958-964Crossref PubMed Scopus (98) Google Scholar Another recent study by Sitaru and colleagues22Sitaru C Mihai S Otto C Chiriac MT Hausser I Dotterweich B Saito H Rose C Ishiko A Zillikens D Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen.J Clin Invest. 2005; 115: 870-878Crossref PubMed Scopus (208) Google Scholar showed that the injection of rabbit polyclonal antibodies to the NC1 domain of mouse type VII collagen into adult mice also induced subepidermal skin blisters reminiscent of human EBA. More recently, we affinity-purified anti-NC1 autoantibodies from EBA patients' sera and injected them into hairless mice. The animals developed a subepidermal bullous disease with clinical, histological, immunological, and ultrastructural features similar to human EBA.23Woodley DT Ram R Doostan A Bandyopadhyay P Huang Y Remington J Hou YP Keene DR Liu Z Chen M Induction of epidermolysis bullosa acquisita in mice by passive transfer of autoantibodies from patients.J Invest Dermatol. 2006; 126: 1324-1330Crossref Scopus (83) Google Scholar These results provide evidence that human EBA autoantibodies to the NC1 domain of type VII collagen are pathogenic and capable of inducing epidermal-dermal separation of skin. In this study, an additional recombinant fusion protein corresponding to the N-terminal 227 amino acids (residues 1 to 227) of NC1 and homologous to CMP was generated and analyzed by immunoblot and enzyme-linked immunosorbent assay (ELISA) for reactivity with autoantibodies from 32 EBA patients. We found that 26 of 32 EBA sera and two of three bullous systemic lupus erythematosus (BSLE) sera reacted with the CMP domain in both assays. We then affinity-purified anti-CMP autoantibodies from the serum of one EBA patient and injected them intradermally into adult immunocompetent hairless mice. The injected autoantibodies consistently induced a subepidermal blistering disease resembling the clinical, histological, and immunological features of human EBA. These results demonstrate that EBA autoantibodies to the CMP subdomain of type VII collagen are pathogenic and likely play an important role in the induction of epidermal dermal disadherence featured in EBA. Serum samples were collected from 32 patients with EBA. These EBA patients had 1) an active, chronic, mechanobullous disorder; 2) subepidermal blisters as assessed by routine light microscopy of lesional skin; 3) IgG deposits detected at the dermal-epidermal junction (DEJ) by routine direct immunofluorescence (DIF); 4) IgG deposits localized to the dermal floor of the patient's skin when the DEJ was fractured through the lamina lucida by treatment with 1 mol/L NaCl24Gammon WR Kowalewski C Chorzelski TP Kumar V Briggaman RA Beutner EH Direct immunofluorescence studies of sodium chloride-separated skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita.J Am Acad Dermatol. 1990; 22: 664-679Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 25Woodley DT Sauder D Talley MJ Silver M Grotendorst G Qwarnstrom E Localization of basement membrane components after dermal-epidermal junction separation.J Invest Dermatol. 1983; 81: 149-153Crossref PubMed Scopus (150) Google Scholar; 5) immunoreactivity to the NC1 domain of type VII collagen by ELISA and immunoblot analysis26Chen M Chan LS Cai X O'Toole EA Samples JC Woodley DT Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita.J Invest Dermatol. 1997; 108: 68-72Crossref PubMed Scopus (137) Google Scholar; and 6) IgG deposits detected within the sublamina densa region of the DEJ using direct immunoelectron microscopy.27Yaoita H Briggaman RA Lawley TJ Provost TT Katz SI Epidermolysis bullosa acquisita: ultrastructural and immunological studies.J In-vest Dermatol. 1981; 76: 288-292PubMed Scopus (221) Google Scholar Because sera from patients with BSLE have been shown to have autoantibodies to type VII collagen,28Gammon WR Woodley DT Dole K Briggaman RA Evidence that antibasement membrane zone antibodies in bullosa eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen.J Invest Dermatol. 