In 1953 Lever described the disease entity known as bullous

Transcription

1 ORIGINAL ARTICLE Bullous Pemphigoid: Using Animal Models to Study the Immunopathology Zhi Liu Departments of Dermatology, Microbiology, and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Bullous pemphigoid was rst described by Lever in 1953 as a subepidermal blistering disease. Its immunohistological features include dermal^epidermal junction separation, an in ammatory cell in ltrate 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 identi ed, including IgG binding, complement activation, mast cell degranulation, and neutrophil in ltration 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. Key words: animal model/autoimmune bullous disease/basement membrane/hemidesmosome/in ammation. J Investig Dermatol Symp Proc 9:41 ^46, 2004 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, 1953). Light microscopic examination of lesional/perilesional skin of BP shows subepidermal vesiculation due to detachment of the epidermis from the underlying dermis. In ammatory 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 in ammatory mediators that can activate mast cells or leukocytes have been identi ed in lesional skin and/or blister uids 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 et al, 1976; Czech et al, 1993; Borrego et al, 1996) and (2) chemoattractants and cytokines, such Manuscript received October 1, 2002; revised January 21, 2003; accepted for publication January 24, 2003 Correspondence and reprint requests to: Zhi Liu, Department of Dermatology, University of North Carolina, 3100 Thurston-Bowles, Chapel Hill, NC 27599, USA. zhiliu@med.unc.edu Abbreviations: BMZ, basement membrane zone; BP: bullous pemphigoid; DEJ: dermal-epidermal junction; eos: eosinophil; GB: gelatinase B, hbp180: human BP180 antigen; Mf: macrophage; MC: mast cell; mbp180: murine BP180 antigen; NE: neutrophil elastase, PMN: neutrophil. as C5a fragments (Diaz-Perez and Jordon, 1976), histamine (Katayama et al, 1984), leukotriene B4 (Kawana et al, 1990), IL-1,2,4,5,6,8,15, TNF-a, IFN-g, RANTES, and eotaxin (reviewed by D Auria et al, 1999; Wakugawa et al, 2000). Several proteinases are also found in BP blister uid, including plasmin, collagenase, elastase, and 92-kDa gelatinase (Oikarinen et al, 1983; Lauharanta et al, 1989; Baird et al, 1990; Gissler et al, 1992; Kramer and Reinartz, 1993; StÔhle-BÌckdahl et al, 1994; Schaefer et al, 1996; Verraes et al, 2001). BP AS AN AUTOIMMUNE DISEASE BP autoantibodies bind to the basement membrane zone (BMZ) and activate complement (Jordon et al, 1967). 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 et al, 1981, 1984; Mutasim et al, 1985; Labib et al, 1986). Both BP180 and BP230 have been cloned and characterized at the molecular level (Stanley et al, 1988; Diaz et al, 1990; Sawamura et al, 1991; Tanaka et al, 1991; Giudice et al, 1992). BP230 is an intracellular protein that localizes to the hemidesmosomal plaque (Klatte et al, 1989; Tanaka et al, 1990; Ishiko et al, 1993) and has signi cant homology with plectin and desmoplakins I and II (Tanaka et al,1991;sawamuraet al, 1991; Green et al, 1992). In contrast, BP180 is a transmembrane protein with a type II orientation (Fig 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 et al,1992; Hopkinson et al, 1992; Ishiko et al, 1993). The extracellular region consists of 15 collagen domains separated from one another by /04/$ Copyright r 2004 by The Society for Investigative Dermatology, Inc. 41

2 42 LIU JID SYMPOSIUM PROCEEDINGS noncollagen sequences. BP180 as a transmembrane antigen makes it a preferred target for pathogenic autoantibodies in BP. 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 et al, 1993; Zillikens et al, 1997). These BP180NC16A-speci c autoantibodies are predominantly IgE and IgG isotypes and IgG1 and IgG4 subclasses (Bernard et al, 1990; Dopp et al, 2000; La tte et al, 2001). The serum levels of autoantibodies to BP180NC16A are correlated with the severity of BP (Haase et al,1998;doppet al, 2000). The majority of BP patients have autoreactive T cells that recognize epitopes located in the extracellular region of BP180 (Budinger et al, 1998) and mainly in the NC16A domain (Lin et al, 2000). These T lymphocytes express CD4 memory T cell surface markers and exhibit a TH1/TH2 mixed cytokine pro- le. 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 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 in ammatory cell in ltration, consisting of neutrophils, lymphocytes, and monocytes/macrophages, with neutrophils being the predominant cells (Fig 2A) (Chen et al, 2002). Using mice de cient in these cells, we demonstrated that mast cells, macrophages, and neutrophils play a role in subepidermal blistering (Fig 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 et al, 1995a). Signi cantly, this epitope overlaps the region of the human BP180 NC16A, which contains the immunodominant epitopes recognized by human BP autoantibodies. The critical role of complement activation in experimental BP is established by the following approaches: (1) C5-de cient 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 0 )2 fragments generated from the pathogenic anti-mbp180 IgG cannot induce subepidermal blisters in C5- 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 (Fig 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 puri ed rabbit anti-mbp180 IgG into neonatal BALB/c mice. The injected animals developed a blistering disease that closely mimicked human BP (Fig 1C) (Liu et al, 1993). Direct immuno uorescence Figure 1. Human 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 COOHterminal 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 (a-mbp180) and rabbit antihuman BP180 (a-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 immuno uourescence (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 immuno uorescence 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 in ammatory in ltrate (panel d).

