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Bullous Pemphigoid: Using Animal Models to Study the Immunopathology

  • Zhi Liu
    Correspondence
    Department of Dermatology, University of North Carolina, 3100 Thurston-Bowles, Chapel Hill, NC 27599, USA
    Affiliations
    Departments of Dermatology, Microbiology, and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA
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      Bullous pemphigoid was first described by Lever in 1953 as a subepidermal blistering disease. Its immunohistological features include dermal–epidermal junction separation, an inflammatory cell infiltrate in the upper dermis, and basement membrane zone–bound autoantibodies. These autoantibodies show a linear staining at the dermal–epidermal junction, activate complement, and recognize two major hemidesmosomal antigens, BP230 (BPAG1) and BP180 (BPAG2 or type XVII collagen). An IgG passive transfer mouse model of BP was developed by administering rabbit antimurine BP180 antibodies to neonatal mice. This model recapitulates the key features of human bullous pemphigus. Using this in vivo model system, several key cellular and molecular events leading to the bullous pemphigus disease phenotype were identified, including IgG binding, complement activation, mast cell degranulation, and neutrophil infiltration and activation. Proteinases and reactive oxygen species released by neutrophils work together to damage the basement membrane zone, causing dermal–epidermal junction separation. Recent experimental data from human bullous pemphigus studies suggest that human bullous pemphigus and its mouse IgG passive transfer model counterpart may well share not only common immunohistological features but also pathological mechanisms underlying the development of this antibody-mediated disease.

      Keywords

      Abbreviations

      BMZ
      basement membrane zone
      BP
      bullous pemphigoid
      DEJ
      dermal-epidermal junction
      eos
      eosinophil
      GB
      gelatinase B
      hBP180
      human BP180 antigen
      macrophage
      MC
      mast cell
      mBP180
      murine BP180 antigen
      NE
      neutrophil elastase
      PMN
      neutrophil

      BP as an inflammatory disease

      In 1953 Lever described the disease entity known as bullous pemphigoid (BP) as a subepidermal bullous dermatosis seen in elderly individuals (
      • Lever W.F.
      Pemphigus.
      ). Light microscopic examination of lesional/perilesional skin of BP shows subepidermal vesiculation due to detachment of the epidermis from the underlying dermis. Inflammatory cells, including eosinophils, neutrophils, lymphocytes, and monocytes/macrophages, are present in the upper dermis and bullous cavity, with eosinophils being the predominant cell type. Electron microscopic analysis of BP lesional skin reveals a split at the level of the lamina lucida (the electron-lucent region immediately beneath the basal keratinocyte) and decreased numbers of hemidesmosomes. Intact and degranulating eosinophils and mast cells are seen in the dermis.
      Various inflammatory mediators that can activate mast cells or leukocytes have been identified in lesional skin and/or blister fluids of BP patients, including (1) granular proteins derived from degranulated leukocytes, such as eosinophil cationic protein (ECP), eosinophil major basic protein (MBP), and neutrophil-derived myeloperoxidase (MPO); (
      • Baba T.
      • Sonozaki H.
      • Seki K.
      • Uchiyama M.
      • Ikesawa Y.
      • Torisu M.
      An eosinophil chemotactic factor present in blister fluids of bullous pemphigoid patients.
      ;
      • Czech W.
      • Schaller J.
      • Schopf E.
      • Kapp A.
      Granulocyte activation in bullous diseases. Release of granular proteins in bullous pemphigoid and pemphigus vulgaris.
      ;
      • Borrego L.
      • Maynard B.
      • Peterson E.A.
      • et al.
      Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins.
      ) and (2) chemoattractants and cytokines, such as C5a fragments (
      • Diaz-Perez J.L.
      • Jordon R.E.
      The complement system in bullous pemphigoid. IV. Chemotactic activity in blister fluid.
      ), histamine (
      • Katayama I.
      • Doi T.
      • Nishioka K.
      High histamine level in the blister fluid of bullous pemphigoid.
      ), leukotriene B4 (
      • Kawana S.
      • Ueno A.
      • Nishiyama S.
      Increased levels of Immunorective leukotriene B4 in blister fluids of bullous pemphigoid patients and effects of a selective 5-lipoxygenase inhibitor on experimental skin lesions.
      ), IL-1,2,4,5,6,8,15, TNF-α, IFN-γ, RANTES, and eotaxin (reviewed by
      • D’Auria L.
      • Cordiali Fei P.
      • Ameglio F.
      Cytokines and bullous pemphigoid.
      ;
      • Wakugawa M.
      • Nakamura K.
      • Hino H.
      • et al.
      Elevated levels of eotaxin and interleukin-5 in blister fluid of bullous pemphigoid: correlation with tissue eosinophilia.
      ). Several proteinases are also found in BP blister fluid, including plasmin, collagenase, elastase, and 92-kDa gelatinase (
      • Oikarinen A.I.
      • Zone J.J.
      • Ahmed A.R.
      • Kiistala U.
      • Uitto J.
      Demonstration of collagenase and elastase activities in blister fluids from bullous skin diseases. Comparison between dermatitis herpetiformis and bullous pemphigoid.
      ;
      • Lauharanta J.
      • Salonen E.M.
      • Vaheri A.
      Plasmin-like proteinase associated with high molecular weight complexes in blister fluid of bullous pemphigoid.
      ;
      • Baird J.
      • Lazarus G.S.
      • Belin D.
      • Vassalli J.D.
      • Busso N.
      • Gubler P.
      • Jensen P.J.
      MRNA for tissue-type plasminogen activator is present in lesional epidermis from patients with psoriasis, pemphigus, or bullous pemphigoid, but is not detected in normal epidermis.
      ;
      • Gissler H.M.
      • Simon M.M.
      • Kramer M.D.
      Enhanced association of plasminogen/plasmin with lesional epidermis of bullous pemphigoid.
      ;
      • Kramer M.D.
      • Reinartz J.
      The autoimmune blistering disease bullous pemphigoid. The presence of plasmin/ 2-antiplasmin complexes in skin blister fluid indicates plasmin generation in lesional skin.
      ;
      • Ståhle-Bäckdahl M.
      • Inoue M.
      • Giudice G.J.
      • Parks W.C.
      92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.
      ;
      • Schaefer B.M.
      • Jaeger C.
      • Drepper E.
      • Kramer M.D.
      Plasminogen activation in bullous pemphigoid immunohistology reveals urokinase type plasminogen activator, its receptor and plasminogen activator inhibitor type-2 in lesional epidermis.
      ;
      • Verraes S.
      • Hornebeck W.
      • Polette M.
      • Borradori L.
      • Bernard P.
      Respective contribution of neutrophil elastase and matrix metalloproteinase 9 in the degradation of BP180 (type XVII collagen) in human bullous pemphigoid.
      ).

