Pemphigoid nodularis associated with autoantibodies to the NC16A domain of BP180 and a hyperproliferative integrin profile

CASE

REPORTS

Pemphigoid nodularis associated with autoantibodies to the NC16A domain of BP180 and a hyperproliferative integrin profile Marianne Schachter, MD,a Joaquin C. Brieva, MD,a Jonathan C. R. Jones, PhD,b Detlef Zillikens, MD,d Christian Skrobek, MD,d and Lawrence S. Chan, MDa,c Chicago, Illinois, and Wuerzburg, Germany Pemphigoid nodularis, a rare variant of bullous pemphigoid, has clinical features resembling prurigo nodularis, with blisters arising from normal-appearing or nodular skin. The fine antigenic epitope of the autoantibodies and the mechanism accounting for the nodular phenotype has not been delineated. We describe a patient with pemphigoid nodularis that fulfilled the criteria of bullous pemphigoid by histopathologic examination and direct and indirect immunofluorescence studies. Immunopathologic examination also revealed in situ deposition and circulating autoantibodies of all IgG subclasses, except IgG3, and both light chains to the patient’s skin basement membrane. By immunoblotting, the patient’s IgG autoantibodies labeled BP180, BP230, and an unidentified 150-kd epidermal protein and mapped the BP180 epitope to the MCW-1, region 2 of the NC16A domain. The nodular plaque skin showed expression of α-6 and β-1 integrin subunits, mediators of matrix-cell signaling and proliferation, at the basal and the suprabasal epidermis, a pattern found in psoriasis, which is the prototype of hyperproliferative dermatoses. (J Am Acad Dermatol 2001;45:747-54.)

B

ullous pemphigoid (BP) is a chronic, autoimmune, subepidermal, blistering skin disease that is characterized by large, tense blisters on normal or erythematous skin and by the deposition of IgG at the basement membrane zone.1 These autoantibodies target the hemidesmosomal components bullous pemphigoid antigens BP230 (BPAg1) and BP180 (BPAg2) synthesized by keratinocytes.2-4 Autoantibodies to the BP230 antigen bind only to the intracellular domain of the hemidesmosome, whereas those to BP180 bind predominantly extracellularly, along the plasma membrane of the hemidesmosome.5 Although the pathogenic role of BP230 is not yet clear, BP180 has been implicated as the patho-

From the Departments of Dermatologya and Cell and Molecular Biology,b Northwestern University Medical School, and the Medicine Service, Section of Dermatology, Lakeside Division, Veterans Affairs Chicago Health Care System,c Chicago; and the Department of Dermatology, University of Wuerzburg.d Supported in part by a Clinical Investigator Award (K08 AR01961, National Institutes of Health, to L. S. Chan) and a grant (No. 98.073.1) from the Wilhelm Sander-Stiftung, Munich, Germany (to D. Zillikens). Reprint requests: Lawrence S. Chan, MD, Department of Dermatology, Tarry 4-721, Mail Code T225, 300 E Superior St, Chicago, IL 60611-3010. E-mail: [email protected]. 16/91/116229 doi:10.1067/mjd.2001.116229

genic antigen in passive transfer experiments.6 There is also epidemiologic evidence showing that antibodies to the BP180 antigen may be a marker for poor prognosis.7 Furthermore, serum levels of autoantibodies to BP180 correlate with disease severity.8 In addition to a generalized blister form, BP has several minor variants: vegetative, vesicular, erythrodermic, and urticarial.9-12 A nodular form of BP, termed pemphigoid nodularis, is the newest variant reported.13-23 Pemphigoid nodularis, a rare variant, is characterized by features of both prurigo nodularis and BP. Although BP230 and BP180 were identified as target antigens in this nodular BP variant, the fine specificity of the autoantibodies and the mechanism of hyperproliferation accounting for the nodular phenotype are not known.19,20 We have encountered a patient with a clinical phenotype of pemphigoid nodularis and performed extensive immunologic studies to identify the autoantibodies’ subclasses, light chain composition, fine antigenic epitope mapping, and the epidermal integrin profile.