3 VOL. 9, NO. 1 JANUARY 2004 IgG PASSIVE TRANSFER MODEL OF BULLOUS PEMPHIGOID 43 Figure 2. Cellular events in the IgG passive transfer model of BP. (A) Quanti cation of in ammatory 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 in ltrating cells. The di erent populations of in ammatory cells were identi ed by staining and ow cytometry. The data shown are the mean7se. po0.05 and po0.01, Student t-test for paired samples. PMN, neutrophils; Mf, macrophages; T, T lymphocytes; B, B lymphocytes; eos, eosinophils. (B) Clinical and histological examination of neonatal mice de cient in di erent in ammatory 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^de cient (T & B(^)) mice developed subepidermal blisters. In contrast, mast cell^de cient (MC(^)), macrophage-de cient (MF(^)), and neutrophil-de cient (PMN(^)) mice injected i.d. with pathogenic IgG showed no evidence of skin disease. su cient mice; and (4) C5-de cient mice reconstituted with C5a become susceptible to experimental BP (Liu et al, 1995b). Further studies reveal that the major function of complement activation is to generate C5a that in turn activates mast cells (Fig 3A). Extensive mast cell (MC) degranulation is seen in the lesional skin of mice injected with pathogenic anti-mbp180 antibodies. MC activation precedes neutrophil in ltration (Fig 3B), and inhibition of MC degranulation blocks neutrophil in ltration and subsequent blister formation. Furthermore, MC-de cient 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-de cient mice reconstituted with neutrophils (Fig 3C),TNF-a, or IL-8 (data not shown). These results suggest that mast cells play a critical role in recruiting neutrophils (Chen et al, 2001). Figure 3. Functional 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 di erent time points. The skin sections at each time point were analyzed by mast cell (MC)-speci c staining (toluidine blue staining) for MC identi cation and quanti cation and by myeloperoxidase (MPO) activity assay for neutrophil in ltration. (A) Complement (C 0 ) activation causes MC degranulation. C5-su cient mice (panel a) show extensive MC degranulation at 2 hours and develop blisters at 12 hours post-injection. In contrast, C5-de cient mice (panel b) exhibit minimal MC degranulation and no skin lesions. d, dermis. (B) Time course of MC degranulation and neutrophil in ltration. MC degranulation reaches the peak level at 1^2 hours after IgG injection (panel a), whereas PMN in ltration 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-de cient mice. Injection of pathogenic anti-mbp180 IgG causes BP in MC-su cient, but not MC-de cient, mice. MC-de cient mice reconstituted with neutrophils (by intradermal administration) injected with anti-mbp180 IgG develop BP. MC-de cient mice pretreated intradermally with IL-8 or TNF-a also become susceptible to experimental BP (data not shown).