      BP as an autoimmune disease

      BP autoantibodies bind to the basement membrane zone (BMZ) and activate complement (
      • Jordon R.E.
      • Beutner E.H.
      • Witebsky E.
      • Blumental G.
      • Hale W.C.
      • Lever W.F.
      Basement zone antibodies in bullous pemphigoid.
      ). These autoantibodies are directed against two major hemidesmosomal antigens of 230 kDa (BP230 or BPAG1) and 180 kDa (BP180, BPAG2, or type XVII collagen) (
      • Stanley J.R.
      • Hawley-Nelson P.
      • Yuspa S.H.
      • Shevach E.M.
      • Katz S.I.
      Characterization of bullous pemphigoid antigen: A unique basement membrane protein of stratified epithelia.
      ,
      • Stanley J.R.
      • Woodley D.T.
      • Katz S.I.
      Identification and partial characterization of pemphigoid antigen extracted from normal human skin.
      ;
      • Mutasim D.F.
      • Takahashi Y.
      • Labib R.S.
      • Anhalt G.J.
      • Patel H.P.
      • Diaz L.A.
      A pool of bullous pemphigoid antigen (s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex.
      ;
      • Labib R.S.
      • Anhalt G.J.
      • Patel H.P.
      • Mutasim D.F.
      • Diaz L.A.
      Molecular heterogeneity of bullous pemphigoid antigens as detected by immunoblotting.
      ). Both BP180 and BP230 have been cloned and characterized at the molecular level (
      • Stanley J.R.
      • Tanaka T.
      • Mueller S.
      • Klaus-Kovtun V.
      • Roop D.
      Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.
      ;
      • Diaz L.A.
      • Ratrie H.
      • Saunders W.S.
      • Futamura S.
      • Squiquera H.L.
      • Anhalt G.J.
      • Giudice G.J.
      Isolation of a human epidermal cNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.
      ;
      • Sawamura D.
      • Li K.
      • Chu M-L.
      • Uitto J.
      Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains.
      ;
      • Tanaka T.
      • Parry D.A.D.
      • Klaus-Kovtun V.
      • Steinert P.M.
      • Stanley J.R.
      Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a new family of cell adhesion junction plaque proteins.
      ;
      • Giudice G.J.
      • Emery D.J.
      • Diaz L.A.
      Cloning and primary structural analysis of the bullous pemphigoid autoantigen, BP-180.
      ). BP230 is an intracellular protein that localizes to the hemidesmosomal plaque (
      • Klatte D.H.
      • Kurpakus M.A.
      • Grelling K.A.
      • Jones J.C.R.
      Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.
      ;
      • Tanaka T.
      • Korman N.J.
      • Shimizu H.
      • Eady A.J.
      • Klaus-Kovtun V.
      • Cehrs K.
      • Stanley J.R.
      Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA.
      ;
      • Ishiko A.
      • Shimizu H.
      • Kikuchi A.
      • Ebihara T.
      • Hashimoto T.
      • Nishikawa T.
      Human autoantibodies against the 230-kD bullous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-kD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin.
      ) and has significant homology with plectin and desmoplakins I and II (
      • Tanaka T.
      • Parry D.A.D.
      • Klaus-Kovtun V.
      • Steinert P.M.
      • Stanley J.R.
      Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a new family of cell adhesion junction plaque proteins.
      ;
      • Sawamura D.
      • Li K.
      • Chu M-L.
      • Uitto J.
      Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains.
      ;
      • Green K.J.
      • Virata M.L.
      • Elgart G.W.
      • Stanley J.R.
      • Parry D.A.D.
      Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: Members of a new gene family involved in organization of intermediate filaments.
      ). In contrast, BP180 is a transmembrane protein with a type II orientation (Figure 1A). Its amino-terminal region localizes to the intracellular hemidesmosomal plaque, and its carboxyl-terminal portion projects into the extracellular milieu of the BMZ (
      • Giudice G.J.
      • Emery D.J.
      • Diaz L.A.
      Cloning and primary structural analysis of the bullous pemphigoid autoantigen, BP-180.
      ;
      • Hopkinson S.B.
      • Riddelle K.S.
      • Jones J.C.R.
      Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: Molecular and cell biologic characterization.
      ;
      • Ishiko A.
      • Shimizu H.
      • Kikuchi A.
      • Ebihara T.
      • Hashimoto T.
      • Nishikawa T.
      Human autoantibodies against the 230-kD bullous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-kD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin.
      ). The extracellular region consists of 15 collagen domains separated from one another by noncollagen sequences. BP180 as a transmembrane antigen makes it a preferred target for pathogenic autoantibodies in BP.
      Figure thumbnail gr1
      Figure 1Human and mouse BP180. (A) Sequence comparison of human and murine BP180 proteins. A schematic representation of the structural organization of the human BP180 protein is shown at the top. The orange arch designates the transmembrane domain. The red oval designates the NC16A antigenic site recognized by BP autoantibodies. The COOH-terminal extracellular region is made up of 15 interrupted collagen-like domains (yellow rectangles). The box (in red) at the bottom shows the amino acid sequence alignment of the human and murine BP180 within the BP epitope. Identical residues are designated by double dots, and conservative substitutions are marked by a single dot. An unusually high degree of sequence divergence is seen in the epitope region. (B) Characterization of anti-BP180 antibody cross-species reactivity. Rabbit antimurine BP180 (α-mBP180) and rabbit antihuman BP180 (α-hBP180) NC16A domain antisera were analyzed by immunoblotting (IB) using recombinant human BP180 (H, lanes 1 and 3) or recombinant murine BP180 (M, lanes 2 and 4) and indirect immunofluourescence (IIF) using human (panels a and b) or murine (panels c and d) skin sections. Anti-hBP180 autoantibodies react with the recombinant human BP180 (lane 1) and human skin (panel a), but fail to cross-react with recombinant mBP180 (lane 2) and mouse skin (panel c). Conversely, anti-mBP180 antibodies react with mBP180 (lane 4) and stain mouse skin (panel d), but do not bind hBP180 (lane 3) or human skin (panel b). (C) Passive transfer of rabbit anti-mBP180 IgG. Neonatal Balb/c mice were injected intradermally with rabbit antimurine BP180 IgG. After 24 hours, gentle friction elicits persistent epidermal wrinkling, producing the “epidermal detachment” sign (panel a). Direct immunofluorescence analysis shows deposition of anti-BP180 IgG (panel b) and murine C3 (panel c) at the basement membrane zone. Histological examination of lesional skin reveals dermal–epidermal junction separation with an inflammatory infiltrate (panel d).
      The humoral response in BP is polyclonal. Epitope-mapping studies show that BP autoantibodies recognize multiple epitopes that cluster within the largest noncollagen domain (referred to as NC16A) of the BP180 antigen (
      • Giudice G.J.
      • Emery D.J.
      • Zelickson B.D.
      • Anhalt G.J.
      • Liu Z.
      • Diaz L.A.
      Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.
      ;
      • Zillikens D.
      • Rose P.A.
      • Balding S.D.
      • et al.
      Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies.
      ). These BP180NC16A-specific autoantibodies are predominantly IgE and IgG isotypes and IgG1 and IgG4 subclasses (
      • Bernard P.
      • Aucouturier P.
      • Denis F.
      • Bonnetblanc J.M.
      Immunoblot analysis of IgG subclasses of circulating antibodies in bullous pemphigoid.
      ;
      • Dopp R.
      • Schmidt E.
      • Chimanovitch I.
      • Leverkus M.
      • Brocker E.B.
      • Zillikens D.
      IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in bullous pemphigoid: Serum levels of these immunoglobulins reflect disease activity.
      ;
      • Laffitte E.
      • Skaria M.
      • Jaunin F.
      • Tamm K.
      • Saurat J.H.
      • Favre B.
      • Borradori L.
      Autoantibodies to the extracellular and intracellular domain of bullous pemphigoid 180, the putative key autoantigen in bullous pemphigoid, belong predominantly to the IgG1 and IgG4 subclasses.
      ). The serum levels of autoantibodies to BP180NC16A are correlated with the severity of BP (
      • Haase C.
      • Budinger L.
      • Borradori L.
      • Yee C.
      • Merk H.F.
      • Yancey K.
      • Hertl M.
      Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoid: ELISA studies utilizing a baculovirus-encoded form of bullous pemphigoid antigen 2.
      ;
      • Dopp R.
      • Schmidt E.
      • Chimanovitch I.
      • Leverkus M.
      • Brocker E.B.
      • Zillikens D.
      IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in bullous pemphigoid: Serum levels of these immunoglobulins reflect disease activity.
      ).
      The majority of BP patients have autoreactive T cells that recognize epitopes located in the extracellular region of BP180 (
      • Budinger L.
      • Borradori L.
      • Yee C.
      • et al.
      Identification and characterization of autoreactive CD4+ T cell responses to the extracellular domain of bullous pemphigoid antigen 2 in patients with bullous pemphigoid and normals.
      ) and mainly in the NC16A domain (
      • Lin M.S.
      • Fu C.L.
      • Giudice G.J.
      • Olague-Marchan M.
      • Lazaro A.M.
      • Stastny P.
      • Diaz L.A.
      Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.
      ). These T lymphocytes express CD4 memory T cell surface markers and exhibit a TH1/TH2 mixed cytokine profile. These studies suggest that BP is a T cell–dependent, B cell–dependent, and antibody-mediated skin autoimmune disease.

