Materials and methods

Patients' sera

Serum samples were obtained from 13 BP patients, prior to the initiation of therapy and from 13 healthy volunteers. All BP patients were characterized by: (i) blisters on the skin; (ii) subepidermal blisters by histopathology; (iii) linear deposits of IgG and C3 at the DEJ by direct immunofluorescence (IF) microscopy; (iv) circulating IgG autoantibodies that fixed complement to the epidermal side of 1 M NaCl-split human skin by indirect IF microscopy; and (v) reactivity to BP180 NC16A as detected by immunoblot analysis and enzyme-linked immunosorbent assay. Findings in patients' sera by indirect IF microscopy on 1 M NaCl-split skin, reactivity with BP180, BP230, and LAD-1 (as analyzed by immunoblotting of keratinocyte extracts, conditioned concentrated HaCaT medium, and a recombinant form of the BP180 C-terminus), and enzyme-linked immunosorbent assay reactivity against recombinant BP180 NC16A are shown in Table I.

Table I - Characterization of sera from patients included in this study.

Full table

Polyclonal rabbit antibodies to BP180 and BP230

Two rabbit sera, R2296 and SA8010, were raised against a glutathione-S-transferase fusion protein containing a 42 amino acid stretch of human BP180 NC16A (NC16A2–4). Preimmune and anti-GST rabbit serum (Sigma, St Louis, MO) were used as controls. Rabbit serum SA6760 was generated against a fusion protein spanning amino acids 61–360 of the intracellular domain of BP180 (^{Schäcke et al, 1998}). Rabbit serum to BP230 was generated against a 165 kDa recombinant fragment of the C-terminal domain of BP230 (^{Tanaka et al, 1990}). The characteristics of these antibodies are shown in Table II

Table II - Monoclonal and polyclonal antibodies to BP180 and BP230 included in this study.

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Preparation of cryosections, indirect IF microscopy, and complement-fixation test

Neonatal human foreskin, obtained from routine circumcision, was washed in cold phosphate-buffered (PBS), cut in pieces of 5  15 mm, embedded in optimum cutting temperature compound (Sakura Finetek Europe BV, Zoeterwonde, the Netherlands), and stored at -80°C. Four cryosections of 6 m were placed in the center of a Superfrost Plus microscope slide (Menzel-Gläser, Braunschweig, Germany) (^{Gammon et al, 1982}). Indirect IF microscopy on 1 M NaCl-split human skin followed a previous protocol (^{Zillikens et al, 1996}). The complement-fixation test was performed on cryosections of human skin (^{Katz et al, 1976}). Bound C3 was visualized using a fluorescein isothiocyanate-labeled goat anti-human C3 antibody (Kallestad Diagnostics, Austin, TX). For antigen mapping studies, mouse monoclonal antibodies to human type IV collagen (clone CIV 22, Sigma), laminin 5 (clone GB3, Loughborough Leicestershire, U.K), and BP230 (1E5;^{Hirako et al, 1998}) were used.

Affinity purification of total IgG and IgG specific for epitopes on BP180

IgG from serum samples of patients, rabbits, and controls was isolated using protein G sepharose fast flow affinity column chromatography (Pharmacia AB, Uppsala, Sweden) as reported (^{Schmidt et al, 2000b)}. Antibodies to BP180 were affinity purified from sera of BP patients and immunized rabbits using an AminoLink Plus immobilization kit following the manufacturer's instructions (Pierce, Rockford, IL). Briefly, recombinant GST fusion proteins containing the NC16A2–4 segment or a cocktail of fusion proteins containing NC16A1, NC16A1–3, and NC16A2–5 fragments (ratio 1:1:3) were covalently coupled to 4% beaded agarose at pH 10. Similarly, the GST-BP180 4575 representing a recombinant form of the C-terminus of BP180 was immobilized on the agarose matrix. Serum samples were incubated with peptide loaded matrix; antibodies were eluted with 0.1 M glycine buffer (pH 2.5) and neutralized with Tris–HCl. Eluted antibodies were concentrated under extensive washing with PBS (pH 7.2) using Ultrafree 15 filters (Millipore, Bradford, MA). After immunoaffinity purification against NC16A, total IgG of the flow-through was purified using protein G. Reactivity of flow-through and eluted fractions was analyzed by indirect IF microscopy on salt-split skin and by immunoblotting of recombinant NC16A, GST-BP180 4575 and both full-length and soluble (LAD-1) keratinocyte-derived BP180. F(ab')₂ fragments of autoantibodies to BP180 NC16A were prepared using a standard protocol (^{Parham, 1983}). The completeness of fragmentation and the reactivity of the F(ab')₂ preparations was tested by indirect IF microscopy on 1 M salt split-skin using fluorescein isothiocyanate-labeled secondary antibodies specific to Fab (Sigma) and Fc (Serotec Ltd, Oxford, U.K.) portions of IgG, respectively.

