- Email: [email protected]
Lichen planus pemphigoides: Case report and results of immunofluorescence and immunoelectron microscopic study
Lichen planus pemphigoides: Case report and results of immunofluorescence and immunoelectron microscopic study Hitoshi Okochi, MD, Kiyoko Nashiro, Tetsuya Tsuchida, MD, Yoshihito Seki, MD, and Kunihiko Tamaki, MD Tokyo, Japan A Japanese woman with lichen planus pemphigoides is reported. Immunologic characteristics of lichen planus pemphigoides antigen in the patient were investigated by indirect immunofluorescence and compared with those of bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen. Ultrastructural localization of lichen planus pemphigoides antigen was studied with the use of immunoelectron microscopic techniques. Lichen planus pemphigoides antigen showed localization similar to that of bullous pemphigoid antigen but different from that of epidermolysis bullosa acquisita antigen. The antigenic stability of lichen planus pemphigoides antigen was different from that of bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen. Thus this study demonstrates that lichen planus pemphigoides antigen is different from bullous pemphigoid antigen. (J AM ACAD DERMATOL 1990;22:62631.)
Lichen planus pemphigoides (LPP) has been well recognized clinically, histopathologically, and immunologically.I Schreiner2 divided bullous lichen planus into two groups. The first group, lichen planus vesiculosis, describes the occurrence of vesicles or bullae on typicallesions oflichen planus (LP), and the second, LPP, is defined as the appearance of bullae on clinically normal skin as well as on lesional erythematous skin. Further studies with immunofluorescence techniques have revealed that bullous lesions of LPP may be indistinguishable from bullous pemphigoid (BP). Thus LPP is considered to be the coexistence of LP and BP by most authors. 3• IO Recent investigations by Lang and Maize II and by Prost et a1. 12 challenged this concept and suggested that BP antigen and LPP antigen might be different. Recently we saw a Japanese woman who had typical LPP clinically, histologically, and immunopathologically. This patient also had autoantibodies against epidermal basement membrane zone From the Department of Dermatology, Faculty of Medicine, Tokyo University Branch Hospital, University of Tokyo. Acccpted for publication June 16, 1989. Reprint requests: H. Okochi, MD, Departmcnt of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongou, Bunkyo·ku, Tokyo, Japan, 113.
16/1/14957
626
(BMZ). In this report we briefly describe this patient and detail our investigations into the nature of the LPP antigen.
CASE REPORT An 85-year-old Japanese woman was referred to the Department of Dermatology at Tokyo University Branch Hospital on Oct. 8, 1986, because of a bullous eruption. Three months before her hospitalization, she noted pruritic brownish maculopapular lesions on the face and dorsum of the hands. Two months later, blisters began to develop. On admission, a number of tense bullae were symmetrically located on the extremities; a few were also observed on the trunk. The bullae arose on normalappearing skin and on erythematous skin (Fig. I). The patient had Hashimoto's thyroiditis and stomach cancer. A biopsy specimen from a reddish plaque on the dorsal aspect of the right hand showed hyperkeratosis, irregular acanthosis associated with sawtooth elongation of rete ridges and colloid body formation. A cellular infiltrate of lymphocytes, histiocytes, and eosinophils formed a bandlike pattern below the dermoepidermaljunction (Fig. 2). The histologic findings were consistent with LP. A specimen taken from a bullous lesion on normal-appearing skin of the anterior chest wall showed subepidermal blister formation with a moderate dermal infiltrate consisting of lymphocytes, eosinophils, and neutrophils (Fig. 3). These findings were consistent with BP. On direct immunofluorescence, linear IgG deposits were noted in both the bullous and the LP lesions. The
Volume 22 Number 4 April 1990
Lichen planus pemphigoides 627
Fig. 1. Bullae and erosions on left foot.
colloid bodies in the dermis of the LP lesions showed marked fluorescence for IgM. Circulating anti-BMZ antibodies were detected by the indirect immunofluorecence technique with normal human skin sections as substrate ata titer of 1:1280. A diagnosis ofLPP was made, and th~ patient was treated with 0.05% clobetasol-17-propionate ointment. The bullae faded gradually, leaving a brownish erythema at their sites. The patient was almost free of lesions by the end of October 1986, although her anti-BMZ titer remained at 1:2560.