CASE REPORT A 69-year-old Indian man presented with a blistering disease of 1 year in duration. He was initially treated with dapsone for 3 months, with some improvement. When the lesions worsened on his hands about 4 months later, he was then diagnosed 747

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Table I. In situ deposition and circulating IgG autoantibody subclasses and light chain composition as detected in the patient’s skin and serum, respectively

Skin Serum

IgG1

IgG2

IgG3

IgG4

κ

λ

2+ 2+

3+ 2+

0 0

4+ 4+

3+ 3+

3+ 3+

METHODS AND RESULTS Histopathology Skin biopsy specimens were fixed in formalin, processed in paraffin, and stained with hematoxylineosin. A non-nodular blistering skin lesion revealed a subepidermal blister with numerous eosinophils. A nodular lesion revealed moderate hyperkeratosis, and the epidermis showed a mild acanthosis with elongation of the rete ridges and mild perivascular infiltrate of mononuclear cells (data not shown).

Fig 1. Clinical manifestation of patient with pemphigoid nodularis. Extensive nodular plaques and interspersed blisters are observed on dorsum of the left foot.

with bullous pemphigoid, and a treatment regimen including dapsone, tetracycline, and nicotinamide was initiated. The initial clinical presentation was pruritic, erythematous, edematous papules, which were subsequently converted to nodular plaques and blisters in 6 months. Because the patient’s lesions and the pruritus persisted, the patient was sent to the Department of Dermatology at Northwestern University for further evaluation. On physical examination, the patient had confluent, hyperkeratotic plaques and nodules with erosions on his dorsal feet, hands, extensor arms, and face, affecting about 40% of his body surface (Fig 1). No lesions were observed in the interscapular areas. Tense bullae were present interspersed within the nodular plaques. Dapsone, tetracycline, and nicotinamide were stopped, and the treatment was changed to a daily regimen of 60 mg of prednisone and 50 mg of azathioprine. The lesions and symptoms (including nodular plaques, blisters, and pruritus) cleared completely after 10 weeks of combined prednisone and azathioprine treatment.

Immunofluorescence studies Direct immunofluorescence microscopy (DIF) was performed on 6 µm thick cryosections of the patient’s perilesional skin biopsy specimen, with fluorescein-conjugated goat anti-human IgG, IgA, IgM, C3, and fibrinogen (Immco, Buffalo, NY).24 For in situ IgG subclass composition, the patient’s skin sections were incubated with monoclonal antibodies against human IgG1, IgG2, IgG3, IgG4 (Sigma, St Louis, Mo), followed by fluorescein-conjugated goat anti-mouse IgG (Kirkeggard & Perry, Gaithersburg, Md). For in situ light chain composition, DIF was performed with fluorescein-conjugated goat antihuman immunoglobulin κ-light chain and λ-light chain (Caltag Laboratories, San Francisco, Calif). DIF revealed linear depositions of IgG(3+), C3(2+), and fibrinogen(2+) at the dermoepidermal junction (Fig 2). The subclass and light chain studies detected in vivo–bound immunoglobulins of IgG1, IgG2, and IgG4, and both light chains (κ and λ) (Table I). Indirect immunofluorescence microscopy (IIF) was performed on 20 mmol/L EDTA-split normal human skin substrate by first incubating with diluted serum of the patient on 6-µm thick cryosections, followed by incubation with fluorescein-conjugated goat anti-human IgG.25 Controls included sera from patients with bullous pemphigoid, epidermolysis bullosa acquisita, and normal subjects. The control IgG from patients with bullous pemphigoid labeled the epidermal roof and the control IgG from patients with epidermolysis bullosa acquisita labeled the dermal floor. IgG from the patient with pemphigoid nodularis labeled the epidermal roof (up to 1:80 dilution), as did the bullous pemphigoid control (Fig

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Fig 2. Upper panel: DIF. Linear IgG deposition at patient’s skin basement membrane underlying a hyperproliferative epidermis. Lower panel: IIF. Patient’s circulating IgG autoantibodies bind exclusively to epidermal roof of salt-split skin. (Original magnification ×200.)

