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Ultrastructural localization of immunoglobulin and fibrin in lichen planus
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Ultrastructural localization of immunoglobulin and fibrin in lichen planus K. Konrad, M.D., H. Pehamberger, M.D., and K. Holubar, M.D.
Vienna, Austria To demonstrate the exact localization of immunoglobulin and fibrin deposits in skin lesions of lichen planus, we employed the peroxidaseantiperoxidase technique, both light and electron microscopically. IgM is shown to be deposited preferentially in and on the cytoid bodies all over their cut surface in a typical stippled pattern, partly obscuring the characteristic fibrillar substructure. All other structures such as the basal lamina are devoid of IgM deposits. Fibrin (F) is also present in cytoid bodies but in a lesser amount; it is characteristically deposited on and along the basal lamina and its reduplications in the uppermost strata of the dermis. Our results indicate that IgM deposition in cytoid bodies occurs at a very early stage during the formation of these structures and that fibrinogen exhibits a specific affinity not only to cytoid bodies but also to basal lamina material. (J AM ACAODE•MATOL 1:233-239, 1979.)
In lichen planus, immunogtobulin (Ig) deposits can be demonstrated, by direct immunofluorescence, in so-called cytoid bodies (CB) located in small groups in the uppermost layers of the dermis and in the basal layer of the epidermis. The CB belong predominantly to the IgM class. IgG, IgA, and fibrin can, however, also be demonstrated in these structures. ''~ In addition, fibrin (F) deposits are observed in an almost bandlike pattern along the dermoepidermal junction and extending as irregular strands into the upper dermis. s5 Since the significance of these findings for the pathogenesis of lichen planus has not yet been established and an exact correlation of these deposits with preexisting structures is not possible by means of direct immunofluorescence, we decided to investigate the fine structural localization of Ig From the Department of Dermatology (I), University of Vienna. Supported, in part, by a grant fl'om Schering AG, Berlin. Reprint requests to: Doz. Klaus Konrad, M.D., Department of Dermatology (I), University of Vienna, Alserstrasse 4, A1090, Vienna, Austria. 0190-9622/79/030233+07500.70/0 9 1979 Am Acad Dermatol
and F deposits in lichen planus skin by employing the peroxidase-antiperoxidase technique, ~ both light and electron microscopically.
MATERIAL AND METHODS Patients Three patients with typical clinical picture and histologic findings of lichen planus were investigated. Biopsies were obtained from lesional skin under local anesthesia by surgical excision for routine histopathology and immunofluorescence and by shave biopsy with a razor blade for immunoelectron microscopy. Direct immunofluorescence Tissue specimens were snap-frozen in liquid nitrogen and processed for the detection of in vivobound IgG, IgM, IgA, C3, and F with the use of standard methods. 7 Peroxidase-antiperoxidase technique The preparation and incubation procedure for the detection of Ig and C3 by light and electron 233
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Fig. 1. Direct immunofluorescence, a, IgM. Numerous cytoid bodies exhibiting bright fluorescence along the BMZ. (Original magnification, x400.) b, Fibrin. Irregular bandlike fluorescence in the BMZ and a few cytoid bodies intraepidermally. (Original magnification, x250.) c, Fibrin. Bandlike fluorescence pattern along the basement membrane zone with irregular strands extending into the upper papillary dermis. (Original magnification, x250.)
microscopy has been described previously. 6 For the demonstration of F depositions, a modification of the incubation chain was employed. Due to the rabbit origin of the F antiserum, a goat antirabbit serum was used as bridging antibody to bind the rabbit anti-F (first step) to the rabbit antigoat serum (third step). Controls. Appropriate controls as outlined in a previous paper s were done, including the use of normal goat or rabbit sera as a first step of the immunologic reaction chain.