Immunofluorescence studies For skin separation and pretreatment with fixatives, samples of normal skin were obtained. Each sample was divided in half. One half was used for separation of the skin at the dermoepidermal junction. The other half was kept at -70 0 C in ornithine carbamyltransferase (OCT) compound and used for experiments with different fixatives. Sera were obtained from our patient with LPP, from four patients with BP, and from a patient with EBA. Each of these sera was shown to contain IgG-class anti-BMZ antibodies, with normal human skin as substrate. The titers of antibodies were as follows: LPP, 1:1280; BP-l, 1:1280; BP-2, 1:80; BP-3, 1:160; BP-4, 1:2560; and EBA, 1: 1280. Sera from normal subjects were used as controls. Indirect immunofluorescence was performed as described previously.I3 Sera were used at a dilution of 1:20. Fluorescein isothiocyanate-conjugated rabbit antihuman IgG (F(ab)2'-specific) was used at a dilution of 1:40. A 0.5 cm piece was cut from each skin sample, and the subcutaneous fat was scraped off. Two separation methods were used as previously described 13: (1) Pieces of skin were immersed in 1 mol sodium chloride at 4 0 C for 5days as described by Scaletta et a1. 14; or (2) skin was floated in phosphate-buffered saline solution (PBS) at 37 0 C for 5 days as described by Regnier et al. IS The sodium chloride was replaced every 24 hours. After these treatments the epidermis was separated easily from the' dermis. After separation, samples were embedded in OCT compound
Fig. 2. Histologic changes of erythematous plaque on dorsal a~pect of right hand. Hyperkeratosis, irregular acanthosIs, colloid body formation, and bandlike cell infiltration of dermis are noted. (Hematoxylin-eosin stain; XI00.)
and snap-frozen and 6,um sections were processed for indirect immunofluorescence study. Frozen sections (6 ,urn) of each human skin sample were washed for 15 minutes in PBS and then immersed in acetone at -20 0 C, in pure ethanol at room temperature, or in 6% formalin at room temperature, each for 30 minutes. In some experiments sections were immersed in pure ethanol for 10 minutes. The sections were then washed for 20 minutes in PBS and subjected to indirect immunofluorescence. The experiments were performed at least three times, and representative results are presented. To determine the precise localization of the immunoglobulin deposits, we performed indirect immunoelectron microscopy with peroxidase-labeled antibody.16 Peroxidase-labeled goat antihuman IgG (F(abh' -specific) (Cooper Biomedical, Malvern, Pa.) was used. For indirect immunoelectron microscopy, normal human skin was used as substrate and normal human serum was used as a control. RESULTS The results oflocalization of LPP, BP, and EBA antigens in separated skin are summarized in Table I. LPP serum produced a sharp linear band of fluorescence at the epidermal side of the BMZ after
Journal of the American Academy of Dermatology
628 Okochi et al. '
..
9.·' •. ' ..
Fig. 3. Histologic findings of bulla from anterior chest wall. Subepidermal blister with eosinophils, neutrophils, and lymphocytes is noted. (Hematoxylin-eosin stain; XI00.)
Table I. Localization and antigenic stability of LPP, BP, and EBA antigens* Dermoepidermal separation (side) Disease (titer)
PBS
LPP (1:1280) BP-l (1;1280) BP-2 (1:80) BP-3 (1:160) BP-4 (1:2560) EBA (1:1280)
++ (E)
± (E)
+++ (D)
I
Fixation NaCl
None
+++ (E) +++ (E) + (E) + (E) +++ (E) +++ (D)
+++ +++ + + +++ +++
I
Ethanol
I
Acetone
++
+++ +++ + + +++ +++
± ±
+++
I
Formalin
± ± - to ± - to ± ± ±
-, Negative; ±, faint; +, moderate; ++, intense; +++, very intense. D, Dermal side; E, epidermal side. *Results are expressed as intensity of staining evaluated by immunofluorescent microscopy.
Table II. Dilutions of LPP, BP, and EBA sera that still detected LPP, BP, and EBA antigen after fixation of substrate by acetone, ethanol, or formalin Fixation Ethanol Disease
None
10 min
LPP BP-l BP-2 BP-3 BP-4 EBA
1:1280 1:1280 1:80 1:160 1:2560 1:1280
1:1280 1:80 <1:20 <1:20 1:40 1:1280
separation both with sodium chloride and with PBS (Fig. 4). No fluorescence was observed along the dermal side. Each BP serum also produced a linear band of fluorescence on the epidermal side after separation with sodium chloride, but virtually no
I
30 min
Acetone
Formalin
1:320 1:20 <1:20 <1:20 1:40 1:1280
1:1280 1:1280 1:80 1:160 1:2560 1:1280
1:160 1:160 1:20 1:40 1:160 1:160
fluorescence was observed after separation with PBS. EBA serum, however, produced a linear band of fluorescence at the dermal side after both these treatments. The staining patterns were the same in all skin
Volume 22 Number 4 April 1990
Lichen planus pemphigoides 629
Fig. 4. Indirect immunofluorescence staining for LPP antigen in human skin separated by warm PBS (A) and cold sodium chloride (B). Fluorescence is observed on epidermal side of skin. (X400.)
samples tested with each of the sera. N onnal human control sera did not show positive fluorescence. The results of antigenic stability of LPP, BP, and EBA antigens after fixation are summarized in Table 1. After fixation with acetone, all three kinds of antigens were detected as strongly as in unfixed controls. Fixation with formalin reduced the intensity of fluorescence for all these antigens. Fixation with ethanol significantly reduced the intensity of fluorescence for BP antigen, and slightly for LPP antigen, whereas virtually no reduction was noted for EBA antigen. Because a marked difference in antigenic stability was noted after fixation with ethanol among BP, LPP, and EBA antigens, semiquantitative studies were performed by serially titering each serum from I :20 to 1:2560, with human skin as substrate. In addition, we compared the duration of ethanol fixation between 10 minutes and 30 minutes. The results are shown in Table II. The titers ofBP sera were reduced markedly after both ethanol fixations, whereas EBA serum did not show a reduction in titer. The titer of LPP serum was not reduced by 10 minutes of ethanol fixation but was reduced by 30 minutes of ethanol fixation; however, the reduction was not as remarkable as with BP sera.
