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Lichen planus pemphigoides with IgG autoantibodies to the 180 kd bullous pemphigoid antigen (type XVII collagen)
136
Brief reports
WA, therapy Although long-term results are not yet available, WA, therapy might develop into a promising new treatment for patients with chronic sclerodermic GVHD. This observation warrants the initiation of additional detailed studies on the effects of WA, phototherapy with a special focus on comparing investigation of low-, medium-, and high-dose WA, therapy for patients with chronic sclerodermic GVHD. REFERENCES I. Ferrara JLM, Deeg HJ. Graft-versus-host disease. N Engl J Med 1991;324:667-74. 2. Lipsky JJ. Mycophenolate mofetil. Lancet 1996;348:1357-9. 3. Marcellus DC, Altomonte VL, Farmer ER, Horn TD, Freemer CS, Grant J, et al. Etretinate therapy for refractory sclerodermous graft versus host disease. Blood 1999;93:66-70. 4. Volt-Platzer B, Honigsmann H, Hinterberger W, Wolff K. Photochemotherapy improves chronic cutaneous graft versus host disease. J Am Acad Dermatol 1990;23:220-8.
J AM ACAD DERMATOL JANUARY 2000
5. Owsianowski M, Gollnick H, Siegert W, Schwerdtfeger R, Orfanos CE. Successful treatment of chronic graft-versus-host disease with extracorporeal photopheresis. Bone Marrow Transplant 1994;14:845-8. 6. Kerscher M, Dirschka T,Volkenandt M.Treatment of localised scleroderma by UVAl phototherapy. Lancet 1995;346:1166. 7. Kerscher M,Volkenandt M,Gruss C, ReutherT,von Kobyletzki G, Freitag M, et al. Low-dose UVA, phototherapy for treatment of localized scleroderma. J Am Acad Dermatol 1998;38:21-6. 8. Scharffetter K, Wlaschek M, Hogg A, Bolsen K, Schothorst A, Goerz G, et al. UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res 1991;283:506-1 I. 9. Gruss C, Reed JA, Altmeyer R McNutt NS, Kerscher M. Induction of interstitial collagenase by UVA-1 phototherapy in morphea fibroblasts. Lancet 1997;350:1295-6. IO. Stege H, Berneburg M, Humke 5, Klammer M,Grewe M,GretherBeck S, et al. High-dose UVA, radiation therapy for localized scleroderma. J Am Acad Dermatol 1997;36:938-44.
Lichen planus pemphigoides with IgG autoantibodies to the 180 kd bullous pemphigoid antigen (type XVII collagen) Sylvia HSU, MD,a Reza F. Ghohestani, MD, PhD,b and Jouni Uitto, MD, PhDb Houston, Texas, and Philadelphia, Pennsylvania We describe a 75-year-old patient with pruritic papules on her trunk and extremities, typical of lichen planus, who later experienced subepidermal blisters. These clinical features are consistent with lichen planus pemphigoides. Immunofluorescence of perilesional skin showed linear deposits of C3 along the dermoepidermal junction. Circulating IgG autoantibodies were found to be directed against an epidermal component of the dermoepidermal junction because the patient’s serum labeled the epidermal side of 1 mol/L NaCl-split skin. The patient’s IgG autoantibodies were directed exclusively against the 180 kd bullous pemphigoid antigen (BPAg2, type XVII collagen) detected in human keratinocyte lysate by Western blot assay No reactivity was found against the 230 kd bullous pemphigoid antigen, type VII collagen, or the laminin-5 subunits. This study demonstrates that BPAg2is recognized, not only by bullous pemphigoid sera, but also by lichen planus pemphigoides sera. Our findings attest to the similarity of immunopathology in these two subepidermal blistering skin diseases. (J Am Acad Dermatol2000;42:136-41.)
From the Department of Dermatology, Baylor College of Medicine, Houston,a and the Department of Dermatology and Cutaneous Biology,Jefferson Medical College, Philade1phia.b Reprint requests: Sylvia Hsu, MD, Assistant Professor, Department of Dermatology,One Baylor Plaza, FB 800, Houston,TX 77030. Copyright 0 2000 by the American Academy of Dermatology, Inc. 0190-9622/2000/$12.00 + 0 16/54/l 01297
L
ichen planus pemphigoides (LPP) is characterized by a concomitant presence of subepidermal bullae and lichen planus-like lesions.i-9 The clinical presentation of skin blistering is quite similar to bullous pemphigoid (BP), and it has been suggested that LPP may represent coexistence of lichen planus (LP) and BP However, little
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Fig 1. Histopathologic section of papule of LI! (Original magnification x40.)