Antisera All conjugates used for immunofluorescence and peroxidase-antiperoxidase technique were obtained commercially from Hyland Laboratories, Costa Mesa, C A (goat antihuman IgG, specific antibody concentration ( s A B c ) = 2.0 mg/ml, final concentration ( f c ) = a p p r o x i m a t e l y 100 /zg/ml; goat antihuman IgA, sABc = 2.0 mg/ml, f c = approximately 100 ~g/ml; goat antihuman lgM, sABc = 2.3 mg/ml, fc = approximately 130/zg/ml; goat antihuman C3, s A B c = 1.8 mg/ml, f c = a p proximately 90/~g/ml; rabbit antifibrin, total protein concentration, 13.3 mg/ml). The properties of the antisera and the cytochemical reagents have been described previously. ~
RESULTS Direct immunofluorescence Antihuman IgM antiserum, lgM deposits were found in the form of numerous CB with bright
fluorescence along the basement membrane zone (BMZ) (Fig. 1, a). The CB varied in number and size. Usually they appeared to be the size of basal cells, and only a small number of them were of larger size. The majority of the CB were located in the upper part of the dermis adjacent to the BMZ or in the dermal papillae distributed in small clusters or paralleling the course of the basal lamina. Occasionally, they were found singly in the basal layer of the epidermis. IgG and IgA were also present in the CB but in lesser amounts and exhibiting weak fluorescence only. Fibrin. F deposition occurred in a bandlike pattern along the BMZ with irregular strands extending from the bandlike deposition in the BMZ into the upper papillary dermis. In addition, F deposits were found in CB within the epidermis and in the uppermost dermis in the same distribution as IgM but in a smaller number and exhibiting weaker fluorescent intensity (Fig. 1, b and c).
Peroxidase-antiperoxidase technique, light microscopy IgM and F deposits could easily be identified by the presence of the dark brown reaction product. The localization and distribution of the reaction product were identical to results of direct immunofluorescence (Fig. 2). Due to the slight brownish background staining of the epidermal cells and of some dermal fibrillar components, the exact localization of the reaction product can be seen better than in direct immunofluoreseence.
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Fig. 2. Peroxidase-antiperoxidase technique, light microscopy, a, IgM. Reaction product on the cytoid bodies. Identical pattern as in Fig. 1, a. (Original magnification, x400.) b, Fibrin. Reaction product in bandlike arrangement along the dermoepidermal junction and in parallel lines in the uppermost dermis (arrows). (Original magnification, x400.)
The F deposits could be identified more clearly as irregular lines running parallel to the basal lamina (Fig. 2, b).
Peroxidase-antiperoxidase technique, electron microscopy The reaction product indicating in vivo deposition of IgM or F appeared as dense precipitate and therefore could easily be identified. Due to the many incubation procedures, tissue fixation was delayed, and, thus, the preservation of the tissue was below the standard for routine electron microscopy; nevertheless, the subcellular details were still well preserved and easily identifiable. IgM. The CB in the papillary dermis, in the BMZ, and also in the epidermis preferentially exhibited the reaction product. All other structures, such as the basal lamina, were devoid of electrondense deposits (Fig. 3, a). The reaction product was uniformly distributed all over the CB areas; it formed small, round dots of almost equal size between and upon the characteristic fibrillar structures of the CB that were partly obscured but still recognizable (Fig. 3, b). The CB therefore exhibited a typical stippled appearance (Fig. 3, a). There were no definite differences in the amount or in the distribution of the reaction product in CB in the epidermis or in the dermis, although the amount of deposits varied from one CB to another. Fibrin, The reaction product was found in CB and along the basal lamina and its reduplications. Only part of the CB exhibited F depositions. Some
of them were completely free. The amount of the reaction product which was seen in one single CB varied considerably (Fig. 4, a). Again, as described for the IgM deposits, the reaction product was almost uniformly distributed all over the cut surface of the CB. The fine-fibrillar structural details of the CB were easily identifiable in all instances (Fig. 4, b). The reaction product could be seen as small dots and also as thin, short strands which appeared partly interconnected (Fig. 4, b). A more intensive staining by the reaction product could be seen in an almost bandlike pattern in the BMZ. Fibrin was located selectively on and below the basal lamina and also on the multiple reduplications of the basal lamina that were present in the uppermost strata of the papillary dermis, running parallel to each other with reaction product densely aggregated along the undulating course of the basal lamina (Fig. 4, a). In some instances, the newly formed basal lamina below regenerating keratinocytes was free of F deposits, the reaction product being present only along the reduplications of the basal lamina further down in the dermis. All other structures of the BMZ were fi'ee of reaction product. Controls. Control specimens (Fig. 3, c) incubated with phosphate-buffered saline instead of the anti-IgM or antifibrin serum in the first step of the reaction chain were completely devoid of peroxidase reaction product. The CB exhibited their characteristic fibrillar substructure as shown in Fig. 3, c.