Fig. 5. Indirect immunoelectron microscopy shows IgG deposits at basal cell hemidesmosomes.. White stars denote immune deposits. BL, Basal lamina; D, dermis; E, epidermis. (X40,OOO.)
Indirect immunoelectron microscopy revealed that the reaction products of LPP antigen (indicating IgG) were observed in basal cell hemidesmosomes (Fig. 5). DISCUSSION
LPP has been considered by IIlost authors to be the coexistence of LP and BP.3-10 In fact, Okada et
Journal of the American Academy of Dermatology
630 Okochi et al. a1.,17 using the serum of a patient with drug-induced LPP, detected a 240 kD antigen identical to BP antigen by immunoprecipitin assay. Lang and Maize, II however, could not detect BP antigen in LPP serum by immunoprecipitin assay. Furthermore, Prost et al. I2 have reported that LPP antigen is not localized along the roof of the bulla as in BP, but is localized instead along the floor. They concluded that LPP antigen might be different from BP antigen. Our investigations revealed the following: LPP antigen was clearly detected on the epidermal side of the BMZ in PBS-treated skin, but BP antigen was not detected. Localization of LPP antigen was similar to that of BP antigen when studied in sodium chloride-treated skin. This finding suggests that LPP antigen is found at the lamina lucida18 or at the basal cell hemidesmosomes. 19 Our immunoelectron microscopic findings revealed that LPP antigen localized to the basal cell hemidesmosomes. On ethanol fixation, EBA antigen was stable whereas BP antigen was unstable even with brief (10 minutes) fixation. Danno et a1. 20 also reported that BP antigen was not detected after ethanol fixation. LPP antigen, however, was detected without reduction ofserum titer after 10 minutes ofethanol fixation but with slight reduction after 30 minutes of ethanol fixation. Thus the sensitivity to ethanol fixation is different between LPP antigen and BP antigen. Because we used the same titer of serum (1:1280) among the three diseases in our experiments, the difference between LPP antigen and BP antigen could not be attributed to the titer of the serum. The molecular weight of BP antigen has been shown to be 220 kD. 21 In one report22 heterogeneous BP antigens ranging in molecular weight from 77 kD to 240 kD were detected. Zhu and Bystryn23 also reported that BP antigens were heterogeneous as assessed byindirectimmunofluorescencetechniques. Possibly some BP antigens are responsible for LPP. These antigens may exhibit different properties than conventional BP antigens, although they show similar localization ultrastructurally. Recently, Davis et a1. 24 reported that the target antigens in LPP included a 180 leD protein that appeared in a minority of cases of BP, in addition to a unique 200 leD protein. They also reported that the target antigen in LPP had an ultrastructural localization similar to that of BP. In summary, our report reveals that the LPP antigen and the BP antigen have different properties.
These findings suggest that LPP may represent a distinct entity and not a simple coexistence ofLP and BP. However, additional studies are necessary. REFERENCES 1. Black MM. Lichen planus and lichenoid eruptions. In: Rook Al, Wilkinson DS, Ebling FJG, et ai, eds. Textbook of dermatology; vol 2. 4th ed. Oxford: Blackwell Scientific, 1986:1682. 2. Schreiner K. Lichen ruber pemphigoides und lichen ruber vesiculosis. Arch Dermatol Syph 1930;161:647-57. 3. Stingl G, Holubar K. Coexistence oflichen planus and bullous pemphigoid: an immunopathological study. Br 1 DermatoI1975;93:313-20. 4. Sobel S, Millar R, Shatin H. Lichen planus pemphigoides: immunofluorescence findings. Arch Dermatol 1976; 112:1280-3. 5. SauratJH, Guinepain MT, Didierjean L, et al. Coexistence d'un lichen plan et d'une pemphigoide bulleuse: etude en immunofluorescence d'nn "lichen pemphigoide." Ann Dermatol VenereoI1977;104:368-74. 6. Morel P, Saurat JH, Civatte 1. Etude en immunofluorescence d'un nouveau cas de lichen plan bulleux. Ann Dermatol VenereoI1978;105:81-2. 7. Hinter H, Tappeiner G, Honigsmann H, et al. Lichen planus and bullous pemphigoid. Acta Derm Venereol (Stockh) 1979;59(suppI89):71-6. 8. Morel P, Perron J, Crickx B, et al. Lichen plan avec depOts lineaires d'IgG et de C3 a la jonction dermoepidermique. Dermatologica 1981;163:117-24. 9. Souteyrand P, Pierini AM, Bussy RF, et al. Lichen planus pemphigoides: entity or association? Dermatologica 1981; 162:414-6. 10. Mora RG, Nesbitt LT, Brantley lB. Lichen planus pemphigoides: clinical and immunofluorescent findings in four cases. 1 AM ACAD DERMATOL 1983;8:331-6. 11. Lang PG, Maize lC. Coexisting lichen planus and bullous pemphigoid or lichen planus pemphigoides? 1 AM ACAD DERMATOL 1983;9:133-40. 12. Prost C, Tesserand F, Laroche L, et al. Lichen planus pemphigoides: an immuno-electron microscopic study. Br 1 DermatoI1985;113:31-6. 13. Fume M, Iwata M, Tamaki K, et al. Anatomical distribution and immunological characteristics of epidermolysis bullosa acquisita antigen and bullous pemphigoid antigen. Br J DermatoI1986;114:651-9. 