Fig 2. Numerous tense bullae among lesions of LP
infor mation is available about the target molecules impli cated in this unique entity We present a typical case of LPP with IgG autoantibodies directed against the 180 kd BP antigen (BPAg2, type XVII collagen). Our findings provide evidence for similarity of immi lnopathology between LPP and BP
CASE REPORT A 75-year-old black woman presented wit month history of pruritic papules on her trur extremities. The patient had a history of by: sion that had been treated with diltia zen mg/day since 14 months before her prese ntal
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Fig 3. Histopathologic section of tense bulla shows subepidermal cleavage with predominant eosinophilic infiltration.
us. Physical examination revealed hundreds of violaceous, flat-topped papules on the trunk and extremities characteristic of LF! There was no involvement of the mucous membranes. Histologic examination of one of the papules revealed a lichenoid lymphohistiocytic infiltrate at the dermoepidermal junction, dyskeratotic keratinocytes, wedge-shaped hypergranulosis, and orthokeratosis, consistent with LP (Fig 1). High-potency topical steroids and antihistamines were of little benefit to the patient. She returned 6 weeks later with a history of recent onset of new lesions on her trunk and extremities. At this time, physical examination revealed numerous tense bullae either on an urticarial base or on normal-appearing skin, among lesions characteristic of LP (Fig 2). Histologic examination of one of the bullous lesions revealed a subepidermal blister with numerous eosinophils (Fig 3). Direct immunofluorescence of perilesional skin showed strong linear deposition of C3 along the skin basement membrane zone (BMZ). The patient’s bullous lesions cleared within 2 weeks after starting prednisone (60 mg/day). The LP-like lesions had not cleared, although associated pruritus had improved significantly. The prednisone was tapered over a 2-month period with no recurrence of the bullae. The patient is now completely clear of both her LP and BP-like lesions for 10 months after discontinuing steroid therapy. Her only medication is diltiazem.
METHODS Sera and antibodies Control sera included (1) 5 sera from patients with BE reacting either with the BPAgl (N = 3) or BPAg2 (N = 2), (2) one epidermolysis bullosa acquisita (EBA) serum reacting with the 290 kd antigen (type VII collagen), (3) 4 sera of patients with pemphigus vulgaris (PV) reacting with the 130 kd antigen, (4) 11 sera of patients with other inflammatory skin disorders with no specific circulating autoantibodies, and (5) 5 sera from normal healthy individuals. All sera were stored at -20°C until used. In addition, a rabbit polyclonal antibody raised against the NCl6a domain of BPAg2,1° a human monoclonal antibody (mAb) to BPAgl,ll a guinea pig polyclonal antibody to the carboxyl terminal part of BPAgl,12 a rabbit polyclonal antibody to laminin-5, and a commercially available mouse mAb to type VII collagen (Serotec, Oxford, UK) were also used in this study. Dermoepidermal separation Strips (3 x 2 cm) of normal human skin were obtained from patients undergoing mammoplasty, and washed in phosphate-buffered saline (PBS) (0.126 mol/L NaCl, 0.008 mol/L Na,HPO*, and 0.002 mol/L KH,P04, pH 7.2). Dermoepidermal separation was performed by incubating the skin pieces with a 1 mol/L NaCl solution containing 10 mmol/L phenylmethylsulfonylfluoride (PMSF) at 4°C for 72 hours.
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Fig 4. Indirect immunofluorescence microscopy of patient’s serum on 1 mol/L. NaCl-split skin (SSS).Patient’s serum reacts with epidermal side of SSS.D, Dermis; E, epidermis.
The mAb to SPAgl labeled the epidermal side, whereas the mAb to type VII collagen labeled the dermal side of the salt-split skin (SSS). Immunofluorescence microscopy Circulating anti-BMZ antibodies were detected by standard immunofluorescence, performed on 4-pm cryostat-cut sections of human SSS.13The sections were incubated for 30 minutes at 20°C with the sera studied (diluted 1:lO in PBS), then washed with PBS, and incubated for 30 minutes either with filtered fluorescein isothiocyanate-conjugated rabbit antihuman IgG (Dako, Glostrup, Denmark) diluted 1:60 in PBS, or tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG (Dako) diluted 1:40 in PBS. Protein lysates To ensure that all the recently described skin antigens were present in the protein lysates, we used two different antigenic preparation protocols.14 Epidermal antigens were obtained from human cultured keratinocytes by extracting the proteins by means of incubation for 30 minutes at 4°C with an extraction buffer consisting of 10 mmol/L TRIS-HCl, pH 7.8, with 2% sodium dodecyl sulfate (SDS), 5% pmercaptoethanol, 100 mmol/L EDTA, 200 mmol/L PMSF, 1 mmol/L leupeptin, and 1 mmol/L. pepstatin. After dermoepidermal separation by either 1 mol/L NaCl or 20 mmol/‘L EDTA, the epidermis was physically peeled from the dermis, and the dermal
proteins were extracted with 8 moliL urea and 0.3 mol/L P-mercaptoethanol in 25 mmol/L TRIS buffer (pH 7.8) with 10 mmol/L PMSF by incubating for 45 minutes in an agitator at 4°C. The tissue remnants were removed by centrifugation at 13,OOOgfor 25 minutes at 4°C. The supernatants were stored at -20°C until used. SDS-polyacrylamide gel electrophoresis and immunoblot analysis Epidermal and dermal proteins were separated by SDS-polyacrylamide gel electrophoresis on 6% acrylamide gels under reducing conditions, as previously described. The proteins were electrophoretically transferred onto nitrocellulose membranes. Nitrocellulose strips were then sequentially incubated with: (1) 5% dried milk in 10 mmol/L TRIS-HCl, 140 mmol/L NaCl, pH 7.4 (TBS/milk) for 1 hour at 37°C or overnight at 4°C; (2) 1: 100 dilution of each serum in TBS/milk for 1 hour at 37°C; (3) 1:500 dilution of a biotinylated goat anti-human IgG (Amersham, Buckinghamshire, UK) in cases of human sera, in TBS/milk for 30 minutes at room temperature; antiguinea pig, anti-rabbit, or anti-mouse conjugates were used for guinea pig, rabbit and mouse antibodies, respectively; (4) 1:3000 dilution of streptavidinalkaline phosphatase substrate solution (Amersham). The reaction was revealed with 5-bromo-gchloro3-indolylphosphate/nitro tetrazolium buffered substrate tablets (Sigma Chemical, St Louis, MO).