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Fig. 3. Peroxidase-antiperoxidase technique, electron microscopy. IgM. a, The reaction product is present in a stippled pattern all over the cut surface of the cytoid body in the uppermost dermis. The basal lamina(arrows) and a reduplication of the basal lamina below the CB (double arrow) are free of reaction product. K, Keratinocyte. (Original magnification, x 12,000.) b, Higher magnification of CB shown in a. The reaction product forms small dots (arrows) between and upon the characteristic fibrillar substructures of the CB. (Original magnification, x75,000.) c, Control specimens. Incubation in antMgM conjugate was omitted. Note the typical fine filaments of a CB without the reaction product. Compare with b. (Original magnification, x75,000.) DISCUSSION Eosinophilic, periodic acid-Schiff (PAS)-positire, round structures, approximately the size of epidermal keratinocytes, can be found quite frequently at the dermoepidermal junction in skin lesions of lichen planus. T h e y are synonymously
called CB, colloid bodies, hyaline bodies, or Civatte bodies. 'a It has been shown by electron microscopy that these CB consist of a dense meshwork of 70 A filaments that most likely correspond to the tonofilaments in epidermal keratinocytes. 1~ It is, therefore, the general belief
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Fig. 4. Peroxidase-antiperoxidase technique. Fibrin. a, Lower magnification of the dermoepidermal junction. Reaction product is present in variable amounts on the CB (l, 2, 3) and also in an undiluting bandlike pattern in the basal lamina and its reduplications in the uppermost dermis (arrows); K, keratinocytes; A, artifacts due to the delayed fixation of the tissue after completion of the immunologic incubation procedures; D, dermis. (Original magnification, x5,400.) b, Higher magnification of CB 2 in a. Reaction product appearing as small dots and as interconnected thin, short strands (arrows). (Original magnification, x23,800.) that they represent degenerated, dyskeratotic basal keratinocytes that are eliminated into the uppermost strata of the papillary dermis. 1~a4 This downward movement and extrusion of damaged keratinocytes from the epidermis into the dermis is also called "apoptosis" according to a suggestion by Kerr, Wyllie, and Currie in 1972. m CB are characteristic but not pathognomonic for lichen planus; they can be seen also in many other skin diseases in which damage to the basal cells occurs? ,'~ It has been shown by immunofluorescence that Ig, especially IgM and to a lesser degree also IgG and IgA,
are deposited in these CB.1,2,'~ lmmunoelectron microscopic investigations disclose that not only are the IgM deposits present on the surface of the CB but also that the entire structure, even the centrally located parts in cross sections of CB, exhibits evenly distributed deposits of IgM. Since a penetration of the Ig molecules into a fully developed CB with its densely interwoven masses of fine fibrils seems to be very unlikely, we have to assume that the deposition of the IgM in CB occurs at a very early stage during the formation of these structures. Recently, SumegP ~ demonstrated, by
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histochemical methods, various paraproteins in necrotic epithelial cells of the liver, around malignant tumors o f various organs, 17 and also in the skin of lichen p l a n u s ? s He found fibrinoid necrosis and keratinization in necrotic keratinocytes and suggested the term "keratofibriuoid" for the pathologic substance that is present in C B ? 8 This mixture o f proteins and keratin in the CB is welt in accordance with our findings. It should be pointed out that we were unable to detect IgM deposits in association with the basal lamina, as has been described b y Baart de la Faille-Kuyper. z This further supports the findings of Abbel et al. z In addition to the IgM deposits, fibrinogen and F can regularly be detected b y immunofluorescence in lichen planus skin lesions, both in association with CB and also in a more bandlike pattern along the BMZ. 3,5 The presence of F could further be substantiated by an immunohistochemical technique using a peroxidase anti-F conjugate? 