Production of recombinant and cell-derived forms of BP180

GST fusion proteins containing full-length BP180 NC16A (NC16A1–5) (Figure 1), various fragments of this domain, including NC16A1, NC16A3, NC16A2–4, NC16A2–5, and the BP180 4575 C-terminal segment (^{Balding et al, 1996}), were expressed in Escherichia coli DH5 (^{Zillikens et al, 1997b)}. Fusion proteins were purified by glutathione agarose affinity chromatography (^{Giudice et al, 1993}). The soluble ectodomain of BP180 (LAD-1) and full-length BP180 were prepared from cultured keratinocytes (^{Zillikens et al, 1999}). Immunoblotting and enzyme-linked immunosorbent assay using BP180 NC16A were performed as described (^{Zillikens et al, 1997a,b)}.

Figure 1.

Schematic diagram of human BP180. The cytoplasmatic globular domain of BP180 is shown at the top. The transmembrane region of BP180 is labeled TM. The ectodomain consists of 15 interrupted collagen domains (bright boxes) and forms a central rod that spans the lamina lucida (LL); the carboxy terminal flexible tail protrudes into the lamina lucida (LL)/lamina densa (LD) interface (^{Nonaka et al, 2000}). The recombinant forms of the NC16A domain are depicted to the left. Amino acid residue numbers are shown next to the boxes. The NC16A domain has been subdivided into five regions, each of approximately 15 amino acids in length.

Full figure and legend (110K)

Peripheral blood leukocytes and complement

Peripheral blood leukocytes from healthy volunteers were isolated by a sedimentation gradient containing sodium diatrizoate and dextran 500 following the instructions of the manufacturer (Nycomed, Oslo, Norway). Cells were harvested, washed twice in RPMI 1640 (Life Technologies, Karlsruhe, Germany) and resuspended in the same medium at a density of 6  10⁷ cells per ml. The cell suspension was kept on ice and cell viability was tested using trypan blue; preparations with a viability greater than 95% were used. Serum samples from healthy volunteers served as a source of human complement (^{Gammon et al, 1980}) and were diluted 2-fold with RPMI.

Treatment of cryostat sections

Sera from BP patients and controls were heat inactivated for 30 min at 56°C and diluted 5-fold in PBS. Protein G affinity purified IgG was diluted in PBS resulting in a final concentration of 4 mg per ml. Rabbit antibodies to BP180 and BP230 were used at an indirect IF titer of 320. Cryostat sections were washed with PBS for 5 min to remove embedding medium before incubation with 50 l of diluted sera or antibody preparations for 90 min at room temperature. After washing the sections with PBS twice, chambers were prepared as described elsewhere (^{Gammon et al, 1982a)} with minor modifications. Briefly, tissue containing slides were covered with a second slide to which transparent adhesive tape (Beiersdorf AG, Hamburg, Germany) had been placed around each end. The tape prevented contact of the skin sections with the covering slide thus creating a chamber of approximately 0.3 mm in thickness and a volume of 0.5 ml. Both slides were taped together and the leukocyte suspension, mixed 1:1 with either diluted fresh or heated serum or medium alone, was injected into the chambers. Chambers were incubated in a humidified air incubator containing 5% CO₂ (Memmert, Schwabach, Germany) for 3 h at 37°C. Subsequently, chambers were disassembled and sections were washed in PBS for 10 min to remove excess serum and nonadherent cells, air dried for 10 min, fixed in formalin, and finally stained with hematoxylin and eosin. Sections were examined by two blinded independent investigators at 50, 100, and 200 magnifica

Results

Sera and purified IgG from BP patients recruit neutrophils to the DEJ and induce subepidermal splits in cryosections of human skin