2). Normal serum did not label either side. For circulating IgG subclass and light chain composition, salt-split IIF was also performed by incubating the patient’s serum (1:10 dilution) with 6-µm thick cryosections of EDTA-split normal human skin substrate, followed by the methods already described in the DIF paragraph. The salt-split skin IIF revealed circulating IgG (IgG1, IgG2, IgG4, κ and λ light chains) binding to the epidermal roof of salt-split skin. The intensity of fluorescence staining of DIF and IIF was graded by two of us (M. S. and L. S. C.) using the following scales: 0 = no staining; 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining; 4+ = very strong staining. IgG4 predominated the IgG subclass autoantibodies (Table I). Immunoblot studies Immunoblot studies were performed to identify the target antigens of autoantibodies in this patient with pemphigoid nodularis. The source of antigens was normal human keratinocytes cultured in lowcalcium medium in the absence of serum. After nor-

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Fig 3. Western blot analysis demonstrates serum from patient with pemphigoid nodularis labels 3 epidermal protein bands. Patient’s circulating IgG autoantibodies recognized a 230-kd (upper dot), a 180-kd (lower dot), and a patient-specific 150-kd protein band in extracts of the human keratinocytes on a 6% gel (lane 1). The 180-kd band recognized by our patient’s serum comigrated with a protein band recognized by serum from a typical patient with bullous pemphigoid (lane 2). A normal control serum does not label the 230-kd, the 180-kd, or the 150kd band (lane 3).

mal human keratinocytes had been washed with phosphate-buffered saline 3 times, the cells were solubilized in extraction buffer consisting of 8 mol/L urea, 1% sodium dodecyl sulfate in 10 mmol/L TRISHCl, pH 6.8, and 15% β-mercaptoethanol. Then the solubilized cellular proteins were collected in a microcentrifuge tube. The solution is then sonnicated on ice (10 pulses, 1-2 seconds each). The substrates are then loaded onto a 4% loading gel over a separating gel (6%) and vertically separated by a Novex SDSPAGE system (Novex, La Jolla, Calif) under reducing conditions.24 The separated proteins were then horizontally transferred to a supported nitrocellulose membrane (Bio-Rad, Hercules, Calif).24 After the transfer efficiency was examined by a reversible Ponceau S stain (Sigma), the membranes were cut into strips, and blocked by 5% nonfat powdered milk. The membranes were first incubated with primary antibodies (diluted sera, 1:50) overnight at 4°C, followed by incubation at room temperature for 1 hour with peroxidase-conjugated goat antibodies to human IgG (Kirkeggard & Perry). The immunoreactions were

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Fig 4. Schematic diagram of recombinant form of BP180. Transmembranous BP180 of basal keratinocytes (BK) spans lamina lucida and projects into the lamina densa of the dermoepidermal junction. The N-terminus (NH2) is situated within the cytoplasm, whereas the C-terminus (COOH) with its 15 interrupted collagenous domains (light gray boxes) projects into the extracellular space. The NC16A domain has been subdivided into 5 regions. Recombinant NC16A fragments used in this study are shown at right. Amino acid residue numbers are indicated above boxes.

Fig 5. Patient’s serum reacts with region 2 of BP180 NC16A domain (NC16A2, MCW-1). Immunoblot analysis of recombinant GST-NC16A1-5, GST-NC16A1-3, GST-NC16A3, GSTNC16A2.5, GST-NC16A2, GST-NC16A1 (lanes 1-6, respectively) with the patient’s serum (BPDa), a polyclonal rabbit serum raised against recombinant GST (RaGST), and a normal human serum (NHS). The migration positions of molecular weight markers of 45 kd (upper) and 31 kd (lower) are shown at left.

visualized with enterochromaffin-like (ECL) Western blotting detection reagents (Amersham Pharmacia, Buckinghamshire, England) on radiographic films (Kodak, Rochester, NY). The patient’s serum contained IgG autoantibodies that recognized a 230-kd band (presumed to be BP230), a 180-kd band, and a 150-kd band. This 180-kd band comigrated with a protein band recognized by the serum of a typical patient with bullous pemphigoid (Fig 3). Five normal human sera did not label these protein bands. Immunoblot was also performed with recombinant proteins of the