      The IgG passive transfer model of BP as an in vivo system to study the disease immunopathogenesis

      Blister formation in BP was thought to be an IgG autoantibody–mediated process; however, previous attempts to demonstrate the pathogenicity of patient autoantibodies were unsuccessful. BP autoantibodies that react with an immunodominant and potentially pathogenic epitope in NC16A of BP180 fail to cross-react with the murine form of this autoantigen and thus cannot be assayed for pathogenicity in a conventional passive transfer mouse model (Figure 1a and 1b). As an alternative, my colleagues and I generated rabbit polyclonal antibodies against a segment of the murine BP180 protein homologous with the human BP180 NC16A (mBP180 NC14A), and passively transferred the purified rabbit anti-mBP180 IgG into neonatal BALB/c mice. The injected animals developed a blistering disease that closely mimicked human BP (Figure 1C) (
      • Liu Z.
      • Diaz L.A.
      • Troy J.L.
      • Taylor A.F.
      • Emery D.E.
      • Fairley J.A.
      • Giudice G.J.
      A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.
      ). Direct immunofluorescence analysis of the lesional/perilesional skin of the diseased mice revealed in vivo deposition of rabbit IgG and mouse C3 at the basement membrane. Routine histology of the lesional skin revealed a DEJ separation and an extensive inflammatory cell infiltration, consisting of neutrophils, lymphocytes, and monocytes/macrophages, with neutrophils being the predominant cells (Figure 2A) (
      • Chen R.
      • Fairley J.A.
      • Zhao M.
      • Giudice G.J.
      • Zillikens D.
      • Diaz L.A.
      • Liu Z.
      Macrophages, but not T and B lymphocytes are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation.
      ). Using mice deficient in these cells, we demonstrated that mast cells, macrophages, and neutrophils play a role in subepidermal blistering (Figure 2B). Epitope-mapping studies revealed that pathogenic anti-mBP180 antibodies recognize a 9–12 amino acid stretch within the murine BP180 NC14A region of the antigen (
      • Liu Z.
      • Diaz L.A.
      • Swartz S.J.
      • Fairley J.A.
      • Troy J.L.
      • Giudice G.J.
      Molecular mapping of a pathogenically relevant BP180 epitope associated with experimentally induced murine bullous pemphigoid.
      ). Significantly, this epitope overlaps the region of the human BP180 NC16A, which contains the immunodominant epitopes recognized by human BP autoantibodies.
      Figure thumbnail gr2
      Figure 2Cellular events in the IgG passive transfer model of BP. (A) Quantification of inflammatory cell recruitment in the dermis of mice injected with pathogenic anti-BP180 IgG. Neonatal mice were injected i.d. with pathogenic (black bars) or control IgG (gray bars). The skin sections at the IgG injection site were obtained 24 hours postinjection and processed to recover infiltrating cells. The different populations of inflammatory cells were identified by staining and flow cytometry. The data shown are the mean±SE. *p<0.05 and **p<0.01, Student t-test for paired samples. PMN, neutrophils; Mφ, macrophages; T, T lymphocytes; B, B lymphocytes; eos, eosinophils. (B) Clinical and histological examination of neonatal mice deficient in different inflammatory cells injected with pathogenic anti-mBP180 IgG. Neonatal mice were injected i.d. with anti-mBP180 IgG. Twelve hours later, wild-type C57BL/6J (WT), T and B cell–deficient (T & B(–)) mice developed subepidermal blisters. In contrast, mast cell–deficient (MC(–)), macrophage-deficient (MΦ(–)), and neutrophil-deficient (PMN(–)) mice injected i.d. with pathogenic IgG showed no evidence of skin disease.
      The critical role of complement activation in experimental BP is established by the following approaches: (1) C5-deficient mice are resistant to experimental BP; (2) Balb/c mice pretreated with cobra venom factor to deplete complement are resistant to experimental BP; (3) F(ab′)2 fragments generated from the pathogenic anti-mBP180 IgG cannot induce subepidermal blisters in C5-sufficient mice; and (4) C5-deficient mice reconstituted with C5a become susceptible to experimental BP (
      • Liu Z.
      • Giudice G.J.
      • Swartz S.J.
      • Fairley J.A.
      • Till G.O.
      • Troy J.L.
      • Diaz L.A.
      The role of complement in experimental bullous pemphigoid.
      ). Further studies reveal that the major function of complement activation is to generate C5a that in turn activates mast cells (Figure 3A).
      Figure thumbnail gr3
      Figure 3Functional relationship between complement, mast cells, and neutrophils in the IgG passive transfer model of BP. Neonatal mice were injected i.d. with pathogenic anti-mBP180 IgG and clinically examined at different time points. The skin sections at each time point were analyzed by mast cell (MC)-specific staining (toluidine blue staining) for MC identification and quantification and by myeloperoxidase (MPO) activity assay for neutrophil infiltration. (A) Complement (C′) activation causes MC degranulation. C5-sufficient mice (panel a) show extensive MC degranulation at 2 hours and develop blisters at 12 hours post-injection. In contrast, C5-deficient mice (panel b) exhibit minimal MC degranulation and no skin lesions. d, dermis. (B) Time course of MC degranulation and neutrophil infiltration. MC degranulation reaches the peak level at 1–2 hours after IgG injection (panel a), whereas PMN infiltration appears at 2 hours and reaches the peak level at 8 hours post-injection. (C) Local reconstitution of neutrophils restores the pathogenic activity of anti-mBP180 antibodies in MC-deficient mice. Injection of pathogenic anti-mBP180 IgG causes BP in MC-sufficient, but not MC-deficient, mice. MC-deficient mice reconstituted with neutrophils (by intradermal administration) injected with anti-mBP180 IgG develop BP. MC-deficient mice pretreated intradermally with IL-8 or TNF-α also become susceptible to experimental BP (data not shown).