14. Scaletta Ll, Occhino lC, MacCallum DK, et al. Isolation and immunologic identification of basement membrane zone antigens from human skin. Lab Invest 1978;39:1-9. 15. Regnier M, Prunieras M, Woodley D. Growth and differentiation of adull human epidermal cells on dermal substrate. Front Matrix Bioi 1981;9:4-35. 16. Nakane PK, Pieree GB. Enzyme-labeled antibodies: preparation and application for the localization of antigens. 1 Histochem Cytochem 1966;14:929-31. 17. Okada N, Kitano Y, Miyagawa S, et al. Expression of pemphigoid antigen by SV40-transformed human keralinocytes.l Invest Dermatol 1986;86:399-401. 18. Holubar K, Wolff K, Konrad, et al. Ultrastructural localization of immunoglobulins in bullous pemphigoid skin. 1 Invest Dermatol 1975;64:47-9. 19. Westgate GE, Weaver AC, Couchman JR. Bullous pemphigoid antigen localization suggests an intracellular asso-
Volume 22 Number 4 April 1990
Lichen planus pemphigoides
dation with hemidesmosomes. J Invest DermatoI 1985; 84:218-224. 20. Danno K, Toyama K, Imamura S. Immunofluorescent studies of bullous pemphigoid antigen. Acta Dermatologica 1982;77:25-8 (in Japanese). 21. Stanley JR, Hawley-Nelson P, Yuspa SR, et al. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 1981;24:897-903.
22. Labib RS, Anhalt GJ, Patel HP, et al. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J ImrnunoI1986;136:1231-5. 23. Zhu XJ, Bystryn Jc. Heterogeneity of pemphigoid antigens. J Invest Dermatol 1983;80:16-20. 24. Davis A, Wojnarowska F, Bhogal B, et al. Lichen planus pemphigoides and its relationship to bullous pemphigoid [Abstract). Br J Dermato11989;120:296.
Contact urticaria and anaphylaxis to natural latex C. Pecquet, MD, F. Leynadier, MD, and J. Dry, MD Paris, France Immediate allergic reaction to natural latex of Hevea brasiliensis has been well documented. We report 17 cases, all in WOmen. Twelve had an anaphylactic reaction during surgery and five had contact urticaria. Reactions to prick tests done through a washed surgical glove and with natural latex were positive in 15 of 17 patients and in 16 of 16 patients, respectively; specific IgE was found in 12 of 17 patients. In 206 control subjects, results ofa prick test through a surgical glove were negative. (J AM ACAD DERMATOL 1990;22:631-3.)
Since the first case of contact urticaria from natural rubber gloves was observed by Nutter l in 1979, approximately 60 cases have been reported. Natural latex is used in the manufacture of surgical gloves, rubber cleaning gloves, balloons, dental dams, and contraceptives, all of which may induce sensitization. The symptoms are usually limited to localized itching and redness but severe systemicreactions can occur. 2 Specific IgE antibodies to latex rubber have been demonstrated. 3 During the past 20 months we have encountered 17 patients with latex-induced contact urticaria. PATIENTS AND METHODS
Seventeen patients with a reaction to natural rubber gloves were examined. Twelve had an anaphylactic reaction during surgery and five had contact urticaria. Prick tests were performed in all patients on the skin of the forearm with disposable needles (Stallerpoint, Labo-
From the Department of Internal Medicine and the Center for Anergy, Hopital Rothschild. Accepted for publication June 16, 1989. Reprint requests: Dr. C, Pecquet, Service du Pr, Dry., Hopital Rothschild, 33 BId. de Picpus, 75012 Paris, France. 16/1/14958
ratoire des Stallergenes, Fresnes, France), through a washed latex surgical glove (Assistance Publique Paris), and with natural latex from the Ivory Coast without accelerators. The natural latex was filtered on Millipore (Molsheim, France). Onemilliliter ofthe filtered solution was diluted in 9 ml of phenolated saline solution. A dilution of 1: 1000 was used for the skin prick test; if the results were negative, 1:100 was used. The mean diameters of the wheals and flares were measured and compared with those ofthe positive control test (9% codeine phosphate) and with those of the negative control test (saline solution). Levels of total 19B and specific 19B to latex in the serum were determined with the Phadezym technique (Pharmacia, St. Quentin en Yvelines, France). Specific 19B values were expressed in radioallergosorbent test score. Skin prick tests were performed with the same protocol in 206 control subjects. j
RESULTS
Seventeen women reported contact urticaria to latex. Their mean age was 35 years (range 26 to 56 years). Daily occupational contact with natural rubber gloves was determined in 10 patients. The contact urticaria to latex had been present from 2 months to more than 12 years. When latex gloves were worn, itching, redness, and/or swelling of the hands began within 1 to 60 minutes (average 15
631