BPAg2 NC16A domain26 (Fig 4). Recombinant glutathione-S-transferase fusion proteins, including GSTNC16A1, GST-NC16A2, GST-NC16A2.5, GST –NC16A3, GST-NC16A1-3, and GST-NC16A1-5, were expressed in Escherichia coli DH5 and affinity-purified by means of glutathione agarose beads (Sigma).26 Recombinant proteins were fractionated by 15% SDSPAGE and electrophoretically transferred to nitrocellulose. Immunoblot analysis with the patient’s sera was performed as described.26,27 The patient’s serum strongly reacted with GST-NC16A1-3 and GST-

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Fig 6. Expression of α-6 and β-1 integrins are detected in basal and superbasal layers of lesional epidermis in pemphigoid nodularis. IIF, using a rat monoclonal anti-human α-6 integrin subunit antibody, demonstrates α-6 integrin expression in the basal layer of normal epidermis (a). In contrast, the nodular plaque skin of the patient with pemphigoid nodularis shows intense suprabasal, in addition to basal, expression (b). A mouse monoclonal antibody to human β-1 integrin subunit labeled the basal layer of normal human skin (c), but stains both basal and suprabasal layers of the nodular plaque skin of the pemphigoid nodularis patient (d). Scale bar = 25 µm (a-d).

NC16A1-5; weaker reactivity was seen with GSTNC16A2 (MCW-1), whereas no reactivity was found with other recombinant fragments of the N-terminal 45 amino acids of NC16A (Fig 5). Thus the dominant epitope targeted by our patient’s IgG autoantibodies is located in the MCW-1 region. Tissue expression of integrin To explore possible mechanisms accounting for the epidermal hyperproliferation of our patient’s skin, IIF was performed on 6-µm thick cryosections of 3 normal skin biopsy specimens and the patient’s nodular plaque skin biopsy specimen with the use of a rat monoclonal antibody against the α-6 integrin subunit (clone No. GoH3, Immunotech, Westbrook, Me) as the primary antibody, followed by incubation with fluorescein-conjugated goat anti-rat IgG (Kirkeggaard & Perry).25 Similarly, tissue expression of β-1 integrin was performed with a mouse monoclonal antibody against the β-1 integrin subunit (clone No. DF7, Tetralink, Amherst, NY), followed by incubation with fluorescein-conjugated goat antimouse IgG (Becton Dickinson, Franklin Lakes, NJ). In contrast to the normal skin that showed expression of α-6 and β-1 integrin subunits at the basal epi-

dermis, the nodular plaque skin showed expression of these subunits not only at the basal, but also at the suprabasal, epidermis (Fig 6). Blocking studies Immunoblot studies showed that this patient’s IgG autoantibodies recognized an extra 150-kd protein band of the keratinocyte extracts (see Fig 3). Because α-6 and β-1 integrins exhibited molecular sizes of approximately 140 kd and 150 kd, respectively, under reducing conditions,28 we examined whether this 150-kd protein represents either α-6 or β-1 integrins. Because antibodies reactive to denatured integrin (α6 and β-1) are not available, blocking experiments were performed on normal human skin, with or without the preincubation with this patient’s serum (1:5 dilution), followed by incubation with a rat monoclonal antibody against the α-6 integrin or a mouse monoclonal antibody against the β-1 integrin. This was followed by incubation with fluorescein-conjugated secondary antibodies as described in the earlier sections. For positive control, 6-µm thick frozen sections of normal human skin were incubated with the patient’s serum (1:10 dilution), followed by incubation with fluorescein-conjugated goat anti-human

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Table II. Pemphigoid nodularis: Clinical and immunologic characteristics in published cases Provost et al13 (1979)

Sex Age (y) at onset Clinical findings Upper extremity Lower extremity Trunk Scalp +/– neck Mucous membranes Histologic findings Subepidermal blister Eosinophils Hyperkeratosis Acanthosis Elongated rete ridges DIF (BMZ deposits)

Case 1

Case 2

F 19

F 46

F 45

F 58

M 52

+ + NA NA –

+ + + NA –

+ + + + –

+ + + + NM

+ – NA + – IgG, IgM, C3 1:401:160 ND ND

NA + + + + IgG, C3

ND None

+ + + + + IgG, IgA, IgM, C3 IIF (BMZ autoantibodies) 1:401:160 SsIIF ND IEM ND Target antigen (kd) Treatment (effective)