1985; 84: 472-476Crossref PubMed Scopus (137) Google Scholar three sera from BSLE patients were also tested. Control serum samples were collected from 12 normal individuals (normal human sera, NHS) and 15 patients who had clinical, histological, and immunofluorescence findings consistent with the diagnosis of BP. Patient and normal control sera were stored frozen at −20°C before analysis. Plasma was collected from one patient with EBA during the early phase of their disease (before treatment). This patient was plasmaphoresed for therapeutic purposes, which generated large volumes of plasma rich in anti-CMP antibodies. This EBA patient met the EBA criteria outlined above. In addition, she had both IgG and C3 deposits detected at the DEJ by routine DIF and indirect immunofluorescence (IIF) titers ranging from 1:1280 to 1:5120 as analyzed on salt-split normal human skin substrate. The study was conducted according to Declaration of Helsinki Principles. The fragment corresponding to the 700-bp CMP subdomain of human type VII collagen cDNA was generated by reverse transcriptase-polymerase chain reaction amplification using human amniotic epithelial cell (WISH) cDNA as a template as described previously.14Chen M Marinkovich MP Veis A O'Toole EA Rao CN Cai XY Woodley DT Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components: a potential role in epidermal-dermal adherence in human skin.J Biol Chem. 1997; 272: 14516-14522Crossref PubMed Scopus (159) Google Scholar The insert was then subcloned into a TA vector and pGEX expression vector (Pharmacia, Inc., Piscataway, NJ), as modified by Dr. George Giudice, Medical College of Wisconsin, Milwaukee, WI.29Smith DB Johnson KS Single-step purification of polypeptides expressed in Escherichia coli of fusions with glutathione S-transferase.Gene. 1988; 67: 31-40Crossref PubMed Scopus (5039) Google Scholar The correct ligation and in-frame insertion of the DNA fragment was confirmed by DNA sequence analysis. Bacterial fusion proteins corresponding to discrete segments within the NC1 domain of type VII collagen were developed and purified by a glutathione-Sepharose 4B column (Pharmacia, Uppsala, Sweden) as described.18Lapiere JC Woodley DT Parente MG Iwasaki T Wynn KC Christiano AM Uitto J Epitope mapping of type VII collagen: identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.J Clin Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar These fusion proteins included CMP (residues 1 to 227), FP1 (residues 201 to 602), FP3 (residues 595 to 826), FP7 (residues 814 to 1028), and FP8 (residues 1022 to 1253). Ninety-six-well microtiter plates (Immulon-4; Dynatch Laboratory Inc., Alexandria, VA) were coated with purified glutathione S-transferase (GST)-CMP at a concentration of 1.5 μg/ml (0.15 μg/well) in 20 mmol/L carbonate buffer, pH 9.3, overnight at 4°C. ELISA was performed as previously described.26Chen M Chan LS Cai X O'Toole EA Samples JC Woodley DT Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita.J Invest Dermatol. 1997; 108: 68-72Crossref PubMed Scopus (137) Google Scholar The patients' sera dilutions ranged from 1:100 to 1:1250. Purified GST-CMP protein (100 ng/well) was run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then electrotransferred to a nitrocellulose membrane. Cut strips of nitrocellulose were blocked for 60 minutes at room temperature with 10% nonfat dry milk or overnight at 4°C with 5% bovine serum albumin in 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 0.1% Tween 20 (TTBS). After washing with TTBS buffer, the strips were incubated for 1 hour at room temperature with individual patient sera or control sera diluted in TTBS with 1% bovine serum albumin (1:100). The strips were then washed as before with TTBS three times. The immunoreactivity was detected with a horseradish peroxidase-conjugated goat anti-human IgG (Organon Teknika-Cappel, Durham, NC) diluted in TTBS with 1% bovine serum albumin (1:5000) for 30 minutes at room temperature and enhanced chemiluminescence (Amersham, Buckinghamshire, UK). SKH1 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and hosted at the University of Southern California Facility. These are hairless mice with an intact immune system. Four- to 10-week-old animals were injected with affinity-purified anti-CMP antibodies or flow-through IgG (depleted of reactivity to CMP) from the EBA patients' sera or control IgG fractions from the sera of normal human patients at the same IgG concentrations. All animal studies were conducted using protocols