Lichen planus pemphigoides (LPP) has been well recognized clinically, histopathologically, and immunologically.I Schreiner2 divided bullous lichen planus into two groups. The first group, lichen planus vesiculosis, describes the occurrence of vesicles or bullae on typicallesions oflichen planus (LP), and the second, LPP, is defined as the appearance of bullae on clinically normal skin as well as on lesional erythematous skin. Further studies with immunofluorescence techniques have revealed that bullous lesions of LPP may be indistinguishable from bullous pemphigoid (BP). Thus LPP is considered to be the coexistence of LP and BP by most authors. 3• IO Recent investigations by Lang and Maize II and by Prost et a1. 12 challenged this concept and suggested that BP antigen and LPP antigen might be different. Recently we saw a Japanese woman who had typical LPP clinically, histologically, and immunopathologically. This patient also had autoantibodies against epidermal basement membrane zone From the Department of Dermatology, Faculty of Medicine, Tokyo University Branch Hospital, University of Tokyo. Acccpted for publication June 16, 1989. Reprint requests: H. Okochi, MD, Departmcnt of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongou, Bunkyo·ku, Tokyo, Japan, 113.
16/1/14957
626
(BMZ). In this report we briefly describe this patient and detail our investigations into the nature of the LPP antigen.
CASE REPORT An 85-year-old Japanese woman was referred to the Department of Dermatology at Tokyo University Branch Hospital on Oct. 8, 1986, because of a bullous eruption. Three months before her hospitalization, she noted pruritic brownish maculopapular lesions on the face and dorsum of the hands. Two months later, blisters began to develop. On admission, a number of tense bullae were symmetrically located on the extremities; a few were also observed on the trunk. The bullae arose on normalappearing skin and on erythematous skin (Fig. I). The patient had Hashimoto's thyroiditis and stomach cancer. A biopsy specimen from a reddish plaque on the dorsal aspect of the right hand showed hyperkeratosis, irregular acanthosis associated with sawtooth elongation of rete ridges and colloid body formation. A cellular infiltrate of lymphocytes, histiocytes, and eosinophils formed a bandlike pattern below the dermoepidermaljunction (Fig. 2). The histologic findings were consistent with LP. A specimen taken from a bullous lesion on normal-appearing skin of the anterior chest wall showed subepidermal blister formation with a moderate dermal infiltrate consisting of lymphocytes, eosinophils, and neutrophils (Fig. 3). These findings were consistent with BP. On direct immunofluorescence, linear IgG deposits were noted in both the bullous and the LP lesions. The
Volume 22 Number 4 April 1990
Lichen planus pemphigoides 627
Fig. 1. Bullae and erosions on left foot.
colloid bodies in the dermis of the LP lesions showed marked fluorescence for IgM. Circulating anti-BMZ antibodies were detected by the indirect immunofluorecence technique with normal human skin sections as substrate ata titer of 1:1280. A diagnosis ofLPP was made, and th~ patient was treated with 0.05% clobetasol-17-propionate ointment. The bullae faded gradually, leaving a brownish erythema at their sites. The patient was almost free of lesions by the end of October 1986, although her anti-BMZ titer remained at 1:2560.
Immunofluorescence studies For skin separation and pretreatment with fixatives, samples of normal skin were obtained. Each sample was divided in half. One half was used for separation of the skin at the dermoepidermal junction. The other half was kept at -70 0 C in ornithine carbamyltransferase (OCT) compound and used for experiments with different fixatives. Sera were obtained from our patient with LPP, from four patients with BP, and from a patient with EBA. Each of these sera was shown to contain IgG-class anti-BMZ antibodies, with normal human skin as substrate. The titers of antibodies were as follows: LPP, 1:1280; BP-l, 1:1280; BP-2, 1:80; BP-3, 1:160; BP-4, 1:2560; and EBA, 1: 1280. Sera from normal subjects were used as controls. Indirect immunofluorescence was performed as described previously.I3 Sera were used at a dilution of 1:20. Fluorescein isothiocyanate-conjugated rabbit antihuman IgG (F(ab)2'-specific) was used at a dilution of 1:40. A 0.5 cm piece was cut from each skin sample, and the subcutaneous fat was scraped off. Two separation methods were used as previously described 13: (1) Pieces of skin were immersed in 1 mol sodium chloride at 4 0 C for 5days as described by Scaletta et a1. 14; or (2) skin was floated in phosphate-buffered saline solution (PBS) at 37 0 C for 5 days as described by Regnier et al. IS The sodium chloride was replaced every 24 hours. After these treatments the epidermis was separated easily from the' dermis. After separation, samples were embedded in OCT compound
Fig. 2. Histologic changes of erythematous plaque on dorsal a~pect of right hand. Hyperkeratosis, irregular acanthosIs, colloid body formation, and bandlike cell infiltration of dermis are noted. (Hematoxylin-eosin stain; XI00.)