Serum samples from BP patients (n = 13) labeled the DEJ when incubated with cryosections of human skin (Figure 2a), whereas sera from healthy controls (n = 13) showed no binding (Figure 2d). Incubation of cryosections with serum samples from BP patients, followed by incubation with complement-containing serum and leukocytes from healthy volunteers, led to the attachment of neutrophils to the DEJ, and most importantly, to the induction of subepidermal splits by all of the 13 BP sera (Figure 2b; Table III). In contrast, sera from healthy controls did not induce leukocyte attachment to the DEJ or a subepidermal split (Figure 2e). When the serum as a source of complement was heat inactivated or omitted completely, BP sera still induced subepidermal splits (Figure 2c). Omitting complement, neutrophil attachment to the stratum corneum, induced by both patients' and control sera, was abolished (Figure 2c, f). Using protein G affinity purified IgG from four BP patients (Table V) and four healthy volunteers, we observed the same findings compared with the use of whole serum. IgG from BP patients, in contrast to controls, led to neutrophil attachment and induced subepidermal cleavage. No separation occurred when leukocytes were omitted.

Figure 2.

Autoantibodies from BP patients bind to the DEJ and induce subepidermal splits. Cryosections of human skin that were incubated with serum from a BP patient (a–c) show IgG deposits at the DEJ (a). The addition of normal human leukocytes results in subepidermal splits both in the presence (b) or absence (c) of fresh serum as a source of human complement. In contrast, cryosections incubated with serum from a healthy control (d–f) do not demonstrate binding of IgG to the DEJ (d) and reveal no split formation after incubation with leukocytes irrespective of addition of complement (e, f). Scale bar: 40 m.

Full figure and legend (231K)

Table III - The role of autoantibodies, complement, and leukocytes in DES in the in vitro cryosection model.

Full table

Table V - Characterization of affinity purified immunoglobulin preparations used in this study.

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The extent of neutrophil attachment to the DEJ depends on both the concentration of antibodies to BP180 and the incubation time with the cells

In a further set of experiments, we investigated the time dependency of neutrophil attachment to the DEJ incubating the cryosections with three different BP sera (BP-SA, BP-CE, and BP-HU) for 1, 2, 3, and 4 h, respectively. Leukocyte attachment to the DEJ was strongest after an incubation time of 1 h and declined subsequently, whereas the extent of DES increased after an incubation time of 1 h and reached its maximum after 3 h (Figure 3). To study the dependency of leukocyte attachment on levels of autoantibodies to BP180 NC16A, we purified antibodies specific to NC16A from two BP patients (BP-CE and BP-SA) by immunoaffinity chromatography. Higher concentrations of antibodies to NC16A induced stronger leukocyte attachment compared with incubation with preparations containing less reactivity to NC16A (Table IV).

Figure 3.

Attachment of leukocytes to the DEJ is strongest after short incubation times, whereas DES is more extensive with longer incubation. In the upper panel, cryosections were incubated with serum from BP-SA followed by incubation with leukocytes for 1, 2, and 3 h, respectively. Scale bar: 40 m. The lower panel shows the extent of leukocyte attachment and DES after incubating the cryosections with three BP sera (BP-SA, BP-CE, and BP-HU). The mean values of percent of the total length of DEJ with attached leukocytes () or DES () are demonstrated at incubation times of 1, 2, 3, and 4 h, respectively. The individual readings with the three different sera are also shown (leukocyte attachment, ; DES, +).

Full figure and legend (130K)

Table IV - Neutrophil attachment to the DEJ increases with higher concentrations of antibodies to BP180.

Full table

Splits induced experimentally by autoantibodies from BP patients localize to the lamina lucida of the DEJ

To study the level of split formation, we stained cryosections, that had previously been incubated with sera from BP patients and leukocytes, with monoclonal antibodies to human BP230 (1E5), type IV collagen, and laminin 5. Antibodies to BP230 stained the roof of the split, whereas those to both type IV collagen and laminin 5 labeled the dermal side demonstrating that the level of split formation localizes to the lamina lucida of the DEJ.