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serum that selectively reacted with the epidermal side of SSS,suggesting that they are directed against an epidermal component of the basement membrane (Pig 4). This pattern was quite similar to that found by a rabbit polyclonal antibody raised against the BPAg2 (R67).
1 234 5. Western immunoblotting on protein lysates obtained from cultured human keratinocytes. Lane I, Immunoreactivity of BP serum with IgG antibodies to 230 kd BPAgl; lane 2, BP serum with antibodies to 180 kd BPAg2;lane 3, patient’s serum reacts exclusivelywith 180 kd BPAg2;lane 4, serum from normal individual nonreactive with epidermal antigens.
Fig
Reaction times for all lanes of individual blots were identical. Intervening washes were performed with 0.1% TBS-Tween 20. Immunofluorescence of nitrocellulose immunoaffinity-purified antibodies The protein bands transferred onto nitrocellulose paper were used as immunoaffinity substrates to purify antibodies directed against the protein of interest. After transfer of the proteins of epidermal extract (obtained from SSS) onto a nitrocellulose membrane, vertical lanes from both sides of the membrane were cut and immunochemically stained with sera to identify the band of interest. Horizontal strips of nitrocellulose containing the antigen of interest were then cut and sera applied as a 1:5 dilution in PBS for 2 hours. After being washed in PBSTween 20, the antibodies were eluted in 20 mmol/L sodium citrate (pH 3.2), 0.04% Tween 20 for 3 minutes at 37°C and then immediately neutralized with 2 mol/L ‘IRIS HCl (pH 7.5). The solution was then dialyzed against the buffer, concentrated, and used in immunofluorescence.
RESULTS The patent’s IgG autoantibodies are directed against an epidermal component of the skin BMZ Indirect immunofluorescence microscopy displayed IgG anti-BMZ antibodies in the patient’s
The patient’s serum exclusively identifles the 180 kd BP antigen We next proceeded to identify the target molecule recognized by the patient’s autoantibodies by means of Western blot assay The patient’s serum reacted with the epidermal protein extract and labeled exclusively a 180 kd antigen comigrating with the BPAg2 identified by a polyclonal anti-BPAg2 (R67) (Fig 5). None of the control sera, including those obtained from normal individuals, patients with pemphigus vulgaris, epidermolysis bullosa acquisita, or other nonbullous inflammatory dermatoses labeled the 180 kd antigen. The patient’s serum did not react with the 290 kd dermal antigen (type VII collagen) or the laminin-5 subunits (~3, j33, $2). Immunoaffinity-purified anti-180 kd IgG antibodies further labeled the skin BMZ, and bound to the epidermal side of SSS.