Massa & Borradori Connolly15 Tani et al16 et al17 (1982) (1989) (1990)

Yung et al14 (1981)

Ross et al18 (1992) Case 1

Case 2

Case 3

F 83

F 76

F 61

F 50

+ + + + NM

+ + + + –

+ + + NA NA

+ + NA NA –

+ + NA NA NA

+ + + + + IgG, IgA, C3 1:160

+ + + + NA IgG, C3

+ + NA NA NA IgG, C3

+ NA NA NA NA IgG, C3

+ NA NA NA NA IgG, C3

1:1280

+ + + + NM IgG, C3, fib 1:1280

1:500

+

1:320

1:160

ND ND

ND ND

ND LL

ND ND

ES LL

ND ND

ND LL

ND

ND

ND

ND

ND

220

ND

220

None

P, D, N

P

NM

P

AZ, P

P

P

AZ, Azathioprine; D, dapsone; ES, epidermal side; fib, fibrinogen; HD, hemidesmosome; IEM, immunoelectron microscopy; LL, lamina lucida; N, nicotinamide; NA, not available; ND, not done; NM, not mentioned; P, prednisone; Pn, prednisolone; SsIIF, salt-split indirect immunofluorescence.

IgG. There is no difference in the intensities of staining of α-6 or β-1 integrins with or without preincubation with this patient’s serum. These results suggest that this patient’s autoantibodies did not share the epitopes of the monoclonal antibodies to α-6 or β-1 integrin subunits. Thus the true identity of this 150-kd epidermal protein recognized by this patient’s serum remains to be determined.

DISCUSSION The purpose of this study aims to better characterize pemphigoid nodularis, a rare and intriguing variant of BP. We have reviewed the literature on this subject and tabulated the reported findings in Table II. Three cases were excluded from this table because of lack of specific clinical information presented in the article in which they were reported.23 In the majority of cases of pemphigoid nodularis in the literature, the patients present with hyperkeratotic nodules and/or plaques and have at some point had tense blisters. The lesions are usually located on the extremities and the trunk and are infrequently

observed on the scalp.13-23 Most of the patients demonstrate a positive IgG deposition at the skin basement membrane zone by DIF and circulating IgG autoantibodies binding to the skin basement membrane zone by IIF.13-23 The patient who presented to us has DIF and IIF findings on regular and salt-split skin, along with histologic assessment, that fulfilled the diagnostic criteria of BP. The predominant clinical phenotype of hyperkeratotic nodules and plaques on the patient’s feet, hands, arms, and face led us to the diagnosis of pemphigoid nodularis. BP230 and BP180 had been identified by Western blotting as target antigens of IgG autoantibodies in pemphigoid nodularis and were also recognized by our patient’s autoantibodies. The BP180 NC16A domain contains immunodominant epitopes recognized by the autoantibodies of classic BP.26,27 Here we clearly demonstrate that the BP180 NC16A domain is also the target epitope recognized by the autoantibodies in pemphigoid nodularis. The most intriguing clinical feature of pemphigoid nodularis is the epidermal hyperproliferation.

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Bourke et al21 (1994)

Borradori et al19 (1992)

Gallo et al20 (1993)

Case 1

Case 2

Case 3

Ratnavel et al22 (1994)

F 69

M 63

F 49

M 66

F 34

M 15

M 69

F/M = 2.2:1 Avg age = 53 (range, 15-83)

+ + + – –

NA + NA NA NA

+ + + + NM

+ + + NA NA

+ + NA NA NA

+ + + NM NM

+ + – + –

94% 100% 63% 38% 0%

+ + + + NA IgG, C3

+ + NA + NM IgG, C3

NA NA NA NA NA C3

+ NA NA NA NA IgG, C3

+ NA + NA NA IgG, C3

1:500

NA + + + + IgG, IgM, C3 1:640

+

+

ND

–

+ + + + + IgG, C3, fibrin +

ES HD

ES ND

ES ND

ES ND

ND ND

ND ND

ES ND

230

160, 180, 200, 230 AZ, P

ND

ND

ND

220

150, 180, 230

81% 63% 56% 63% 31% IgG recognized in 94% of cases, C3 in 100% When done, IIF was positive in 93% of cases When done, ES labeled in 100% of cases When done, LL/HD seen in 100% of cases 150, 160, 200, 220, 230