Immunoadsorption against recombinant BP180 NC16A abolishes the capacity of BP patients' sera to induce DES

Sera from two BP patients (BP-SA and BP-CE) were subjected to affinity purification using a cocktail of recombinant fragments covering the entire NC16A domain of BP180 that was covalently coupled to an agarose matrix. Total IgG from flow-through fractions was isolated by protein G chromatography and concentrated to the same volume as the eluted fraction. The eluted IgG strongly reacted with recombinant NC16A by immunoblot analysis. In contrast, IgG from flow-through fractions showed no reactivity with NC16A demonstrating the complete elimination of antibodies to this portion of BP180 (Figure 4). Subsequently, IgG specific for BP180 NC16A and flow-through fractions were tested by indirect IF microscopy on 1 M NaCl-split skin. Interestingly, both eluted and flow-through fractions still contained IgG that bound to the epidermal side of the split (Table V). When concentrated to the same volume, indirect IF titers of flow-through fractions were 2-fold lower compared with the eluted fractions. Both eluted and flow-through fractions also reacted with cell-derived full-length BP180 and LAD-1 (Figure 4) indicating that the immunoadsorption procedure only abolished antibodies to NC16A from the sera, whereas IgG directed to other autoantigens or to other portions of the BP180 molecule remained unaffected. Importantly, IgG specific to the NC16A domain of BP180 induced subepidermal splits in the cryosections (Figure 5c), whereas the flow-through fractions of BP sera lost their blister-inducing capacity (Figure 5d). When we concentrated the IgG from flow-through fractions to an indirect IF titer 2-fold higher compared with the eluted fraction, these preparations still did not induce leukocyte attachment to the DEJ or subepidermal cleavage. Under these conditions, the IgG concentration of the flow fraction was 80-fold (BP-SA) and 280-fold (BP-CE) higher, respectively, compared with the eluted fraction and the IgG preparations had the same complement-fixing ability. In a further set of experiments, we purified antibodies specific to the C-terminus of BP180 from serum of three BP patients (BP-CE, BP-SA, and BP-PR) using recombinant GST-BP180 4575. Antibody preparations to this fragment of BP180, which strongly fixed complement, were concentrated to a titer of 80 (Table V). While at this titer, patient antibodies specific to NC16A induced DES, antibodies specific to the BP180 C-terminus induced no leukocyte attachment and no DES.

Figure 4.

Immunoblot analysis using IgG from BP sera affinity purified against BP180 NC16A Purification of autoantibodies to BP180 NC16A1–5 from two BP sera (BP-SA and BP-CE) was performed using an agarose beaded gel covalently coupled with a cocktail consisting of NC16A1, NC16A1–3, and NC16A2–5 encompassing the entire NC16A domain. Left panel, immunoblotting of recombinant BP180 NC16A with BP-SA (lanes 1–3) and BP-CE (lanes 4–6). IgG autoantibodies eluted from the column (lanes 3 and 6) react, like reference control serum BP-CO (lane 7), with NC16A, whereas flow-through fractions (lanes 2 and 5) or normal serum (lane 8) show no reactivity. Right panel, Immunoblotting of keratinocyte-derived full-length BP180. Serum samples from BP-SA and BP-CE (lanes 1 and 4), eluted fractions (lanes 3 and 6), but also IgG in flow-through fractions (lanes 2 and 5) react with full-length BP180 (arrow). Reactivity of control sera (BP-CO and NHS) is shown in lanes 7 and 8. Migration position of molecular weight markers are depicted on the left and right.

Full figure and legend (143K)

Figure 5.

Antibodies to BP180 NC16A induce subepidermal splits in cryosections of human skin. Serum from rabbit SA8010, immunized against recombinant human GST-NC16A2–4 (a), and autoantibodies from patient BP-SA, affinity purified against BP180 NC16A (c), induce DES in cryosections of human skin when coincubated with leukocytes from healthy donors. In contrast, incubation with serum from a rabbit immunized against GST (b) or the flow-through fraction of serum BP-SA (d) do not lead to split formation. Scale bar: 40 m.

Full figure and legend (363K)

F(ab')₂ fragments of autoantibodies to NC16A do not induce subepidermal cleavage in cryosections of human skin

To address the question, if the Fc portion of autoantibodies is of importance for blister induction in our in vitro model, we prepared F(ab')₂ fragments of antibodies that had been eluted from NC16A coupled to agarose. IgG specific to NC16A from two BP patients (BP-SA and BP-CE) was digested with pepsin. F(ab')₂ fragments, lacking the Fc portion of the antibody, were used at the same indirect IF titer (80) as the original unfragmented antibody preparations to BP180 NC16A that were pathogenic; however, F(ab')₂ preparations did not induce subepidermal cleavage when incubated with the cryosections.