DISCUSSION LPP is a rare inflammatory disease characterized by tense bullae coexisting with violaceous, LP-like lesions. Histologic examination of the bullous lesions reveals subepidermal cleavage with an abundance of eosinophils, as in BP whereas histologic examination of the violaceous papules reveals findings characteristic of LP Direct immunofluorescence of peribullous skin reveals linear deposits of C3 and IgG along the dermoepidermal junction. LPP can be differentiated from bullous LP where blisters occur in long-standing lesions as a result of intense lichenoid inflammation and extensive liquefaction degeneration of basal keratinocytes. In a case-controlled study, neuroleptics and diuretics have been suggested as risk factors for BE15 Our patient, however, was not receiving these medications. In fact, she had been taking diltiazem, a calcium channel blocker. Although nifedipine, another calcium channel blocker, has been reported to induce acantholysis and subepidermal blisters in an in vitro skin explant culture system, this effect is, primarily mechanical rather immunologic without the need of autoantibodies The classification of LPP has been controversial. Some authors believe that it is merely a coexistence of LP and BP; others have suggested that LPP is a separate, unique bullous disease.3,5>7To date, there have been fewer than a dozen reports on the identifica-
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tion of the antigen or antigens in LPP by immunoblot analyses. Most of these reports have identified the 230 kd or the 180 kd BP antigens. However, Davis et al3 reported the identification of a 200 kd protein in 2 patients and suggested that the target antigen in LPP may be different from that in BP The 190-200 kd antigen is, however, recognized by some sera from patients with BP or from normal individuals.14 To better understand the immunopathology of this unique entity, we attempted to determine the antigenie specificity of autoantibodies in serum from a patient with a typical form of LPI? By immunofluorescence microscopy, the circulating IgG antibodies were found to react with the normal skin BMZ, specifically on the epidermal side. These IgG autoantibodies uniquely bound the 180 kd BPAg2 by Western blot assay This finding supports the previous suggestions that LPP is a combination of BP and LP because BPAg2 was identified as the target molecule. Thus far, BPAg2 is recognized by autoantibodies in at least 4 different entities: BP, pemphigoid gestationis, cicatricial pemphigoid, and linear IgA dermatosis. The pathogenesis of LPP remains unknown. Damage to the basal cells by the inflammatory infiltrate in LP-like lesions may expose hidden antigens to the autoreactive lymphocytes, leading to formation of autoantibodies and subepidermal bullae indistinguishable from those in BE7 This may explain why in LPP,the blisters usually appear after onset of LP-like lesions. LPP,however, tends to have a more benign course than that of BP REFERENCES 1. Swale VJ, Black MM, Bhogal BS. Lichen planus pemphigoides: two case reports. Clin Exp Dermatol 1998;23:132-5. 2. Tamada Y,Yokochi K, Nitta Y, lkeya T, Hara K, Owaribe K. Lichen planus pemphigoides: identification of 180 kd hemidesmosome antigen. J Am Acad Dermatol 1995;32:883-7. 3. Davis AL, Bhogal BS, Whitehead P, et al. Lichen planus pemphigoides: its relationship to bullous pemphigoid. Br J Dermatol 1991;125:263-71.
4. Paige DG, Bhogal BS, Black MM, Harper JI. Lichen planus pemphigoides in a child: immunopathological findings. Clin Exp Dermatol 1993;18:552-4. 5. Ogg GS, Bhogal BS, Hashimoto T, Coleman R, Barker JN. Ramipril-associated lichen planus pemphigoides. Br J Dermatol 1997;136:412-4. 6. Hofmann-Wellenhof R,SalmhoferW,Kerl H.Lichen planus pemphigoides in a g-year-old child: successful treatment with topical corticosteroids. Pediatrics 1999;16:70-1. 7. Joshi RK, Atukorala DN, Abanmi A, al Awadi T. Lichen planus pemphigoides: Is it a separate entity? [letter] Br J Dermatol 1994;130:537-8. 8. Mora RG, Nesbitt LT Jr, Brantley JB. Lichen planus pemphigoides: clinical and immunofluorescent findings in four cases. J Am Acad Dermatol 1983;8:331-6. 9. Grolleau-Rochiccioli P, Cadilhac H, Boulinguez S, et al. Lichen planus pemphigoides. Eur J Dermatol 1994;4:295-8. 10. Ghohestani RF, Cozzani E, Delaporte E, Nicolas JF, Parodi A, Claudy A. IgE antibodies in sera from patients with bullous pemphigoid are autoantibodies preferentially directed against the 230-kDa epidermal antigen (BP230). J Clin lmmunol 1998; 18:202-g. 11. Peyron E, Nicolas JF, Reano A, et al. Human monoclonal autoantibodies specific for the bullous pemphigoid antigen 1 (BPAg l).J lmmunol 1994;153:1333-9. 12. Gaucherand M, Nicolas JF, Paranhos Baccala G, et al. Major antigenie epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein: evidence using a 55 kDa recombinant protein. Br J Dermatol 1995;132:190-6. 13. Ghohestani R, Cozzani E, Kanitakis J, Nicolas JF, Faure M, Claudy A. BPAg2 antibodies are present at lower levels than BPAgl antibodies in serum patients with bullous pemphigoid. Eur J Dermatol 1996;6:491-4. 14. Ghohestani R, Kanitakis J, Nicolas JF, Cozzani E, Claudy A. Comparative sensitivity of indirect immunofluorescence to immunoblot assay for the detection of circulating antibodies to bullous pemphigoid antigens 1 and 2. Br J Dermatol 1996; 135:74-g. 15. Bastuji-Garin 5, Joly P, Picard-Dahan C, Bernard P, Vaillant L, Pauwels C, et al. Drugs associated with bullous pemphigoid: a case-control study.Arch Dermatol 1996;132:272-6. 16. Brenner S, Ruocco V, Bialy-Golan A,Tur E, Flaminio C, Ruocco E, et al. Pemphigus and pemphigoid-like effects of nifedipine on in vitro cultured normal human skin explants. Int J Dermatol 1999;38:36-40.