      Extensive mast cell (MC) degranulation is seen in the lesional skin of mice injected with pathogenic anti-mBP180 antibodies. MC activation precedes neutrophil infiltration (Figure 3B), and inhibition of MC degranulation blocks neutrophil infiltration and subsequent blister formation. Furthermore, MC-deficient mice are resistant to experimental BP and, when reconstituted with MC, restore the pathogenic activity of anti-mBP180 IgG. In addition, pathogenic anti-mBP180 antibodies also induce BP skin lesions in MC-deficient mice reconstituted with neutrophils (Figure 3C), TNF-α, or IL-8 (data not shown). These results suggest that mast cells play a critical role in recruiting neutrophils (
      • Chen R.
      • Ning G.
      • Zhao M.L.
      • Fleming M.G.
      • Diaz L.A.
      • Werb Z.
      • Liu Z.
      Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid.
      ).
      Neutrophil infiltration is a prerequisite for experimental BP, and disease severity is directly correlated to the number of infiltrating neutrophils (
      • Liu Z.
      • Giudice G.J.
      • Zhou X.
      • et al.
      A major role for neutrophils in experimental bullous pemphigoid.
      ). Mice depleted of neutrophils are resistant to experimental BP. Blocking neutrophil infiltration abolishes subepidermal blistering. Therefore, infiltrating neutrophils are the cells that directly cause tissue injury in the basement membrane zone, leading to subepidermal blistering.
      Infiltrating neutrophils, upon activation through molecular interaction between Fc of pathogenic anti-mBP180 IgG and Fc receptors on the cell surface of neutrophils, release several proteolytic enzymes, including neutrophil elastase (NE) and gelatinase B (GB). Mice lacking NE or GB are resistant to experimental BP (
      • Liu Z.
      • Shipley J.M.
      • Vu T.H.
      • Zhou X.
      • Diaz L.A.
      • Werb Z.
      • Senior R.M.
      Gelatinase B-deficient mice are resistant to experimental BP.
      ,
      • Liu Z.
      • Zhou X.
      • Shapiro S.D.
      • et al.
      The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo.
      ). In vitro, although both GB and NE are capable of degrading the recombinant BP180 protein, only NE produces DEJ separation when incubated with skin sections (
      • Ståhle-Bäckdahl M.
      • Inoue M.
      • Giudice G.J.
      • Parks W.C.
      92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.
      ;
      • Liu Z.
      • Shapiro S.D.
      • Zhou X.
      • et al.
      Neutrophil elastase plays a direct role in dermal-epidermal junction separation in experimental bullous pemphigoid.
      ) (Figure 4A). In vivo, NE compensates for the lack of GB whereas GB cannot compensate for NE deficiency in the development of BP. In addition, the degradation of BP180 in vivo depends on NE activity (Figure 4B). These findings demonstrate that GB acts upstream of NE. Further dissection of this proteolytic event in experimental BP reveals a functional relationship between GB and NE; GB proteolytically inactivates α1-proteinase inhibitor, the physiological inhibitor of NE. Unchecked NE then cleaves extracellular matrix proteins, including BP180 antigen, resulting in DEJ separation (
      • Liu Z.
      • Shapiro S.D.
      • Zhou X.
      • et al.
      Neutrophil elastase plays a direct role in dermal-epidermal junction separation in experimental bullous pemphigoid.
      ).
      Figure thumbnail gr4
      Figure 4Proteolytic events in the IgG passive transfer model of BP. (A) Neutrophil elastase produces DEJ separation in mouse skin in vitro. Neonatal mouse skin sections were incubated in medium alone (panel a), with purified neutrophil elastase (NE) (panel b), or with gelatinase B (GB) (panel c) at 37°C for 24 hours and were examined by routine histology. DEJ separation is seen in sections treated with NE but not GB or medium control. Epidermis (e), dermis (d), blister vesicle (v), site of basal keratinocytes (arrow). (B) In vivo BP180 degradation depends on neutrophil elastase (NE) activity. Neonatal wild-type (WT), NE-deficient (NE-), and GB-deficient (GB-) mice were injected i.d. with pathogenic IgG alone or with neutrophils and were examined 12 hours after injection. The skin extracts were analyzed by immunoblotting using the anti-mBP180 IgG. The wild-type mice injected with pathogenic IgG develop BP blisters and both intact and degraded BP180 are present in lesional skin extracts (lane 1). In contrast, NE-deficient mice injected with pathogenic IgG show no skin lesions and no degraded BP180 was found in skin samples (lane 2). GB-deficient mice locally reconstituted with 2.5 million GB-deficient (but NE-sufficient) neutrophils and then injected i.d. with pathogenic IgG develop BP, and cleaved BP180 is detected in their lesional skin extracts (lane 3). In contrast, GB-deficient mice locally reconstituted with the same number of NE-deficient (but GB-sufficient) neutrophils and then injected i.d. with pathogenic IgG fail to develop BP, and extracts from their skin show only intact BP180 (lane 4). As a negative control, wild-type mice injected i.d. with normal IgG show no skin lesions and no degraded BP180 is seen in skin extract samples (data not shown). mPMN: mouse neutrophils.