approved by the University of Southern California Institutional Animal Use Committee. The patient's plasma was first diluted with antibody binding buffer (20 mmol/L sodium phosphate, pH 7.0) at a 1:5 dilution and then centrifuged at 4000 rpm to remove insoluble particulate material. The supernatants were then subjected to chromatography using a protein G-Sepharose Fast Flow column following the manufacturer's recommendation (Amersham Biosciences, Uppsala, Sweden). Control IgG fractions were prepared in an identical manner from a commercial lot of human gamma globulins obtained from several hundred normal donors (Sigma, St. Louis, MO). IgG fractions from the EBA patient were further affinity-purified using recombinant GST-CMP fusion protein covalently coupled to a CNBr-activated Sepharose 4B column following the manufacturer's instructions (Amersham Biosciences). Affinity-purified anti-CMP EBA antibodies were dialyzed against phosphate-buffered saline, concentrated by Centricon Plus-20 ultrafiltration (Amicon, Lexington, MA) to 20 to 50 mg/ml, filter-sterilized, and stored at −20°C. By IIF, antibody titers ranged from 1:5000 to 1:10,000 on normal human skin, mouse skin, and salt-split skin substrate. The affinity-purified autoantibodies were also assessed by Western blot analyses and ELISA as described.26Chen M Chan LS Cai X O'Toole EA Samples JC Woodley DT Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita.J Invest Dermatol. 1997; 108: 68-72Crossref PubMed Scopus (137) Google Scholar F(ab′)2 fragments of affinity-purified anti-CMP IgG were prepared by digestion with pepsin as described.23Woodley DT Ram R Doostan A Bandyopadhyay P Huang Y Remington J Hou YP Keene DR Liu Z Chen M Induction of epidermolysis bullosa acquisita in mice by passive transfer of autoantibodies from patients.J Invest Dermatol. 2006; 126: 1324-1330Crossref Scopus (83) Google Scholar Undigested IgG and Fc fragments were removed by affinity chromatography using a protein G-Sepharose Fast Flow column (Amersham Biosciences). Purified F(ab′)2 fragments migrated as a 100-kd band under nonreduced SDS-PAGE. The completeness of digestion was assessed by IIF on mouse skin substrate. The purified F(ab′)2 preparations showed reactivity only with a fluorescein isothiocyanate (FITC)-labeled goat anti-human-F(ab′)2 but not with a FITC-labeled goat anti-human-Fc secondary antibodies. SKHl mice were injected intradermally with EBA anti-CMP IgG (n = 8), EBA IgG depleted of reactivity to the CMP domain of NC1 (flow-through fractions from the CMP affinity column) (n = 7), or normal human control IgG (n = 8) once every day for 8 days and observed every day. IgG doses ranged from 10 to 400 μg/g body weight/per day. The animals were photographed daily. Skin erythema, blisters, and erosions were recorded. Mice that developed blisters had skin biopsies from the blisters and nonblistered normal-appearing skin within 0.5 cm of a blister. Skin samples were fixed in 10% buffered formalin and stained with hematoxylin and eosin. Both lesional and perilesional tissues were subjected to DIF staining as previously described.2Woodley DT Briggaman RA O'Keefe EJ Inman AQ Queen LL Gammon WR Identification of the skin basement membrane autoantigen in epidermolysis bullosa acquisita.New Engl J Med. 1984; 310: 1007-1013Crossref PubMed Scopus (434) Google Scholar, 24Gammon WR Kowalewski C Chorzelski TP Kumar V Briggaman RA Beutner EH Direct immunofluorescence studies of sodium chloride-separated skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita.J Am Acad Dermatol. 1990; 22: 664-679Abstract Full Text PDF PubMed Scopus (159) Google Scholar Monospecific FITC-conjugated sera were obtained commercially: goat anti-human IgG (Sigma), monospecific goat anti-mouse C3 (Cappel Laboratories, Durham, NC), goat anti-mouse neutrophils (Cedarlane, Ontario, ON, Canada), and goat anti-human F(ab′)2 and Fc (Cappel Laboratories). Photographs of immunolabeled tissues were obtained with a Zeiss Axioplan fluorescence microscope equipped with a Zeiss Axiocam MRM digital camera system (Carl Zeiss, Thornwood, NY). We previously produced a series of GST fusion proteins encompassing the complete FNIII and VWFA regions of NC1 domain (Figure 1).18Lapiere JC Woodley DT Parente MG Iwasaki T Wynn KC Christiano AM Uitto