Brief reports
WA, therapy Although long-term results are not yet available, WA, therapy might develop into a promising new treatment for patients with chronic sclerodermic GVHD. This observation warrants the initiation of additional detailed studies on the effects of WA, phototherapy with a special focus on comparing investigation of low-, medium-, and high-dose WA, therapy for patients with chronic sclerodermic GVHD. REFERENCES I. Ferrara JLM, Deeg HJ. Graft-versus-host disease. N Engl J Med 1991;324:667-74. 2. Lipsky JJ. Mycophenolate mofetil. Lancet 1996;348:1357-9. 3. Marcellus DC, Altomonte VL, Farmer ER, Horn TD, Freemer CS, Grant J, et al. Etretinate therapy for refractory sclerodermous graft versus host disease. Blood 1999;93:66-70. 4. Volt-Platzer B, Honigsmann H, Hinterberger W, Wolff K. Photochemotherapy improves chronic cutaneous graft versus host disease. J Am Acad Dermatol 1990;23:220-8.
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5. Owsianowski M, Gollnick H, Siegert W, Schwerdtfeger R, Orfanos CE. Successful treatment of chronic graft-versus-host disease with extracorporeal photopheresis. Bone Marrow Transplant 1994;14:845-8. 6. Kerscher M, Dirschka T,Volkenandt M.Treatment of localised scleroderma by UVAl phototherapy. Lancet 1995;346:1166. 7. Kerscher M,Volkenandt M,Gruss C, ReutherT,von Kobyletzki G, Freitag M, et al. Low-dose UVA, phototherapy for treatment of localized scleroderma. J Am Acad Dermatol 1998;38:21-6. 8. Scharffetter K, Wlaschek M, Hogg A, Bolsen K, Schothorst A, Goerz G, et al. UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res 1991;283:506-1 I. 9. Gruss C, Reed JA, Altmeyer R McNutt NS, Kerscher M. Induction of interstitial collagenase by UVA-1 phototherapy in morphea fibroblasts. Lancet 1997;350:1295-6. IO. Stege H, Berneburg M, Humke 5, Klammer M,Grewe M,GretherBeck S, et al. High-dose UVA, radiation therapy for localized scleroderma. J Am Acad Dermatol 1997;36:938-44.
Lichen planus pemphigoides with IgG autoantibodies to the 180 kd bullous pemphigoid antigen (type XVII collagen) Sylvia HSU, MD,a Reza F. Ghohestani, MD, PhD,b and Jouni Uitto, MD, PhDb Houston, Texas, and Philadelphia, Pennsylvania We describe a 75-year-old patient with pruritic papules on her trunk and extremities, typical of lichen planus, who later experienced subepidermal blisters. These clinical features are consistent with lichen planus pemphigoides. Immunofluorescence of perilesional skin showed linear deposits of C3 along the dermoepidermal junction. Circulating IgG autoantibodies were found to be directed against an epidermal component of the dermoepidermal junction because the patient’s serum labeled the epidermal side of 1 mol/L NaCl-split skin. The patient’s IgG autoantibodies were directed exclusively against the 180 kd bullous pemphigoid antigen (BPAg2, type XVII collagen) detected in human keratinocyte lysate by Western blot assay No reactivity was found against the 230 kd bullous pemphigoid antigen, type VII collagen, or the laminin-5 subunits. This study demonstrates that BPAg2is recognized, not only by bullous pemphigoid sera, but also by lichen planus pemphigoides sera. Our findings attest to the similarity of immunopathology in these two subepidermal blistering skin diseases. (J Am Acad Dermatol2000;42:136-41.)
From the Department of Dermatology, Baylor College of Medicine, Houston,a and the Department of Dermatology and Cutaneous Biology,Jefferson Medical College, Philade1phia.b Reprint requests: Sylvia Hsu, MD, Assistant Professor, Department of Dermatology,One Baylor Plaza, FB 800, Houston,TX 77030. Copyright 0 2000 by the American Academy of Dermatology, Inc. 0190-9622/2000/$12.00 + 0 16/54/l 01297
L
ichen planus pemphigoides (LPP) is characterized by a concomitant presence of subepidermal bullae and lichen planus-like lesions.i-9 The clinical presentation of skin blistering is quite similar to bullous pemphigoid (BP), and it has been suggested that LPP may represent coexistence of lichen planus (LP) and BP However, little
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Fig 1. Histopathologic section of papule of LI! (Original magnification x40.)