      Relevance of the IgG Passive Transfer Model to Human BP

      Experimental BP reproduces key characteristics of human BP at the clinical, histological, and immunological levels (
      • Liu Z.
      • Diaz L.A.
      • Troy J.L.
      • Taylor A.F.
      • Emery D.E.
      • Fairley J.A.
      • Giudice G.J.
      A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.
      ). Since the development of the IgG passive transfer model of BP in 1993, the pathogenic role of anti-BP180 antibodies has been confirmed by other in vitro and in vivo studies. BP180NC16A-specific autoantibodies purified from human BP sera induce dermal–epidermal separation in cryosections of human skin in the presence of neutrophils (
      • Sitaru C.
      • Schmidt E.
      • Petermann S.
      • Munteanu L.S.
      • Brocker E.B.
      • Zillikens D.
      Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
      ). Like the rabbit anti-mBP180 IgG passive transfer model, rabbit antibodies directed against the extracellular domain of hamster BP180 (NC16A region) trigger BP-like subepidermal blisters in neonatal hamsters (
      • Yamamoto K.
      • Inoue N.
      • Masuda R.
      • et al.
      Cloning of hamster type XVII collagen cDNA, and pathogenesis of anti-type XVII collagen antibody and complement in hamster bullous pemphigoid.
      ).
      Despite the similarities in the immunopathological features of the experimental model of BP and the human disease, there is one striking difference. The inflammatory infiltrate of early lesions in human BP shows large numbers of eosinophils. The majority of BP patients are eosinophil-rich with some patients showing neutrophil-rich or pauci-inflammatory pemphigoid blisters. These different pathological presentations indicate that BP is a heterogeneous disease and that blisters may be initiated and sustained by multiple immunopathological mechanisms. Some of these mechanisms require only pathogenic antibodies; others depend on both pathogenic antibodies and inflammatory cells (eosinophils and neutrophils). Therefore, different BP patients may respond to a particular treatment differently. In the IgG passive transfer mouse model triggered by a well-defined IgG preparation, however, neutrophils are the predominant inflammatory cell type. Time course studies did not uncover signs of eosinophil recruitment into the injection site of the mice up to 24 hours after passive transfer of pathogenic anti-mBP180 IgG, despite extensive subepidermal blistering observed by 12 hours postinjection. Therefore, it is quite clear that in experimental BP, eosinophils do not play an important role in the initial stages of subepidermal blistering.
      We cannot rule out the possibility of fundamental differences in the pathogenic mechanisms of human BP and mouse BP; however, it remains quite possible that eosinophils are not directly involved, or play only an accessory role, in the initiation of human BP. Interestingly, recent studies implicate neutrophils as the possible disease–causing inflammatory cells. In vitro DEJ separation induced by human BP autoantibodies specific for BP180NC16A depends on neutrophils (
      • Sitaru C.
      • Schmidt E.
      • Petermann S.
      • Munteanu L.S.
      • Brocker E.B.
      • Zillikens D.
      Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
      ). This study confirms the findings by Gammon and colleagues two decades ago which showed that BP autoantibodies induce DEJ separation in human skin sections only in the presence of both leukocytes (mostly neutrophils) and complement (
      • Gammon W.R.
      • Merritt C.C.
      • Lewis D.M.
      • Sams Jr., W.M.
      • Carlo J.R.
      • Wheeler Jr., C.E.
      An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement.
      ). In addition, although human BP blister fluid has high levels of both neutrophil elastase (NE) and gelatinase B (GB), its degradation of BP180 depends on neutrophil elastase activity (
      • Verraes S.
      • Hornebeck W.
      • Polette M.
      • Borradori L.
      • Bernard P.
      Respective contribution of neutrophil elastase and matrix metalloproteinase 9 in the degradation of BP180 (type XVII collagen) in human bullous pemphigoid.
      ). These results confirm the findings from experimental BP and strongly suggest that NE plays a key role in direct cleavage of BP180 and subepidermal blister formation in human BP. In fact, neutrophil-predominant forms of BP have been described in which eosinophils are absent in the inflammatory cell infiltrate (
      • Ackerman A.B.
      • Chongchitnant N.
      • Sanchez J.
      • Guo Y.
      • Rennin B.
      • Reichel M.
      • Randal M.B.
      ). It is worth noting that most patient biopsies are obtained days or weeks after the onset of disease activity. Eosinophil predominance may reflect the chronic stage of the inflammatory reaction in BP. Taken together, these findings strongly suggest that, as in the IgG passive transfer model of BP, neutrophils may be responsible for subepidermal blister formation in human BP. The exact role eosinophils play in BP requires further investigation.