IgG from rabbits immunized against recombinant human BP180 NC16A induce subepidermal splits in skin cryo sections

For this study, we immunized two rabbits (R2296 and SA8010) against recombinant NC16A2–4. When cryosections were incubated with sera SA8010 and R2296 or with IgG from SA8010, affinity purified against BP180 NC16A2–4, and subsequently treated with leukocytes from healthy donors, a subepidermal split formation was observed (Figure 5a). In contrast, preimmune rabbit sera, serum from a rabbit immunized against recombinant GST, and the flow-through fraction of serum SA8010, obtained after subjecting the serum to affinity purification with recombinant NC16A2–4, did not bind to the DEJ and did not induce DES in the cryosections (Figure 5b). Although used at the same indirect IF titers as rabbit antibodies to NC16A (320), rabbit antibody SA6760 to the intracellular domain of BP180 and a rabbit antibody to BP230 were not pathogenic in this model.

Discussion

In this study, we show the capacity of antibodies to human BP180 from patients with BP and from rabbits immunized with recombinant BP180 to induce subepidermal splits. This study used, with some modifications, an in vitro model originally described by^{Gammon et al (1982a)} involving cryosections of human skin, sera from BP patients, and leukocytes; however, at the time their model was first reported, the different specificities of autoantibodies in BP sera had not been elucidated. Subsequent work demonstrated that autoantibodies in BP sera are directed to two major autoantigens, BP180 and BP230 (^{Stanley et al, 1981,1988;Labib et al, 1986;Diaz et al, 1990}).

Previous attempts to induce BP by passive transfer of patients' autoantibodies into different animals had failed (^{Sams and Gleich, 1971;Gammon and Briggaman, 1988}). The pathogenic relevance of BP180 was strongly suggested by the use of an animal model where neonatal mice injected with rabbit anti-murine BP180 antibodies developed a BP-like subepidermal blistering disease (^{Liu et al, 1993}); however, the pathogenic effect of antibodies to BP180 from patients with BP has not been demonstrated to date. Recently, we also failed to induce blisters by injection of autoantibodies to BP180 from patients with BP into human skin grafted on to severe combined immunodeficient mice (^{Zillikens et al, 2001}).

In this study, we therefore used the cryosection model to test the hypothesis that antibodies to human BP180 are pathogenic. In a first set of experiments, we reproduced the finding of^{Gammon et al (1982a)} that sera from BP patients induce subepidermal splits in cryosections of human skin. Subsequently, this observation was confirmed with the use of IgG fractions isolated by protein G affinity purification from BP patients, whereas normal human sera or normal IgG caused no DES. The level of experimentally induced split formation localizes to the lamina lucida of the DEJ, the same site where blister formation occurs in the patient's skin (^{Schmidt and Zillikens, 2000}). Interestingly, omission of complement did not change the extent of DES in our assay.^{Gammon et al (1982a)} had also found some degree of DES in cryosections treated with heat-inactivated serum. Their three-step skin chamber method consisted of incubation with BP sera (step 1), followed by leukocytes (step 2), and finally by fresh serum as a source of complement (step 3). Importantly, in their assay, the incubation with leukocytes was performed prior to the addition of complement. In addition, to remove nonadherent leukocytes the sections were then rinsed in PBS before complement was added. This sequence of incubation steps could not undoubtedly clarify the role of complement for the induction of splits in the assay described by^{Gammon et al (1982a)}; however, from these and our own data one may not conclude that complement activation is not essential for blister formation in BP. In the cryosection model, in contrast to the in vivo situation, the leukocytes do not have to migrate along a chemotactic gradient from blood vessels to the DEJ. Instead, by incubating the cryosections with leukocytes, the cells are placed in close contact with the DEJ. As in the cryosection model there is no requirement for complement to induce splits, the role of complement in blister formation of human BP cannot be explored in this in vitro model and will be addressed in future studies using other experimental models. On the other hand, our data clearly show the importance of neutrophils for the induction of DES in the cryosection model: (i) omitting leukocytes resulted in a lack of split formation, and (ii) pepsin digestion of split-inducing autoantibodies, which leads to a loss of the Fc portion (mediating binding of neutrophils to immune complexes), also abolished split formation.