Fig 2. Numerous tense bullae among lesions of LP
infor mation is available about the target molecules impli cated in this unique entity We present a typical case of LPP with IgG autoantibodies directed against the 180 kd BP antigen (BPAg2, type XVII collagen). Our findings provide evidence for similarity of immi lnopathology between LPP and BP
CASE REPORT A 75-year-old black woman presented wit month history of pruritic papules on her trur extremities. The patient had a history of by: sion that had been treated with diltia zen mg/day since 14 months before her prese ntal
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Fig 3. Histopathologic section of tense bulla shows subepidermal cleavage with predominant eosinophilic infiltration.
us. Physical examination revealed hundreds of violaceous, flat-topped papules on the trunk and extremities characteristic of LF! There was no involvement of the mucous membranes. Histologic examination of one of the papules revealed a lichenoid lymphohistiocytic infiltrate at the dermoepidermal junction, dyskeratotic keratinocytes, wedge-shaped hypergranulosis, and orthokeratosis, consistent with LP (Fig 1). High-potency topical steroids and antihistamines were of little benefit to the patient. She returned 6 weeks later with a history of recent onset of new lesions on her trunk and extremities. At this time, physical examination revealed numerous tense bullae either on an urticarial base or on normal-appearing skin, among lesions characteristic of LP (Fig 2). Histologic examination of one of the bullous lesions revealed a subepidermal blister with numerous eosinophils (Fig 3). Direct immunofluorescence of perilesional skin showed strong linear deposition of C3 along the skin basement membrane zone (BMZ). The patient’s bullous lesions cleared within 2 weeks after starting prednisone (60 mg/day). The LP-like lesions had not cleared, although associated pruritus had improved significantly. The prednisone was tapered over a 2-month period with no recurrence of the bullae. The patient is now completely clear of both her LP and BP-like lesions for 10 months after discontinuing steroid therapy. Her only medication is diltiazem.
METHODS Sera and antibodies Control sera included (1) 5 sera from patients with BE reacting either with the BPAgl (N = 3) or BPAg2 (N = 2), (2) one epidermolysis bullosa acquisita (EBA) serum reacting with the 290 kd antigen (type VII collagen), (3) 4 sera of patients with pemphigus vulgaris (PV) reacting with the 130 kd antigen, (4) 11 sera of patients with other inflammatory skin disorders with no specific circulating autoantibodies, and (5) 5 sera from normal healthy individuals. All sera were stored at -20°C until used. In addition, a rabbit polyclonal antibody raised against the NCl6a domain of BPAg2,1° a human monoclonal antibody (mAb) to BPAgl,ll a guinea pig polyclonal antibody to the carboxyl terminal part of BPAgl,12 a rabbit polyclonal antibody to laminin-5, and a commercially available mouse mAb to type VII collagen (Serotec, Oxford, UK) were also used in this study. Dermoepidermal separation Strips (3 x 2 cm) of normal human skin were obtained from patients undergoing mammoplasty, and washed in phosphate-buffered saline (PBS) (0.126 mol/L NaCl, 0.008 mol/L Na,HPO*, and 0.002 mol/L KH,P04, pH 7.2). Dermoepidermal separation was performed by incubating the skin pieces with a 1 mol/L NaCl solution containing 10 mmol/L phenylmethylsulfonylfluoride (PMSF) at 4°C for 72 hours.
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Fig 4. Indirect immunofluorescence microscopy of patient’s serum on 1 mol/L. NaCl-split skin (SSS).Patient’s serum reacts with epidermal side of SSS.D, Dermis; E, epidermis.
The mAb to SPAgl labeled the epidermal side, whereas the mAb to type VII collagen labeled the dermal side of the salt-split skin (SSS). Immunofluorescence microscopy Circulating anti-BMZ antibodies were detected by standard immunofluorescence, performed on 4-pm cryostat-cut sections of human SSS.13The sections were incubated for 30 minutes at 20°C with the sera studied (diluted 1:lO in PBS), then washed with PBS, and incubated for 30 minutes either with filtered fluorescein isothiocyanate-conjugated rabbit antihuman IgG (Dako, Glostrup, Denmark) diluted 1:60 in PBS, or tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG (Dako) diluted 1:40 in PBS. Protein lysates To ensure that all the recently described skin antigens were present in the protein lysates, we used two different antigenic preparation protocols.14 Epidermal antigens were obtained from human cultured keratinocytes by extracting the proteins by means of incubation for 30 minutes at 4°C with an extraction buffer consisting of 10 mmol/L TRIS-HCl, pH 7.8, with 2% sodium dodecyl sulfate (SDS), 5% pmercaptoethanol, 100 mmol/L EDTA, 200 mmol/L PMSF, 1 mmol/L leupeptin, and 1 mmol/L. pepstatin. After dermoepidermal separation by either 1 mol/L NaCl or 20 mmol/‘L EDTA, the epidermis was physically peeled from the dermis, and the dermal
proteins were extracted with 8 moliL urea and 0.3 mol/L P-mercaptoethanol in 25 mmol/L TRIS buffer (pH 7.8) with 10 mmol/L PMSF by incubating for 45 minutes in an agitator at 4°C. The tissue remnants were removed by centrifugation at 13,OOOgfor 25 minutes at 4°C. The supernatants were stored at -20°C until used. SDS-polyacrylamide gel electrophoresis and immunoblot analysis Epidermal and dermal proteins were separated by SDS-polyacrylamide gel electrophoresis on 6% acrylamide gels under reducing conditions, as previously described. The proteins were electrophoretically transferred onto nitrocellulose membranes. Nitrocellulose strips were then sequentially incubated with: (1) 5% dried milk in 10 mmol/L TRIS-HCl, 140 mmol/L NaCl, pH 7.4 (TBS/milk) for 1 hour at 37°C or overnight at 4°C; (2) 1: 100 dilution of each serum in TBS/milk for 1 hour at 37°C; (3) 1:500 dilution of a biotinylated goat anti-human IgG (Amersham, Buckinghamshire, UK) in cases of human sera, in TBS/milk for 30 minutes at room temperature; antiguinea pig, anti-rabbit, or anti-mouse conjugates were used for guinea pig, rabbit and mouse antibodies, respectively; (4) 1:3000 dilution of streptavidinalkaline phosphatase substrate solution (Amersham). The reaction was revealed with 5-bromo-gchloro3-indolylphosphate/nitro tetrazolium buffered substrate tablets (Sigma Chemical, St Louis, MO).