      Conclusions

      Recent experimental evidence strongly suggests that human BP and mouse BP share not only a common disease phenotype but also a common underlying immunopathogenesis. This model has proved to be very useful in dissecting the anti-BP180 IgG-mediated inflammatory cascade in BP and could be used to test new therapeutic strategies. Preclinical data demonstrate that blocking any one of these steps can abolish the BP disease phenotype. Findings from the IgG passive transfer model of BP may also have significant implications for cicatricial pemphigoid, herpes gestationis, linear IgA bullous dermatosis, and lichen planus pemphigoides, all of which are autoimmune subepidermal blistering disorders that have an anti-BP180 immune response (
      • Morrison L.H.
      • Labib R.S.
      • Zone J.J.
      • Diaz L.A.
      • Anhalt G.J.
      Herpes gestationis autoantibodies recognize a 180-kD human epidermal antigen.
      ;
      • Bernard P.
      • Prost C.
      • Durepaire N.
      • Basset-Seguin N.
      • Didierjean L.
      • Saurat J.H.
      The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen.
      ;
      • Tamada Y.
      • Yokochi K.
      • Nitta Y.
      • Toshihiko I.
      • Hara K.
      • Owaribe K.
      Lichen planus pemphigoides. Identification of 180 kD hemidesmosome antigen.
      ;
      • Zone J.J.
      • Taylor T.B.
      • Meyer L.J.
      • Petersen M.J.
      The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2.
      ).
      I am indebted to Drs Luis A. Diaz and David S. Rubenstein for their thoughtful review of this manuscript. This work was supported by US Public Health Service grant R01 AI40768.