and snap-frozen and 6,um sections were processed for indirect immunofluorescence study. Frozen sections (6 ,urn) of each human skin sample were washed for 15 minutes in PBS and then immersed in acetone at -20 0 C, in pure ethanol at room temperature, or in 6% formalin at room temperature, each for 30 minutes. In some experiments sections were immersed in pure ethanol for 10 minutes. The sections were then washed for 20 minutes in PBS and subjected to indirect immunofluorescence. The experiments were performed at least three times, and representative results are presented. To determine the precise localization of the immunoglobulin deposits, we performed indirect immunoelectron microscopy with peroxidase-labeled antibody.16 Peroxidase-labeled goat antihuman IgG (F(abh' -specific) (Cooper Biomedical, Malvern, Pa.) was used. For indirect immunoelectron microscopy, normal human skin was used as substrate and normal human serum was used as a control. RESULTS The results oflocalization of LPP, BP, and EBA antigens in separated skin are summarized in Table I. LPP serum produced a sharp linear band of fluorescence at the epidermal side of the BMZ after
Journal of the American Academy of Dermatology
628 Okochi et al. '
..
9.·' •. ' ..
Fig. 3. Histologic findings of bulla from anterior chest wall. Subepidermal blister with eosinophils, neutrophils, and lymphocytes is noted. (Hematoxylin-eosin stain; XI00.)
Table I. Localization and antigenic stability of LPP, BP, and EBA antigens* Dermoepidermal separation (side) Disease (titer)
PBS
LPP (1:1280) BP-l (1;1280) BP-2 (1:80) BP-3 (1:160) BP-4 (1:2560) EBA (1:1280)
++ (E)
± (E)
+++ (D)
I
Fixation NaCl
None
+++ (E) +++ (E) + (E) + (E) +++ (E) +++ (D)
+++ +++ + + +++ +++
I
Ethanol
I
Acetone
++
+++ +++ + + +++ +++
± ±
+++
I
Formalin
± ± - to ± - to ± ± ±
-, Negative; ±, faint; +, moderate; ++, intense; +++, very intense. D, Dermal side; E, epidermal side. *Results are expressed as intensity of staining evaluated by immunofluorescent microscopy.
Table II. Dilutions of LPP, BP, and EBA sera that still detected LPP, BP, and EBA antigen after fixation of substrate by acetone, ethanol, or formalin Fixation Ethanol Disease
None
10 min
LPP BP-l BP-2 BP-3 BP-4 EBA
1:1280 1:1280 1:80 1:160 1:2560 1:1280
1:1280 1:80 <1:20 <1:20 1:40 1:1280
separation both with sodium chloride and with PBS (Fig. 4). No fluorescence was observed along the dermal side. Each BP serum also produced a linear band of fluorescence on the epidermal side after separation with sodium chloride, but virtually no
I
30 min
Acetone
Formalin
1:320 1:20 <1:20 <1:20 1:40 1:1280
1:1280 1:1280 1:80 1:160 1:2560 1:1280
1:160 1:160 1:20 1:40 1:160 1:160
fluorescence was observed after separation with PBS. EBA serum, however, produced a linear band of fluorescence at the dermal side after both these treatments. The staining patterns were the same in all skin
Volume 22 Number 4 April 1990
Lichen planus pemphigoides 629
Fig. 4. Indirect immunofluorescence staining for LPP antigen in human skin separated by warm PBS (A) and cold sodium chloride (B). Fluorescence is observed on epidermal side of skin. (X400.)
samples tested with each of the sera. N onnal human control sera did not show positive fluorescence. The results of antigenic stability of LPP, BP, and EBA antigens after fixation are summarized in Table 1. After fixation with acetone, all three kinds of antigens were detected as strongly as in unfixed controls. Fixation with formalin reduced the intensity of fluorescence for all these antigens. Fixation with ethanol significantly reduced the intensity of fluorescence for BP antigen, and slightly for LPP antigen, whereas virtually no reduction was noted for EBA antigen. Because a marked difference in antigenic stability was noted after fixation with ethanol among BP, LPP, and EBA antigens, semiquantitative studies were performed by serially titering each serum from I :20 to 1:2560, with human skin as substrate. In addition, we compared the duration of ethanol fixation between 10 minutes and 30 minutes. The results are shown in Table II. The titers ofBP sera were reduced markedly after both ethanol fixations, whereas EBA serum did not show a reduction in titer. The titer of LPP serum was not reduced by 10 minutes of ethanol fixation but was reduced by 30 minutes of ethanol fixation; however, the reduction was not as remarkable as with BP sera.
Fig. 5. Indirect immunoelectron microscopy shows IgG deposits at basal cell hemidesmosomes.. White stars denote immune deposits. BL, Basal lamina; D, dermis; E, epidermis. (X40,OOO.)