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serum that selectively reacted with the epidermal side of SSS,suggesting that they are directed against an epidermal component of the basement membrane (Pig 4). This pattern was quite similar to that found by a rabbit polyclonal antibody raised against the BPAg2 (R67).
1 234 5. Western immunoblotting on protein lysates obtained from cultured human keratinocytes. Lane I, Immunoreactivity of BP serum with IgG antibodies to 230 kd BPAgl; lane 2, BP serum with antibodies to 180 kd BPAg2;lane 3, patient’s serum reacts exclusivelywith 180 kd BPAg2;lane 4, serum from normal individual nonreactive with epidermal antigens.
Fig
Reaction times for all lanes of individual blots were identical. Intervening washes were performed with 0.1% TBS-Tween 20. Immunofluorescence of nitrocellulose immunoaffinity-purified antibodies The protein bands transferred onto nitrocellulose paper were used as immunoaffinity substrates to purify antibodies directed against the protein of interest. After transfer of the proteins of epidermal extract (obtained from SSS) onto a nitrocellulose membrane, vertical lanes from both sides of the membrane were cut and immunochemically stained with sera to identify the band of interest. Horizontal strips of nitrocellulose containing the antigen of interest were then cut and sera applied as a 1:5 dilution in PBS for 2 hours. After being washed in PBSTween 20, the antibodies were eluted in 20 mmol/L sodium citrate (pH 3.2), 0.04% Tween 20 for 3 minutes at 37°C and then immediately neutralized with 2 mol/L ‘IRIS HCl (pH 7.5). The solution was then dialyzed against the buffer, concentrated, and used in immunofluorescence.
RESULTS The patent’s IgG autoantibodies are directed against an epidermal component of the skin BMZ Indirect immunofluorescence microscopy displayed IgG anti-BMZ antibodies in the patient’s
The patient’s serum exclusively identifles the 180 kd BP antigen We next proceeded to identify the target molecule recognized by the patient’s autoantibodies by means of Western blot assay The patient’s serum reacted with the epidermal protein extract and labeled exclusively a 180 kd antigen comigrating with the BPAg2 identified by a polyclonal anti-BPAg2 (R67) (Fig 5). None of the control sera, including those obtained from normal individuals, patients with pemphigus vulgaris, epidermolysis bullosa acquisita, or other nonbullous inflammatory dermatoses labeled the 180 kd antigen. The patient’s serum did not react with the 290 kd dermal antigen (type VII collagen) or the laminin-5 subunits (~3, j33, $2). Immunoaffinity-purified anti-180 kd IgG antibodies further labeled the skin BMZ, and bound to the epidermal side of SSS.