      REFERENCES

        • Ackerman A.B.
        • Chongchitnant N.
        • Sanchez J.
        • Guo Y.
        • Rennin B.
        • Reichel M.
        • Randal M.B.
        Histologic Diagnosis of Inflammatory Skin Diseases. (2nd edn.). Baltimore, Williams & Wilkins1997: 238-239
        • Baba T.
        • Sonozaki H.
        • Seki K.
        • Uchiyama M.
        • Ikesawa Y.
        • Torisu M.
        An eosinophil chemotactic factor present in blister fluids of bullous pemphigoid patients.
        J Immunol. 1976; 116: 112-116
        • Baird J.
        • Lazarus G.S.
        • Belin D.
        • Vassalli J.D.
        • Busso N.
        • Gubler P.
        • Jensen P.J.
        MRNA for tissue-type plasminogen activator is present in lesional epidermis from patients with psoriasis, pemphigus, or bullous pemphigoid, but is not detected in normal epidermis.
        J Invest Dermatol. 1990; 95: 548-552
        • Bernard P.
        • Aucouturier P.
        • Denis F.
        • Bonnetblanc J.M.
        Immunoblot analysis of IgG subclasses of circulating antibodies in bullous pemphigoid.
        Clin Immunol Immunopathol. 1990; 54: 484-494
        • Bernard P.
        • Prost C.
        • Durepaire N.
        • Basset-Seguin N.
        • Didierjean L.
        • Saurat J.H.
        The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen.
        J Invest Dermatol. 1992; 99: 174-179
        • Borrego L.
        • Maynard B.
        • Peterson E.A.
        • et al.
        Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins.
        Am J Pathol. 1996; 148: 897-909
        • Budinger L.
        • Borradori L.
        • Yee C.
        • et al.
        Identification and characterization of autoreactive CD4+ T cell responses to the extracellular domain of bullous pemphigoid antigen 2 in patients with bullous pemphigoid and normals.
        J Clin Invest. 1998; 102: 2082-2089
        • Chen R.
        • Fairley J.A.
        • Zhao M.
        • Giudice G.J.
        • Zillikens D.
        • Diaz L.A.
        • Liu Z.
        Macrophages, but not T and B lymphocytes are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil infiltration depends on mast cell activation.
        J Immunol. 2002; 169: 3987-3992
        • Chen R.
        • Ning G.
        • Zhao M.L.
        • Fleming M.G.
        • Diaz L.A.
        • Werb Z.
        • Liu Z.
        Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid.
        J Clin Invest. 2001; 108: 1151
        • Czech W.
        • Schaller J.
        • Schopf E.
        • Kapp A.
        Granulocyte activation in bullous diseases. Release of granular proteins in bullous pemphigoid and pemphigus vulgaris.
        J Am Acad Dermatol. 1993; 29: 210-215
        • D’Auria L.
        • Cordiali Fei P.
        • Ameglio F.
        Cytokines and bullous pemphigoid.
        Eur Cytokine Net. 1999; 10: 123-134
        • Diaz L.A.
        • Ratrie H.
        • Saunders W.S.
        • Futamura S.
        • Squiquera H.L.
        • Anhalt G.J.
        • Giudice G.J.
        Isolation of a human epidermal cNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.
        J Clin Invest. 1990; 86: 1088-1094
        • Diaz-Perez J.L.
        • Jordon R.E.
        The complement system in bullous pemphigoid. IV. Chemotactic activity in blister fluid.
        Clin Immunol Immunopathol. 1976; 5: 360-370
        • Dopp R.
        • Schmidt E.
        • Chimanovitch I.
        • Leverkus M.
        • Brocker E.B.
        • Zillikens D.
        IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in bullous pemphigoid: Serum levels of these immunoglobulins reflect disease activity.
        J Am Acad Dermatol. 2000; 42: 577-583
        • Gammon W.R.
        • Merritt C.C.
        • Lewis D.M.
        • Sams Jr., W.M.
        • Carlo J.R.
        • Wheeler Jr., C.E.
        An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement.
        J Invest Dermatol. 1982; 78: 285-290
        • Gissler H.M.
        • Simon M.M.
        • Kramer M.D.
        Enhanced association of plasminogen/plasmin with lesional epidermis of bullous pemphigoid.
        Br J Dermatol. 1992; 127: 272-277
        • Giudice G.J.
        • Emery D.J.
        • Diaz L.A.
        Cloning and primary structural analysis of the bullous pemphigoid autoantigen, BP-180.
        J Invest Dermatol. 1992; 99: 243-250
        • Giudice G.J.
        • Emery D.J.
        • Zelickson B.D.
        • Anhalt G.J.
        • Liu Z.
        • Diaz L.A.
        Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.
        J Immunol. 1993; 151: 5742-5750
        • Green K.J.
        • Virata M.L.
        • Elgart G.W.
        • Stanley J.R.
        • Parry D.A.D.
        Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: Members of a new gene family involved in organization of intermediate filaments.
        Int J Macromol. 1992; 14: 145-153
        • Haase C.
        • Budinger L.
        • Borradori L.
        • Yee C.
        • Merk H.F.
        • Yancey K.
        • Hertl M.
        Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoid: ELISA studies utilizing a baculovirus-encoded form of bullous pemphigoid antigen 2.
        J Invest Dermatol. 1998; 110: 282-286
        • Hopkinson S.B.
        • Riddelle K.S.
        • Jones J.C.R.
        Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: Molecular and cell biologic characterization.
        J Invest Dermatol. 1992; 99: 264-270
        • Ishiko A.
        • Shimizu H.
        • Kikuchi A.
        • Ebihara T.
        • Hashimoto T.
        • Nishikawa T.
        Human autoantibodies against the 230-kD bullous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-kD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin.
        J Clin Invest. 1993; 91: 1608-1615
        • Jordon R.E.
        • Beutner E.H.
        • Witebsky E.
        • Blumental G.
        • Hale W.C.
        • Lever W.F.
        Basement zone antibodies in bullous pemphigoid.
        JAMA. 1967; 200: 751-758
        • Katayama I.
        • Doi T.
        • Nishioka K.
        High histamine level in the blister fluid of bullous pemphigoid.
        Arch Dermatol Res. 1984; 276: 126-127
        • Kawana S.
        • Ueno A.
        • Nishiyama S.
        Increased levels of Immunorective leukotriene B4 in blister fluids of bullous pemphigoid patients and effects of a selective 5-lipoxygenase inhibitor on experimental skin lesions.
        Acta Derm Venereol. 1990; 70: 281-285
        • Klatte D.H.
        • Kurpakus M.A.
        • Grelling K.A.
        • Jones J.C.R.
        Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.
        J Cell Biol. 1989; 109: 3377-3390
        • Kramer M.D.
        • Reinartz J.
        The autoimmune blistering disease bullous pemphigoid. The presence of plasmin/ 2-antiplasmin complexes in skin blister fluid indicates plasmin generation in lesional skin.
        J Clin Invest. 1993; 92: 978-983
        • Labib R.S.
        • Anhalt G.J.
        • Patel H.P.
        • Mutasim D.F.
        • Diaz L.A.
        Molecular heterogeneity of bullous pemphigoid antigens as detected by immunoblotting.