4 44 LIU JID SYMPOSIUM PROCEEDINGS Neutrophil in ltration is a prerequisite for experimental BP, and disease severity is directly correlated to the number of in ltrating neutrophils (Liu et al, 1997). Mice depleted of neutrophils are resistant to experimental BP. Blocking neutrophil in ltration abolishes subepidermal blistering. Therefore, in ltrating neutrophils are the cells that directly cause tissue injury in the basement membrane zone, leading to subepidermal blistering. In ltrating 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 et al,1998,2000).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 (StÔhle-BÌckdahl et al,1994; Liuet al,2000)(fig 4A). In vivo, NE compensates for the lack of GB whereas GB cannot compensate for NE de ciency in the development of BP. In addition, the degradation of BP180 in vivo depends on NE activity (Fig 4B). These ndings 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 a1-proteinase inhibitor, the physiological inhibitor of NE. Unchecked NE then cleaves extracellular matrix proteins, including BP180 antigen, resulting in DEJ separation (Liu et al, 2000). RELEVANCE OF THE IGG PASSIVE TRANSFER MODEL TO HUMAN BP Figure 4. Proteolytic 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 puri ed neutrophil elastase (NE) (panel b), or with gelatinase B (GB) (panel c) at 371C 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-de cient (NE-), and GB-de- cient (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 wildtype 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-de cient mice injected with pathogenic IgG show no skin lesions and no degraded BP180 was found in skin samples (lane 2). GB-de cient mice locally reconstituted with 2.5 million GB-de cient (but NE-su cient) 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-de cient mice locally reconstituted with the same number of NE-de cient (but GB-su cient) 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. Experimental BP reproduces key characteristics of human BP at the clinical, histological, and immunological levels (Liu et al, 1993). Since the development of the IgG passive transfer model of BP in 1993, the pathogenic role of anti-bp180 antibodies has been con rmed by other in vitro and in vivo studies. BP180NC16A-speci c autoantibodies puri ed from human BP sera induce dermal^epidermal separation in cryosections of human skin in the presence of neutrophils (Sitaru et al, 2002).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 et al, 2002). Despite the similarities in the immunopathological features of the experimental model of BP and the human disease, there is one striking di erence. The in ammatory in ltrate 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-in ammatory pemphigoid blisters. These di erent 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 in ammatory cells (eosinophils and neutrophils). Therefore, di erent BP patients may respond to a particular treatment di erently. In the IgG passive transfer mouse model triggered by a well-de ned IgG preparation, however, neutrophils are the predominant in- ammatory 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 di erences 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 in ammatory cells. In vitro DEJ separation induced by human BP autoantibodies speci c for BP180NC16A depends on neutrophils (Sitaru et al, 2002).This study con rms the ndings by Gammon and colleagues two decades ago which showed that BP autoantibodies induce DEJ

5 VOL. 9, NO. 1 JANUARY 2004 IgG PASSIVE TRANSFER MODEL OF BULLOUS PEMPHIGOID 45 separation in human skin sections only in the presence of both leukocytes (mostly neutrophils) and complement (Gammon et al, 1982). In addition, although human BP blister uid has high levels of both neutrophil elastase (NE) and gelatinase B (GB), its degradation of BP180 depends on neutrophil elastase activity (Verraes et al, 2001). These results con rm the ndings 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 in ammatory cell in ltrate (Ackerman et al, 1997). It is worth noting that most patient biopsies are obtained days or weeks after the onset of disease activity. Eosinophil predominance may re ect the chronic stage of the in ammatory reaction in BP. Taken together, these ndings 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 IgGmediated in ammatory 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 signi cant 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 et al, 1988; Bernard et al,1992;tamada et al,1995; Zone et al,1998). 