In a second set of our experiments, we studied the time-dependency of leukocyte attachment and split formation in our assay. We demonstrate that leukocyte attachment to the DEJ predominates over DES with shorter incubation times (1 h) and declines with longer incubation, whereas the extent of DES increases gradually and reaches its maximum with longer incubation (3 h). When fresh serum was added as a source of complement, we regularly found attachment of neutrophils to the stratum corneum of the cryosections irrespective of whether the sections were incubated with sera/purified IgG from patients or controls. This observation is in line with the finding that corneocytes may activate the alternative complement pathway thus mediating the adhesion of leukocytes to serum-treated stratum corneum in an antibody-independent manner (^{Terui et al, 1989;Terui et al, 1995}). When fresh serum was omitted or replaced by heat-inactivated serum, we did not observe a major neutrophil adhesion to the stratum corneum.

The importance of antibodies to the NC16A domain of BP180 has been suggested by several lines of evidence: (i) in experimental murine BP, antibodies to the murine homolog of human NC16A induce skin blisters in neonatal mice (^{Liu et al, 1993,1995)}; (ii) in BP patients, serum levels of circulating antibodies to NC16A, in contrast to indirect IF microscopy titers of antibodies to the DEJ, correlate with disease activity (^{Schmidt et al, 2000a)}; and (iii) antibodies to the NC16A domain were identified to trigger expression and secretion of inflammatory mediators when incubated with cultured normal human keratinocytes (^{Schmidt et al, 2000b)}. In a further set of experiments, we therefore tested the pathogenic relevance of antibodies to BP180 NC16A from BP patients in cryosections of human skin. Interestingly, when we affinity purified antibodies specific to BP180 NC16A from the serum of BP patients, this fraction induced a concentration-dependent leukocyte attachment to the DEJ, and subsequently subepidermal splits in the cryosections. The split-inducing potential of autoantibodies specific to BP180 NC16A was shown to be dependent on their Fc portion. In contrast, serum fractions that were depleted of reactivity BP180 NC16A (flow-through fractions) lost their split-inducing capacity, although they remained reactive with full-length BP180 and LAD-1 derived from cultured keratinocytes. Autoantibodies from BP patients, purified against the C-terminus of BP180, and used at the same indirect IF titer and IgG concentration as antibodies to NC16A, were also not pathogenic. These data suggest that the split-inducing ability of autoantibodies is associated with their specificity. This is further supported by findings using the experimental mouse model of BP. By molecular mapping studies it was shown that the blister-inducing ability of rabbit antibodies generated to different recombinant forms of murine BP180 was dependent on their fine specificity (^{Liu et al, 1995}). Evidence that specificity of antibodies may be associated with their pathogenicity is also provided by studies using a mouse model for hemolytic anemia where mice develop autoantibodies to different membrane antigens on erythrocytes. An analysis of autoantibodies and of a panel of anti-red blood cell monoclonal antibodies derived from the diseased mice revealed that pathogenic and nonpathogenic autoantibodies differed with regard to their specificity (^{DeHeer et al, 1978;Shibata et al, 1990;Diiulio et al, 1997}). In these mice, anemia was shown to be mediated by Fc receptor-dependent erythrophagocytosis (^{Shibata et al, 1990;Clynes and Ravetch, 1995}).

In a final set of experiments, the pathogenic relevance of antibodies to the NC16A domain was further confirmed by the finding that treatment of cryosections of human skin with rabbit sera generated against BP180 NC16A2–4 also resulted in subepidermal splits. No split was induced when rabbit serum was depleted of reactivity against NC16A2–4 by affinity chromatography or when cryosections were incubated with preimmune rabbit sera. Interestingly, although used at the same indirect IF titers as rabbit antibodies to NC16A, rabbit antibodies to the intracellular domain of BP180 did not induce subepidermal splits when incubated with the cryosections, nor did rabbit antibodies to BP230.