AZ

AZ, P

AZ

D, Pn

AZ, P

P

Epidermal integrins have been reported to play a role in the mechanism of epidermal hyperproliferation.29,30 Under normal conditions, the epidermal integrins and their expression are under tight control and are observed only in the basal layer.29 In psoriatic skin, the prototype of hyperproliferative dermatoses, there is expression of α-6 integrin, the mediator of matrix-cell signaling, and β-1 integrin, not only at the basal, but also at the suprabasal, epidermis.29 That is precisely what we have found in the nodular plaque skin of our patient with pemphigoid nodularis. α-6 β-4 Integrin interacts with BP180 in vitro and has been shown to be a part of a pathway involved in the regulation of epidermal cell proliferation.30-32 The β-1 integrin subunit has also been linked to the proliferative capacity of basal keratinocytes. In human epidermis, stem cells (which have a high self-renewal capacity and low probability of terminal differentiation) express a two-fold higher level of β-1 integrins than do transit-amplifying cells (the daughter of the stem cell that is destined to differentiate earlier).33 In one study that examined stem cell function in vitro, it was

Present study

Summary

shown that degrees of stimulation of the integrin β1/mitogen-activated protein kinase pathway correlates with the degree of proliferation of the keratinocytes.34 In another study, a transgenic model demonstrated that suprabasal β-1 integrin expression induced a hyperproliferative epidermis.35 In summary, we have described a patient with a rare variant of BP, with a predominantly nodular, rather than blistering, phenotype. We further provide evidence that the patient’s autoantibodies recognize the MCW-1 region within the NC16A domain of BP180 and that the nodular clinical phenotype is associated with a hyperproliferative integrin profile. No specific conclusion regarding the molecular mechanism underlying the epidermal hyperproliferation can be made at this time. Investigation of additional cases is needed to confirm the findings presented in this article. Furthermore, how the binding of autoantibodies to the skin basement membrane might interact with epidermal integrin subunits and what cellular mechanism induces the epidermal hyperproliferation in pemphigoid nodularis remain to be determined by future studies.

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REFERENCES 1. Stanley JR. Bullous pemphigoid. In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI, et al, editors. Fitzpatrick’s Dermatology in general medicine. New York: McGraw-Hill; 1999. p. 666-73. 2. Stanley JR, Hawley-Nelson P, Yuspa SH, Shewach EM, Katz SI. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 1981;24:897-903. 3. Labib RS, Anhalt GJ, Patel HP, Mutasim DF, Diaz LA. Molecular heterogeneity of the bullous pemphigoid antigen as detected by immunoblotting. J Immunol 1986;136:1231-5. 4. Mueller S, Klaus-Kotvun V, Stanley JR. A 230-kd basic protein is the major bullous pemphigoid antigen. J Invest Dermatol 1989; 92:33-8. 5. Ishiko A, Shimizu H, Kikuchi A, Ebinhara 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 and swine skin. J Clin Invest 1993;91:1608-15. 6. Liu Z, Diaz LA,Troy JL,Taylor AF, Emery DJ, Fairley JA, 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;92:2480-8. 7. Bernard P, Bedane C, Bonnetblanc JM. Anti-BP180 autoantibodies as a marker of poor prognosis in bullous pemphigoid: a cohort analysis of 94 elderly patients. Br J Dermatol 1997; 136:694-8. 8. 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 2000;136: 174-8. 9. Chan LS, Droman MA, Agha A, Suzuki T, Cooper KD, Hashimoto K. Pemphigoid vegetans represents a bullous pemphigoid variant: patient’s IgG autoantibodies identify the major bullous pemphigoid antigen. J Am Acad Dermatol 1993;28:331-5. 10. Bean SF, Michel B, Furey N, Thorne G, Meltzer L. Vesicular pemphigoid. Arch Dermatol 1976;112:1402-4. 11. Korman NJ, Woods SG. Erythrodermic bullous pemphigoid is a clinical variant of bullous pemphigoid. Br J Dermatol 1995;133: 967-71. 12. Strohal R, Rappersberger K, Pehamberger H, Wolff K. Nonbullous pemphigoid: prodrome of bullous pemphigoid or a distinct pemphigoid variant? J Am Acad Dermatol 1993;29: 293-9. 13. Provost TT, Maize JC, Ahmed AR, Strauss JS, Dobson RL. Unusual subepidermal bullous diseases with immunologic features of pemphigoid. Arch Dermatol 1979;115:156-60. 14. Yung CW, Soltani K, Lorincz AL. Pemphigoid nodularis. J Am Acad Dermatol 1981;5:54-60. 15. Massa MC, Connolly SM. Bullous pemphigoid with features of prurigo nodularis. Arch Dermatol 1982;118:937-9. 16. Tani M, Murata Y, Masaki H. Pemphigoid nodularis. J Am Acad Dermatol 1989;21:1099-104. 17. Borradori L, Rybojad M, Verola O, Flageul B, Puissant A, Morel P. Pemphigoid nodularis. Arch Dermatol 1990;126:1522-3. 18. Ross JS, McKee PH, Smith NP, Shimizu H, Griffiths WA, Bhogal BS, et al. Unusual variants of pemphigoid: from pruritus to pemphigoid nodularis. J Cutan Pathol 1992;19:212-6.