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 AI REFERENCES Ackerman AB, Chongchitnant N, Sanchez J, Guo Y, Rennin B, Reichel M, Randal MB: Histologic Diagnosis of In ammatory Skin Diseases, (2nd edn.). Baltimore: Williams & Wilkins, 1997; p 238^239 Baba T, Sonozaki H, Seki K, Uchiyama M, IkesawaY, Torisu M: An eosinophil chemotactic factor present in blister uids of bullous pemphigoid patients. JImmunol 116:112^116, 1976 Baird J, Lazarus GS, Belin D, Vassalli JD, Busso N, Gubler P, Jensen PJ: 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 95:548^552, 1990 Bernard P, Aucouturier P, Denis F, Bonnetblanc JM: Immunoblot analysis of IgG subclasses of circulating antibodies in bullous pemphigoid. Clin Immunol Immunopathol 54:484^494, 1990 Bernard P, Prost C, Durepaire N, Basset-Seguin N, Didierjean L, Saurat JH: The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen. J Invest Dermatol 99:174^179, 1992 Borrego L, Maynard B, Peterson EA, et al: Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins. Am J Pathol 148:897^909, 1996 Budinger L, Borradori L, Yee C, et al: Identi cation and characterization of autoreactive CD4þ T cell responses to the extracellular domain of bullous pemphigoid antigen 2 in patients with bullous pemphigoid and normals. JClinInvest 102:2082^2089, 1998 Chen R, Fairley JA, Zhao M, Giudice GJ, Zillikens D, Diaz LA, Liu Z: Macrophages, but not T and B lymphocytes are critical for subepidermal blister formation in experimental bullous pemphigoid: macrophage-mediated neutrophil in ltration depends on mast cell activation. JImmunol169:3987^ 3992, 2002 Chen R, Ning G, Zhao ML, Fleming MG, Diaz LA,Werb Z, Liu Z: Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid. JClin Invest 108:1151, 2001 Czech W, Schaller J, Schopf E, Kapp A: Granulocyte activation in bullous diseases. Release of granular proteins in bullous pemphigoid and pemphigus vulgaris. JAmAcadDermatol29:210^215, 1993 D Auria L, Cordiali Fei P, Ameglio F: Cytokines and bullous pemphigoid. Eur Cytokine Net 10:123^134, 1999 Diaz LA, Ratrie H, Saunders WS, Futamura S, Squiquera HL, Anhalt GJ, Giudice GJ: 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. JClinInvest86:1088^1094, 1990 Diaz-Perez JL, Jordon RE: The complement system in bullous pemphigoid. IV. Chemotactic activity in blister uid. Clin Immunol Immunopathol 5:360^370, 1976 Dopp R, Schmidt E, Chimanovitch I, Leverkus M, Brocker EB, Zillikens D: IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in bullous pemphigoid: Serum levels of these immunoglobulins re ect disease activity. J Am Acad Dermatol 42:577^583, 2000 Gammon WR, Merritt CC, Lewis DM, Sams WM Jr, Carlo JR,Wheeler CE Jr: An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement. JInvest Dermatol 78:285^290, 1982 Gissler HM, Simon MM, Kramer MD: Enhanced association of plasminogen/plasmin with lesional epidermis of bullous pemphigoid. Br J Dermatol 127:272^277, 1992 Giudice GJ, Emery DJ, Diaz LA: Cloning and primary structural analysis of the bullous pemphigoid autoantigen, BP-180. J Invest Dermatol 99:243^250, 1992 Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Liu Z, Diaz LA: Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain. JImmunol151:5742^5750, 1993 Green KJ, Virata ML, Elgart GW, Stanley JR, Parry DAD: Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: Members of a new gene family involved in organization of intermediate laments. Int J Macromol 14:145^153, 1992 Haase C, Budinger L, Borradori L, Yee C, Merk HF, 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 110:282^286, 1998 Hopkinson SB, Riddelle KS, Jones JCR: Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: Molecular and cell biologic characterization. JInvestDermatol99:264^270, 1992 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. JClin Invest 91:1608^1615, 1993 Jordon RE, Beutner EH, Witebsky E, Blumental G, Hale WC, Lever WF: Basement zone antibodies in bullous pemphigoid. JAMA 200:751^758, 1967 Katayama I, Doi T, Nishioka K: High histamine level in the blister uid of bullous pemphigoid. Arch Dermatol Res 276:126^127, 1984 Kawana S, Ueno A, Nishiyama S: Increased levels of Immunorective leukotriene B4 in blister uids of bullous pemphigoid patients and e ects of a selective 5-lipoxygenase inhibitor on experimental skin lesions. Acta Derm Venereol 70:281^285, 1990 Klatte DH, Kurpakus MA, Grelling KA, Jones JCR: Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells. J Cell Biol 109:3377^3390, 1989 Kramer MD, Reinartz J: The autoimmune blistering disease bullous pemphigoid. The presence of plasmin/ 2-antiplasmin complexes in skin blister uid indicates plasmin generation in lesional skin. JClinInvest92:978^983, 1993 Labib RS, Anhalt GJ, Patel HP, Mutasim DF, Diaz LA: Molecular heterogeneity of bullous pemphigoid antigens as detected by immunoblotting. J Immunol 136:1231^1235, 1986 La tte E, Skaria M, Jaunin