In summary, this study demonstrates that antibodies to human BP180, in the presence of leukocytes, induce subepidermal splits in cryosections of human skin, which confirms that BP180 plays a key part in the pathogenesis of BP. We show that the pathogenic mechanisms that lead to subepidermal split formation are mediated by autoantibodies targeting epitopes within the NC16A domain of BP180. Future studies will be aimed at characterizing the pathogenically relevant epitope(s) within NC16A and other potentially relevant epitopes on the BP180 molecule.

References

1. Balding, SD, Prost, C, Diaz, LA, et al: Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain. J Invest Dermatol 1996 106:141–146,  | Article | PubMed | ISI | ChemPort |
2. Clynes, R, Ravetch, JV: Cytotoxic antibodies trigger inflammation through Fc receptors. Immunity 1995 3:21–26,  | Article | PubMed | ISI | ChemPort |
3. DeHeer, DH, Linder, EJ, Edgington, TS: Delineation of spontaneous erythrocyte autoantibody responses of NZB and other strains of mice. J Immunol 1978 120:825–830,  | PubMed | ISI | ChemPort |
4. Diaz, LA, Ratrie, HD, Saunders, WS, et al: Isolation of a human epidermal cDNA 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,  | PubMed | ISI | ChemPort |
5. Diiulio, NA, Fairchild, RL, Caulfield, MJ: The anti-erythrocyte autoimmune response of NZB mice. Identification of two distinct autoantigens. Immunology 1997 91:246–251,  | Article | PubMed | ISI | ChemPort |
6. Gammon, WR, Briggaman, RA: Absence of specific histologic changes in guinea pig skin treated with bullous pemphigoid antibodies. J Invest Dermatol 1988 90:495–500,  | Article | PubMed | ISI | ChemPort |
7. Gammon, WR, Lewis, DM, Carlo, JR, Sams, WM, Wheeler, CE: Pemphigoid antibody mediated attachment of peripheral blood leukocytes at the dermal–epidermal junction of human skin. J Invest Dermatol 1980 75:334–339,  | Article | PubMed | ISI | ChemPort |
8. Gammon, WR, Merritt, CC, Lewis, DM, et al: 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,  | Article | PubMed | ISI | ChemPort |
9. Giudice, GJ, Emery, DJ, Diaz, LA: Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180. J Invest Dermatol 1992 99:243–250,  | Article | PubMed | ISI | ChemPort |
10. Giudice, GJ, Emery, DJ, Zelickson, BD, et al: Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain. J Immunol 1993 151:5742–5750,  | PubMed | ISI | ChemPort |
11. Hirako, Y, Usukura, J, Uematsu, J, et al: Cleavage of BP180, a 180-kDa bullous pemphigoid antigen, yields a 120-kDa collagenous extracellular polypeptide. J Biol Chem 1998 273:9711–9717,  | Article | PubMed | ISI | ChemPort |
12. Katz, SI, Hertz, KC, Yaoita, H: Herpes gestationis. Immunopathology and characterization of the HG factor. J Clin Invest 1976 57:1434–1441,  | PubMed | ISI | ChemPort |
13. Labib, RS, Anhalt, GJ, Patel, HP, Mutasim, DF, Diaz, LA: Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J Immunol 1986 136:1231–1235,  | PubMed | ISI | ChemPort |
14. Liu, Z, Diaz, LA, Troy, JL, et al: 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,  | PubMed | ISI | ChemPort |
15. Liu, Z, Diaz, LA, Swartz, SJ, et al: Molecular mapping of a pathogenically relevant BP180 epitope associated with experimentally induced murine bullous pemphigoid. J Immunol 1995 155:5449–5454,  | PubMed | ISI | ChemPort |
16. Nonaka, S, Ishiko, A, Masunaga, T, et al: The extracellular domain of BPAG2 has a loop structure in the carboxy terminal flexible tail in vivo. J Invest Dermatol 2000 115:889–892,  | Article | PubMed | ISI | ChemPort |
17. Parham, P: On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. J Immunol 1983 131:2895–2902,  | PubMed | ISI | ChemPort |
18. Sams, WM, Jr: Gleich, GJ: Failure to transfer bullous pemphigoid with serum from patients. Proc Soc Exp Biol Medical 1971 136:1027–1031,  | ChemPort |