DISCUSSION LPP is a rare inflammatory disease characterized by tense bullae coexisting with violaceous, LP-like lesions. Histologic examination of the bullous lesions reveals subepidermal cleavage with an abundance of eosinophils, as in BP whereas histologic examination of the violaceous papules reveals findings characteristic of LP Direct immunofluorescence of peribullous skin reveals linear deposits of C3 and IgG along the dermoepidermal junction. LPP can be differentiated from bullous LP where blisters occur in long-standing lesions as a result of intense lichenoid inflammation and extensive liquefaction degeneration of basal keratinocytes. In a case-controlled study, neuroleptics and diuretics have been suggested as risk factors for BE15 Our patient, however, was not receiving these medications. In fact, she had been taking diltiazem, a calcium channel blocker. Although nifedipine, another calcium channel blocker, has been reported to induce acantholysis and subepidermal blisters in an in vitro skin explant culture system, this effect is, primarily mechanical rather immunologic without the need of autoantibodies The classification of LPP has been controversial. Some authors believe that it is merely a coexistence of LP and BP; others have suggested that LPP is a separate, unique bullous disease.3,5>7To date, there have been fewer than a dozen reports on the identifica-
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tion of the antigen or antigens in LPP by immunoblot analyses. Most of these reports have identified the 230 kd or the 180 kd BP antigens. However, Davis et al3 reported the identification of a 200 kd protein in 2 patients and suggested that the target antigen in LPP may be different from that in BP The 190-200 kd antigen is, however, recognized by some sera from patients with BP or from normal individuals.14 To better understand the immunopathology of this unique entity, we attempted to determine the antigenie specificity of autoantibodies in serum from a patient with a typical form of LPI? By immunofluorescence microscopy, the circulating IgG antibodies were found to react with the normal skin BMZ, specifically on the epidermal side. These IgG autoantibodies uniquely bound the 180 kd BPAg2 by Western blot assay This finding supports the previous suggestions that LPP is a combination of BP and LP because BPAg2 was identified as the target molecule. Thus far, BPAg2 is recognized by autoantibodies in at least 4 different entities: BP, pemphigoid gestationis, cicatricial pemphigoid, and linear IgA dermatosis. The pathogenesis of LPP remains unknown. Damage to the basal cells by the inflammatory infiltrate in LP-like lesions may expose hidden antigens to the autoreactive lymphocytes, leading to formation of autoantibodies and subepidermal bullae indistinguishable from those in BE7 This may explain why in LPP,the blisters usually appear after onset of LP-like lesions. LPP,however, tends to have a more benign course than that of BP REFERENCES 1. Swale VJ, Black MM, Bhogal BS. Lichen planus pemphigoides: two case reports. Clin Exp Dermatol 1998;23:132-5. 2. Tamada Y,Yokochi K, Nitta Y, lkeya T, Hara K, Owaribe K. Lichen planus pemphigoides: identification of 180 kd hemidesmosome antigen. J Am Acad Dermatol 1995;32:883-7. 3. Davis AL, Bhogal BS, Whitehead P, et al. Lichen planus pemphigoides: its relationship to bullous pemphigoid. Br J Dermatol 1991;125:263-71.
4. Paige DG, Bhogal BS, Black MM, Harper JI. Lichen planus pemphigoides in a child: immunopathological findings. Clin Exp Dermatol 1993;18:552-4. 5. Ogg GS, Bhogal BS, Hashimoto T, Coleman R, Barker JN. Ramipril-associated lichen planus pemphigoides. Br J Dermatol 1997;136:412-4. 6. Hofmann-Wellenhof R,SalmhoferW,Kerl H.Lichen planus pemphigoides in a g-year-old child: successful treatment with topical corticosteroids. Pediatrics 1999;16:70-1. 7. Joshi RK, Atukorala DN, Abanmi A, al Awadi T. Lichen planus pemphigoides: Is it a separate entity? [letter] Br J Dermatol 1994;130:537-8. 8. Mora RG, Nesbitt LT Jr, Brantley JB. Lichen planus pemphigoides: clinical and immunofluorescent findings in four cases. J Am Acad Dermatol 1983;8:331-6. 9. Grolleau-Rochiccioli P, Cadilhac H, Boulinguez S, et al. Lichen planus pemphigoides. Eur J Dermatol 1994;4:295-8. 10. Ghohestani RF, Cozzani E, Delaporte E, Nicolas JF, Parodi A, Claudy A. IgE antibodies in sera from patients with bullous pemphigoid are autoantibodies preferentially directed against the 230-kDa epidermal antigen (BP230). J Clin lmmunol 1998; 18:202-g. 11. Peyron E, Nicolas JF, Reano A, et al. Human monoclonal autoantibodies specific for the bullous pemphigoid antigen 1 (BPAg l).J lmmunol 1994;153:1333-9. 12. Gaucherand M, Nicolas JF, Paranhos Baccala G, et al. Major antigenie epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein: evidence using a 55 kDa recombinant protein. Br J Dermatol 1995;132:190-6. 13. Ghohestani R, Cozzani E, Kanitakis J, Nicolas JF, Faure M, Claudy A. BPAg2 antibodies are present at lower levels than BPAgl antibodies in serum patients with bullous pemphigoid. Eur J Dermatol 1996;6:491-4. 14. Ghohestani R, Kanitakis J, Nicolas JF, Cozzani E, Claudy A. Comparative sensitivity of indirect immunofluorescence to immunoblot assay for the detection of circulating antibodies to bullous pemphigoid antigens 1 and 2. Br J Dermatol 1996; 135:74-g. 15. Bastuji-Garin 5, Joly P, Picard-Dahan C, Bernard P, Vaillant L, Pauwels C, et al. Drugs associated with bullous pemphigoid: a case-control study.Arch Dermatol 1996;132:272-6. 16. Brenner S, Ruocco V, Bialy-Golan A,Tur E, Flaminio C, Ruocco E, et al. Pemphigus and pemphigoid-like effects of nifedipine on in vitro cultured normal human skin explants. Int J Dermatol 1999;38:36-40.