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19. Borradori L, Prost C, Wolkenstein P, Bernard P, Baccard H, Morel P. Localized pretibial pemphigoid and pemphigoid nodularis. J Am Acad Dermatol 1992;27:863-7. 20. Gallo R, Parodi A, Rebora A. Pemphigoid nodularis. Br J Dermatol 1993;129:744-5. 21. Bourke JF, Berth-Jones J, Gawkrodger DJ, Burns DA. Pemphigoid nodularis: a report of two cases. Clin Exp Dermatol 1994;19:496-9. 22. Ratnavel RC, Shanks AJ, Grant JW, Norris PG. Pemphigoid nodularis. Br J Dermatol 1994;130:125-6. 23. Cliff S, Holden CA. Pemphigoid nodularis: a report of three cases and review of the literature. Br J Dermatol 1997;136:398-401. 24. Chan LS, Fine J-D, Briggaman RA, Woodley DT, Hammerberg C, Drugge RJ, et al. Identification and partial characterization of a novel 105-kDalton lower lamina lucida autoantigen associated with a novel immune-mediated subepidermal blistering disease. J Invest Dermatol 1993;101:262-7. 25. Chan LS, Trazcyk T, Taylor TB, Eramo LR, Woodley DT, Zone JJ. Linear IgA bullous dermatosis: characterization of a subset of patients with concurrent IgA and IgG anti-basement membrane autoantibodies. Arch Dermatol 1995;131:1432-7. 26. Zillikens D, Rose PA, Balding SD, Liu Z, Olague-Marchan M, Diaz LA, et al. Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies. J Invest Dermatol 1997;109:573-9. 27. Zillikens D, Mascaro JM, Rose PM, Liu Z, Ewing SM, Caux F, 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 1997;109:679-83. 28. DiPersio CM, Hodivala-Dilke KM, Jaenisch R, Kreidberg JA, Hynes RO. α3β1 Integrin is required for normal development of the epidermal basement membrane. J Cell Biol 1997;137:729-42. 29. Hertle MD, Kubler M-D, Leigh IM, Watt FM. Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. J Clin Invest 1992;89:1892-901. 30. Hopkinson SB, Baker SE, Jones JCR. Molecular genetic studies of a human epidermal autoantigen (the 180-kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and α6 integrin. J Cell Biol 1995; 130:117-25. 31. Mainiero F, Murgia C, Wary KK, Curatola AM, Pepe A, Blumemberg M, et al. The coupling of α-6 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation. EMBO J 1997;16:2365-75. 32. Hopkinson SB, Findlay K, deHart GW, Jones JCR. Interaction of BP180 (type XVII collagen) and α-6 integrin is necessary for stabilization of hemidesmosome structure. J Invest Dermatol 1998;111:1015-22. 33. Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993;73:713-24. 34. Zhu AJ, Haase I, Watt FM. Signalling via β-1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc Natl Acad Sci U S A 1999;96:6728-33. 35. Carroll JM, Romero MR,Watt FM. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis. Cell 1995;83: 957-68.