19. Schäcke, H, Schumann, H, Hammami-Hauasli, N, Raghunath, M, Bruckner-Tuderman, L: Two forms of collagen XVII in keratinocytes: a full-length transmembrane protein and soluble ectodomain. J Biol Chem 1998 273:25937–25943,  | Article | PubMed | ISI | ChemPort |
20. Schmidt, E, Zillikens, D: Autoimmune and inherited subepidermal blistering diseases: advances in the clinic and the laboratory. Adv Dermatol 2000 16:113–157,  | PubMed | ChemPort |
21. Schmidt, E, Obe, K, Brocker, EB, Zillikens, D: Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid. Arch Dermatol 2000a 136:174–178,  | Article | PubMed | ISI | ChemPort |
22. Schmidt, E, Reimer, S, Kruse, N, et al: Autoantibodies to BP180 associated with bullous pemphigoid release interleukin-6 and interleukin-8 from cultured human keratinocytes. J Invest Dermatol 2000b 115:842–848, 10.1046/j.1523-1747.2000.00141.x | Article | ISI | ChemPort |
23. Shibata, T, Berney, T, Reininger, L, Chicheportiche, Y, Ozaki, S, Shirai, T, Izui, S: Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause autoimmune hemolytic anemia by two distinct pathogenic mechanisms. Int Immunol 1990 2:1133–1141,  | PubMed | ISI | ChemPort |
24. Stanley, JR, Hawley-Nelson, P, Yuspa, SH, Shevach, EM, Katz, SI: Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 1981 24:897–903,  | Article | PubMed | ISI | ChemPort |
25. Stanley, JR, 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,  | PubMed | ISI | ChemPort |
26. Tanaka, T, Korman, NJ, Shimizu, H, et al: Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA. J Invest Dermatol 1990 94:617–623,  | Article | PubMed | ISI | ChemPort |
27. Terui, T, Kato, T, Tagami, H: Stratum corneum activation of complement through the antibody-independent alternative pathway. J Invest Dermatol 1989 92:593–597,  | Article | PubMed | ISI | ChemPort |
28. Terui, T, Zhen, YX, Kato, T, Tagami, H: Mechanism of human polymorphonuclear leukocyte adhesion to serum-treated corneocytes. J Invest Dermatol 1995 104:297–301,  | Article | PubMed | ISI | ChemPort |
29. Zillikens, D, Kawahara, Y, Ishiko, A, et al: A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone. J Invest Dermatol 1996 106:1333–1338,  | Article | PubMed | ChemPort |
30. Zillikens, D, Mascaro, JM, Rose, PA, et al: A highly sensitive enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid. J Invest Dermatol 1997a 109:679–683,  | Article | PubMed | ISI | ChemPort |
31. Zillikens, D, Rose, PA, Balding, SD, et al: Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies. J Invest Dermatol 1997b 109:573–579,  | Article | PubMed | ISI | ChemPort |
32. Zillikens, D, Herzele, K, Georgi, M, et al: Autoantibodies in a subgroup of patients with linear IgA disease react with the NC16A domain of BP1801. J Invest Dermatol 1999 113:947–953, 10.1046/j.1523-1747.1999.00808.x | Article | PubMed | ISI | ChemPort |
33. Zillikens, D, Schmidt, E, Reimer, S, et al: Antibodies to desmogleins 1 and 3, but not to BP180, induce blisters in human skin grafted onto SCID mice. J Pathol 2001 193:117–124, 10.1002/1096-9896(2000)9999:9999<::aid-path742>3.0.co;2-w | Article | PubMed | ISI | ChemPort |

Acknowledgments

This work was supported by grants 98.073.2 from the Whilhelm Sander-Stiftung, Munich (D.Z), GK 560 from the Deutsche Forschungsgemeinschaft (C.S), and 01KS9603 from the Interdisciplinary Center for Clinical Research at the University of Würzburg (E.S). We gratefully acknowledge the following investigators for providing us with antibodies to BP180 and BP230: Dr. Katshushi Owaribe, Nagoya, Japan, Dr. L. Bruckner-Tuderman, Münster, Germany, and Dr. J. R. Stanley, Philadelphia, U.S.A. Dr. G. J. Giudice, Milwaukee, U.S.A., kindly provided us with recombinant GST-BP180 4575. We thank Andrea Knopp, Würzburg, for assisstance with the cryosection experiments.