Indirect immunoelectron microscopy revealed that the reaction products of LPP antigen (indicating IgG) were observed in basal cell hemidesmosomes (Fig. 5). DISCUSSION
LPP has been considered by IIlost authors to be the coexistence of LP and BP.3-10 In fact, Okada et
Journal of the American Academy of Dermatology
630 Okochi et al. a1.,17 using the serum of a patient with drug-induced LPP, detected a 240 kD antigen identical to BP antigen by immunoprecipitin assay. Lang and Maize, II however, could not detect BP antigen in LPP serum by immunoprecipitin assay. Furthermore, Prost et al. I2 have reported that LPP antigen is not localized along the roof of the bulla as in BP, but is localized instead along the floor. They concluded that LPP antigen might be different from BP antigen. Our investigations revealed the following: LPP antigen was clearly detected on the epidermal side of the BMZ in PBS-treated skin, but BP antigen was not detected. Localization of LPP antigen was similar to that of BP antigen when studied in sodium chloride-treated skin. This finding suggests that LPP antigen is found at the lamina lucida18 or at the basal cell hemidesmosomes. 19 Our immunoelectron microscopic findings revealed that LPP antigen localized to the basal cell hemidesmosomes. On ethanol fixation, EBA antigen was stable whereas BP antigen was unstable even with brief (10 minutes) fixation. Danno et a1. 20 also reported that BP antigen was not detected after ethanol fixation. LPP antigen, however, was detected without reduction ofserum titer after 10 minutes ofethanol fixation but with slight reduction after 30 minutes of ethanol fixation. Thus the sensitivity to ethanol fixation is different between LPP antigen and BP antigen. Because we used the same titer of serum (1:1280) among the three diseases in our experiments, the difference between LPP antigen and BP antigen could not be attributed to the titer of the serum. The molecular weight of BP antigen has been shown to be 220 kD. 21 In one report22 heterogeneous BP antigens ranging in molecular weight from 77 kD to 240 kD were detected. Zhu and Bystryn23 also reported that BP antigens were heterogeneous as assessed byindirectimmunofluorescencetechniques. Possibly some BP antigens are responsible for LPP. These antigens may exhibit different properties than conventional BP antigens, although they show similar localization ultrastructurally. Recently, Davis et a1. 24 reported that the target antigens in LPP included a 180 leD protein that appeared in a minority of cases of BP, in addition to a unique 200 leD protein. They also reported that the target antigen in LPP had an ultrastructural localization similar to that of BP. In summary, our report reveals that the LPP antigen and the BP antigen have different properties.
These findings suggest that LPP may represent a distinct entity and not a simple coexistence ofLP and BP. However, additional studies are necessary. REFERENCES 1. Black MM. Lichen planus and lichenoid eruptions. In: Rook Al, Wilkinson DS, Ebling FJG, et ai, eds. Textbook of dermatology; vol 2. 4th ed. Oxford: Blackwell Scientific, 1986:1682. 2. Schreiner K. Lichen ruber pemphigoides und lichen ruber vesiculosis. Arch Dermatol Syph 1930;161:647-57. 3. Stingl G, Holubar K. Coexistence oflichen planus and bullous pemphigoid: an immunopathological study. Br 1 DermatoI1975;93:313-20. 4. Sobel S, Millar R, Shatin H. Lichen planus pemphigoides: immunofluorescence findings. Arch Dermatol 1976; 112:1280-3. 5. SauratJH, Guinepain MT, Didierjean L, et al. Coexistence d'un lichen plan et d'une pemphigoide bulleuse: etude en immunofluorescence d'nn "lichen pemphigoide." Ann Dermatol VenereoI1977;104:368-74. 6. Morel P, Saurat JH, Civatte 1. Etude en immunofluorescence d'un nouveau cas de lichen plan bulleux. Ann Dermatol VenereoI1978;105:81-2. 7. Hinter H, Tappeiner G, Honigsmann H, et al. Lichen planus and bullous pemphigoid. Acta Derm Venereol (Stockh) 1979;59(suppI89):71-6. 8. Morel P, Perron J, Crickx B, et al. Lichen plan avec depOts lineaires d'IgG et de C3 a la jonction dermoepidermique. Dermatologica 1981;163:117-24. 9. Souteyrand P, Pierini AM, Bussy RF, et al. Lichen planus pemphigoides: entity or association? Dermatologica 1981; 162:414-6. 10. Mora RG, Nesbitt LT, Brantley lB. Lichen planus pemphigoides: clinical and immunofluorescent findings in four cases. 1 AM ACAD DERMATOL 1983;8:331-6. 11. Lang PG, Maize lC. Coexisting lichen planus and bullous pemphigoid or lichen planus pemphigoides? 1 AM ACAD DERMATOL 1983;9:133-40. 12. Prost C, Tesserand F, Laroche L, et al. Lichen planus pemphigoides: an immuno-electron microscopic study. Br 1 DermatoI1985;113:31-6. 13. Fume M, Iwata M, Tamaki K, et al. Anatomical distribution and immunological characteristics of epidermolysis bullosa acquisita antigen and bullous pemphigoid antigen. Br J DermatoI1986;114:651-9. 