F, Tamm K, Saurat JH, 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 144:760^768, 2001 Lauharanta J, Salonen EM, Vaheri A: Plasmin-like proteinase associated with high molecular weight complexes in blister uid of bullous pemphigoid. Acta Dermato-Venereologica 69:527^529, 1989 Lever WF: Pemphigus. Medicine 32:1^123, 1953 Lin MS, Fu CL, Giudice GJ, Olague-Marchan M, Lazaro AM, Stastny P, Diaz LA: Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain. JInvest Dermatol 115:955^961, 2000 Liu Z, Diaz LA, Swartz SJ, Fairley JA, Troy JL, Giudice GJ: Molecular mapping of a pathogenically relevant BP180 epitope associated with experimentally induced murine bullous pemphigoid. JImmunol155:5449^5454, 1995a Liu Z, Diaz LA, Troy JL, Taylor AF, Emery DE, Fairley JA, Giudice GJ: A passive transfer model of the organ-speci c autoimmune disease, bullous pemphigoid,

6 46 LIU JID SYMPOSIUM PROCEEDINGS using antibodies generated against the hemidesmosomal antigen, BP180. J Clin Invest 92:2480^2488, 1993 Liu Z, Giudice GJ, Swartz SJ, Fairley JA,Till GO,Troy JL, Diaz LA: The role of complement in experimental bullous pemphigoid. JClinInvest95:1539^1544, 1995b Liu Z, Giudice GJ, Zhou X, et al: A major role for neutrophils in experimental bullous pemphigoid. JClinInvest100:1256, 1997 Liu Z, Shapiro SD, Zhou X, et al: Neutrophil elastase plays a direct role in dermalepidermal junction separation in experimental bullous pemphigoid. JClin Invest 105:113^123, 2000 Liu Z, Shipley JM, Vu TH, Zhou X, Diaz LA, Werb Z, Senior RM: Gelatinase B-de cient mice are resistant to experimental BP. JExpMed188:475^482, 1998 Liu Z, Zhou X, Shapiro SD, et al: The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell 102:647^655, 2000 Morrison LH, Labib RS, Zone JJ, Diaz LA, Anhalt GJ: Herpes gestationis autoantibodies recognize a 180-kD human epidermal antigen. JClinInvest81:2023^ 2026, 1988 Mutasim DF, Takahashi Y, Labib RS, Anhalt GJ, Patel HP, Diaz LA: A pool of bullous pemphigoid antigen (s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex. J Invest Dermatol 84:47^ 53, 1985 Oikarinen AI, Zone JJ, Ahmed AR, Kiistala U, Uitto J: Demonstration of collagenase and elastase activities in blister uids from bullous skin diseases. Comparison between dermatitis herpetiformis and bullous pemphigoid. J Invest Dermatol 81:261^266, 1983 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. JBiolChem266:17784^17790, 1991 Schaefer BM, Jaeger C, Drepper E, Kramer MD: Plasminogen activation in bullous pemphigoid immunohistology reveals urokinase type plasminogen activator, its receptor and plasminogen activator inhibitor type-2 in lesional epidermis. Autoimmunity 23:155^164, 1996 Sitaru C, Schmidt E, Petermann S, Munteanu LS, Brocker EB, Zillikens D: Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin. JInvestDermatol118:664^6671, 2002 StÔhle-BÌckdahl M, Inoue M, Giudice GJ, Parks WC: 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. JClinInvest93:2022^2030, 1994 Stanley JR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI: Characterization of bullous pemphigoid antigen: A unique basement membrane protein of strati- ed epithelia. Cell 24:897^903, 1981 Stanley JR, Tanaka T, Mueller S, Klaus-Kovtun V, Roop D: Isolation of complementary DNA for bullous pemphigoid antigen by use of patients autoantibodies. JClinInvest82:1864^1870, 1988 Stanley JR, Woodley DT, Katz SI: Identi cation and partial characterization of pemphigoid antigen extracted from normal human skin. JInvestDermatol 82:108^111, 1984 Tamada Y, Yokochi K, Nitta Y, Toshihiko I, Hara K, Owaribe K: Lichen planus pemphigoides. Identi cation of 180 kd hemidesmosome antigen. JAmAcad Dermatol 32:883^887, 1995 Tanaka T, Korman NJ, Shimizu H, Eady AJ, Klaus-Kovtun V, Cehrs K, Stanley JR: Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cdna. JInvestDermatol94:617^623, 1990 Tanaka T, Parry DAD, Klaus-Kovtun V, Steinert PM, Stanley JR: Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I con rms that they de ne a new family of cell adhesion junction plaque proteins. J Biol Chem 266:12555^12125, 1991 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. JInvestDermatol 117:1091^1096, 2001 Wakugawa M, Nakamura K, Hino H, et al: Elevated levels of eotaxin and interleukin-5 in blister uid of bullous pemphigoid: correlation with tissue eosinophilia. Bri J Dermatol 143:112^116, 2000 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. JInvestDermatol118:485^492, 2002 Zillikens D, Rose PA, Balding SD, et al: Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies. J Invest Dermatol 109:573^579, 1997 Zone JJ, Taylor TB, Meyer LJ, Petersen MJ: 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 110:207^210, 1998