J Epitope mapping of type VII collagen: identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.J Clin Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar In this study, we produced an additional NC1 subdomain recombinant fusion protein corresponding to the N-terminal 227 amino acids (residues 1 to 227) of NC1 and homologous to CMP. These recombinant type VII collagen fusion proteins were used to identify regions within NC1 recognized by EBA sera. As shown previously, EBA autoantibodies recognize four major NC1 antigenic epitopes confined within FINIII and VWF-A subdomains of NC1 (Figure 1A).18Lapiere JC Woodley DT Parente MG Iwasaki T Wynn KC Christiano AM Uitto J Epitope mapping of type VII collagen: identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.J Clin Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar By ELISA, 26 of 32 EBA sera (81%) exhibited reactivity with CMP with optical density (OD) values ranging from 0.4 to 2 (Figure 2). Two of three BSLE sera also reacted with CMP, consistent with previous studies showing that BSLE sera contain autoantibodies to NC1.28Gammon WR Woodley DT Dole K Briggaman RA Evidence that antibasement membrane zone antibodies in bullosa eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen.J Invest Dermatol. 1985; 84: 472-476Crossref PubMed Scopus (137) Google Scholar In contrast, all control sera (15 BP and 12 NHS) showed background reactivity with CMP with values less than 0.21 OD (0.25 OD is the cutoff for a positive ELISA reading).26Chen M Chan LS Cai X O'Toole EA Samples JC Woodley DT Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita.J Invest Dermatol. 1997; 108: 68-72Crossref PubMed Scopus (137) Google Scholar The 26 EBA and 2 BSLE sera that reacted with CMP in the ELISA were further analyzed by immunoblot analysis. A representative blot is shown in Figure 3. The 50-kd GST-CMP fusion protein was recognized by 22 EBA sera and two BSLE sera but not by BP or normal human sera at the same 1:100 dilution. Four EBA sera were negative by immunoblot analysis even when the serum dilution was decreased to 1:20. These four EBA sera are those sera with the lowest reactivity to CMP by ELISA. Taken together, these data show that like FINIII and VWF-A subdomains, CMP also contains epitopes recognized by the majority of EBA autoantibodies. We recently showed that affinity-purified autoantibodies against type VII collagen NC1 from EBA patients' sera induced subepidermal blisters when injected into adult hairless immunocompetent mice.23Woodley DT Ram R Doostan A Bandyopadhyay P Huang Y Remington J Hou YP Keene DR Liu Z Chen M Induction of epidermolysis bullosa acquisita in mice by passive transfer of autoantibodies from patients.J Invest Dermatol. 2006; 126: 1324-1330Crossref Scopus (83) Google Scholar To determine whether pathogenic EBA autoantibodies are reactive with CMP epitopes, we affinity-purified CMP-specific EBA antibodies and analyzed these antibodies by immunoblotting and immunofluorescence. Using a panel of GST fusion proteins encompassing the complete NC1 domain of type VII collagen (Figure 4A),18Lapiere JC Woodley DT Parente MG Iwasaki T Wynn KC Christiano AM Uitto J Epitope mapping of type VII collagen: identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.J Clin Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar we found that the pathogenic EBA IgG used in this study recognized exclusively only CMP (Figure 4B, lane 2). The antibody reactivity with this antigenic epitope was specific because the EBA IgG did not recognize FP1, FP3, FP7, or FP8 (Figure 4B, lanes 3 to 6). Further, control normal human serum did not react with any of these fusion proteins (data not shown). As expected, the CMP-specific EBA antibodies only reacted with the 50-kd CMP protein and NC1, not with other fus" @default.
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• W2024496190 date "2007-06-01" @default.
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• W2024496190 title "The Cartilage Matrix Protein Subdomain of Type VII Collagen Is Pathogenic for Epidermolysis Bullosa Acquisita" @default.
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• W2024496190 doi "https://doi.org/10.2353/ajpath.2007.061212" @default.
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