14. Scaletta Ll, Occhino lC, MacCallum DK, et al. Isolation and immunologic identification of basement membrane zone antigens from human skin. Lab Invest 1978;39:1-9. 15. Regnier M, Prunieras M, Woodley D. Growth and differentiation of adull human epidermal cells on dermal substrate. Front Matrix Bioi 1981;9:4-35. 16. Nakane PK, Pieree GB. Enzyme-labeled antibodies: preparation and application for the localization of antigens. 1 Histochem Cytochem 1966;14:929-31. 17. Okada N, Kitano Y, Miyagawa S, et al. Expression of pemphigoid antigen by SV40-transformed human keralinocytes.l Invest Dermatol 1986;86:399-401. 18. Holubar K, Wolff K, Konrad, et al. Ultrastructural localization of immunoglobulins in bullous pemphigoid skin. 1 Invest Dermatol 1975;64:47-9. 19. Westgate GE, Weaver AC, Couchman JR. Bullous pemphigoid antigen localization suggests an intracellular asso-
Volume 22 Number 4 April 1990
Lichen planus pemphigoides
dation with hemidesmosomes. J Invest DermatoI 1985; 84:218-224. 20. Danno K, Toyama K, Imamura S. Immunofluorescent studies of bullous pemphigoid antigen. Acta Dermatologica 1982;77:25-8 (in Japanese). 21. Stanley JR, Hawley-Nelson P, Yuspa SR, et al. Characterization of bullous pemphigoid antigen: a unique basement membrane protein of stratified squamous epithelia. Cell 1981;24:897-903.
22. Labib RS, Anhalt GJ, Patel HP, et al. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J ImrnunoI1986;136:1231-5. 23. Zhu XJ, Bystryn Jc. Heterogeneity of pemphigoid antigens. J Invest Dermatol 1983;80:16-20. 24. Davis A, Wojnarowska F, Bhogal B, et al. Lichen planus pemphigoides and its relationship to bullous pemphigoid [Abstract). Br J Dermato11989;120:296.
Contact urticaria and anaphylaxis to natural latex C. Pecquet, MD, F. Leynadier, MD, and J. Dry, MD Paris, France Immediate allergic reaction to natural latex of Hevea brasiliensis has been well documented. We report 17 cases, all in WOmen. Twelve had an anaphylactic reaction during surgery and five had contact urticaria. Reactions to prick tests done through a washed surgical glove and with natural latex were positive in 15 of 17 patients and in 16 of 16 patients, respectively; specific IgE was found in 12 of 17 patients. In 206 control subjects, results ofa prick test through a surgical glove were negative. (J AM ACAD DERMATOL 1990;22:631-3.)
Since the first case of contact urticaria from natural rubber gloves was observed by Nutter l in 1979, approximately 60 cases have been reported. Natural latex is used in the manufacture of surgical gloves, rubber cleaning gloves, balloons, dental dams, and contraceptives, all of which may induce sensitization. The symptoms are usually limited to localized itching and redness but severe systemicreactions can occur. 2 Specific IgE antibodies to latex rubber have been demonstrated. 3 During the past 20 months we have encountered 17 patients with latex-induced contact urticaria. PATIENTS AND METHODS
Seventeen patients with a reaction to natural rubber gloves were examined. Twelve had an anaphylactic reaction during surgery and five had contact urticaria. Prick tests were performed in all patients on the skin of the forearm with disposable needles (Stallerpoint, Labo-
From the Department of Internal Medicine and the Center for Anergy, Hopital Rothschild. Accepted for publication June 16, 1989. Reprint requests: Dr. C, Pecquet, Service du Pr, Dry., Hopital Rothschild, 33 BId. de Picpus, 75012 Paris, France. 16/1/14958
ratoire des Stallergenes, Fresnes, France), through a washed latex surgical glove (Assistance Publique Paris), and with natural latex from the Ivory Coast without accelerators. The natural latex was filtered on Millipore (Molsheim, France). Onemilliliter ofthe filtered solution was diluted in 9 ml of phenolated saline solution. A dilution of 1: 1000 was used for the skin prick test; if the results were negative, 1:100 was used. The mean diameters of the wheals and flares were measured and compared with those ofthe positive control test (9% codeine phosphate) and with those of the negative control test (saline solution). Levels of total 19B and specific 19B to latex in the serum were determined with the Phadezym technique (Pharmacia, St. Quentin en Yvelines, France). Specific 19B values were expressed in radioallergosorbent test score. Skin prick tests were performed with the same protocol in 206 control subjects. j
RESULTS
Seventeen women reported contact urticaria to latex. Their mean age was 35 years (range 26 to 56 years). Daily occupational contact with natural rubber gloves was determined in 10 patients. The contact urticaria to latex had been present from 2 months to more than 12 years. When latex gloves were worn, itching, redness, and/or swelling of the hands began within 1 to 60 minutes (average 15
631