        J Immunol. 1986; 136: 1231-1235
        • Laffitte E.
        • Skaria M.
        • Jaunin F.
        • Tamm K.
        • Saurat J.H.
        • Favre B.
        • Borradori L.
        Autoantibodies to the extracellular and intracellular domain of bullous pemphigoid 180, the putative key autoantigen in bullous pemphigoid, belong predominantly to the IgG1 and IgG4 subclasses.
        Bri J Dermatol. 2001; 144: 760-768
        • Lauharanta J.
        • Salonen E.M.
        • Vaheri A.
        Plasmin-like proteinase associated with high molecular weight complexes in blister fluid of bullous pemphigoid.
        Acta Dermato-Venereologica. 1989; 69: 527-529
        • Lever W.F.
        Pemphigus.
        Medicine. 1953; 32: 1-123
        • Lin M.S.
        • Fu C.L.
        • Giudice G.J.
        • Olague-Marchan M.
        • Lazaro A.M.
        • Stastny P.
        • Diaz L.A.
        Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.
        J Invest Dermatol. 2000; 115: 955-961
        • Liu Z.
        • Diaz L.A.
        • Swartz S.J.
        • Fairley J.A.
        • Troy J.L.
        • Giudice G.J.
        Molecular mapping of a pathogenically relevant BP180 epitope associated with experimentally induced murine bullous pemphigoid.
        J Immunol. 1995; 155: 5449-5454
        • Liu Z.
        • Diaz L.A.
        • Troy J.L.
        • Taylor A.F.
        • Emery D.E.
        • Fairley J.A.
        • Giudice G.J.
        A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.
        J Clin Invest. 1993; 92: 2480-2488
        • Liu Z.
        • Giudice G.J.
        • Swartz S.J.
        • Fairley J.A.
        • Till G.O.
        • Troy J.L.
        • Diaz L.A.
        The role of complement in experimental bullous pemphigoid.
        J Clin Invest. 1995; 95: 1539-1544
        • Liu Z.
        • Giudice G.J.
        • Zhou X.
        • et al.
        A major role for neutrophils in experimental bullous pemphigoid.
        J Clin Invest. 1997; 100: 1256
        • Liu Z.
        • Shapiro S.D.
        • Zhou X.
        • et al.
        Neutrophil elastase plays a direct role in dermal-epidermal junction separation in experimental bullous pemphigoid.
        J Clin Invest. 2000; 105: 113-123
        • Liu Z.
        • Shipley J.M.
        • Vu T.H.
        • Zhou X.
        • Diaz L.A.
        • Werb Z.
        • Senior R.M.
        Gelatinase B-deficient mice are resistant to experimental BP.
        J Exp Med. 1998; 188: 475-482
        • Liu Z.
        • Zhou X.
        • Shapiro S.D.
        • et al.
        The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo.
        Cell. 2000; 102: 647-655
        • Morrison L.H.
        • Labib R.S.
        • Zone J.J.
        • Diaz L.A.
        • Anhalt G.J.
        Herpes gestationis autoantibodies recognize a 180-kD human epidermal antigen.
        J Clin Invest. 1988; 81: 2023-2026
        • Mutasim D.F.
        • Takahashi Y.
        • Labib R.S.
        • Anhalt G.J.
        • Patel H.P.
        • Diaz L.A.
        A pool of bullous pemphigoid antigen (s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex.
        J Invest Dermatol. 1985; 84: 47-53
        • Oikarinen A.I.
        • Zone J.J.
        • Ahmed A.R.
        • Kiistala U.
        • Uitto J.
        Demonstration of collagenase and elastase activities in blister fluids from bullous skin diseases. Comparison between dermatitis herpetiformis and bullous pemphigoid.
        J Invest Dermatol. 1983; 81: 261-266
        • Sawamura D.
        • Li K.
        • Chu M-L.
        • Uitto J.
        Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains.
        J Biol Chem. 1991; 266: 17784-17790
        • Schaefer B.M.
        • Jaeger C.
        • Drepper E.
        • Kramer M.D.
        Plasminogen activation in bullous pemphigoid immunohistology reveals urokinase type plasminogen activator, its receptor and plasminogen activator inhibitor type-2 in lesional epidermis.
        Autoimmunity. 1996; 23: 155-164
        • Sitaru C.
        • Schmidt E.
        • Petermann S.
        • Munteanu L.S.
        • Brocker E.B.
        • Zillikens D.
        Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
        J Invest Dermatol. 2002; 118: 664-6671
        • Ståhle-Bäckdahl M.
        • Inoue M.
        • Giudice G.J.
        • Parks W.C.
        92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.
        J Clin Invest. 1994; 93: 2022-2030
        • Stanley J.R.
        • Hawley-Nelson P.
        • Yuspa S.H.
        • Shevach E.M.
        • Katz S.I.
        Characterization of bullous pemphigoid antigen: A unique basement membrane protein of stratified epithelia.
        Cell. 1981; 24: 897-903
        • Stanley J.R.
        • Tanaka T.
        • Mueller S.
        • Klaus-Kovtun V.
        • Roop D.
        Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.
        J Clin Invest. 1988; 82: 1864-1870
        • Stanley J.R.
        • Woodley D.T.
        • Katz S.I.
        Identification and partial characterization of pemphigoid antigen extracted from normal human skin.
        J Invest Dermatol. 1984; 82: 108-111
        • Tamada Y.
        • Yokochi K.
        • Nitta Y.
        • Toshihiko I.
        • Hara K.
        • Owaribe K.
        Lichen planus pemphigoides. Identification of 180 kD hemidesmosome antigen.
        J Am Acad Dermatol. 1995; 32: 883-887
        • Tanaka T.
        • Korman N.J.
        • Shimizu H.
        • Eady A.J.
        • Klaus-Kovtun V.
        • Cehrs K.
        • Stanley J.R.
        Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA.
        J Invest Dermatol. 1990; 94: 617-623
        • Tanaka T.
        • Parry D.A.D.
        • Klaus-Kovtun V.
        • Steinert P.M.
        • Stanley J.R.
        Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a new family of cell adhesion junction plaque proteins.
        J Biol Chem. 1991; 266: 12125-12555
        • Verraes S.
        • Hornebeck W.
        • Polette M.
        • Borradori L.
        • Bernard P.
        Respective contribution of neutrophil elastase and matrix metalloproteinase 9 in the degradation of BP180 (type XVII collagen) in human bullous pemphigoid.
        J Invest Dermatol. 2001; 117: 1091-1096
        • Wakugawa M.
        • Nakamura K.
        • Hino H.
        • et al.
        Elevated levels of eotaxin and interleukin-5 in blister fluid of bullous pemphigoid: correlation with tissue eosinophilia.
        Bri J Dermatol. 2000; 143: 112-116
        • Yamamoto K.
        • Inoue N.
        • Masuda R.
        • et al.
        Cloning of hamster type XVII collagen cDNA, and pathogenesis of anti-type XVII collagen antibody and complement in hamster bullous pemphigoid.
        J Invest Dermatol. 2002; 118: 485-492
        • Zillikens D.
        • Rose P.A.
        • Balding S.D.
        • et al.
        Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies.
        J Invest Dermatol. 1997; 109: 573-579
        • Zone J.J.
        • Taylor T.B.
        • Meyer L.J.
        • Petersen M.J.
        The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2.
        J Invest Dermatol. 1998; 110: 207-210