9 By routine electron microscopy, these F depositions have been shown to be localized on the surface and within the CB, bet w e e n collagen bundles and also following the BMZ. 4 Our results confirm these previous observations and, in addition, provide further information on the exact localization of F in lichen planus skin lesions. O f special interest seems to be the fact that we could regularly observe a selective deposition of F in a linear arrangement below the basal lamina and its reduplications in the u p p e r m o s t strata of the dermis. This indicates that there is some affinity of the fibrinogen to basal lamina material, which presents evidence against the assumption that the polymerization of fibrinogen takes place unspecifically after contact with any preexisting structures such as collagen bundles, elastic fibers, or cell surface. The number of CB with F deposition is definitely smaller than with IgM deposits. This difference can be o b s e r v e d by immunofluorescence and by the peroxidase-ant[peroxidase technique, both at the light microscopic and at the ultrastructural levels. Th e am ount of reaction product changes very much from one CB to another. The pattern of F deposition in the CB is almost identical as compared to deposition of IgM. The reaction product again appears both on the surface and within the central parts o f these structures. Our findings support the pathogenetic concept that a damage to the basal layer of the epidermis, resulting both in the formation of CB and in fragmentation and reduplication of the basal lamina, is
the basic underlying process in lichen planus. 1~,14 What really initiates this process, the deposition of IgM and/or of F onto or into the basal cells (as a consequence of either circulatory disturbances leading to hypoxia-anoxia of the basal epidermal cells is or a primary injury in the nuclei of the keratinocytes with liberation of an antigen and thus initiation of a cell-mediated delayed type of hypersensitivity reaction), 2~ cannot be concluded on the basis of our results in fully developed lichen ptanus lesions. It will be interesting to investigate experimentally induced lichen planus lesions (for example, by K6bner's phenomenon) to study in detail the sequence of both the morphologic changes and the IgM and F depositions in very early lesions. It will also be interesting to investigate lichenoid eruptions in graft-vs-host disease. The technical assistance of Mrs. Steffi Pogantsch, Mrs. Lotte Polasek, Mrs. Susan Csegezi, and Mr. GfJnther Raffesberg is gratefully acknowledged. REFERENCES
1. UekiH: Hyalinebodies in subepidermalpapillae: Immunohistochcmical studies in several dermatoses. Arch Dermatol 100:610-617, 1969. 2. Baartde la Faille-KuyperEH, Baart de la Fai|le-KuyperH: An immunofluorescentstudy of lichenplanus. Br J Dermatol 90:365-371, 1974. 3. Abbel E, Presbury DGC, Marks R, Ramnarain D: The diagnostic significanceof immunoglobulinand fibrindeposition in lichen planus. Br J Dermatol 93:17-24, 1975. 4. EbnerH, Kraft D: Fibrinablagerungenbeim Lichen ruber planus. Arch Dermatol Res 243:305-317, 1972. 5. Michel B, Erlinda KS: Tissue fixed immunoglobulins in lichen planus, in Beutner EH, Chorzelski TP, Bean SF, Jordon RE, editors: Immunopathology of the skin. Stroudsburg, PA, 1973, Dowden, Hutchinson and Ross, Inc., pp. 182-193. 6. Ho|ubar K, WolffK, Konrad K, Beutner EH: Ultrastructural localization of immunogiobulins in buUous pemphigold skin. J Invest Dermatol 64:220-227, 1975. 7. Beutner EH, Nisengard RJ, Hale MA, Chorzelski TP, Holubar K: Defined immunofluorescencein clinical immunopathology,in BeutnerEH, ChorzelskiTP, Bean SF, Jordon RE, editors: Immunopathology of the skin. Stroudsburg, PA, 1973, Dowden, Hutchinson and Ross, Inc., pp. 197-247. 8. PehambergerH, Konrad K, Holubar K: Immunoelectron microscopyof skin in lupus erythematosus.J Cutan Pathol 5:319-328, I978. 9. Gebhart W: Zytoide K6rperchen in der menschlichen Haut. Wien Klin Wochenschr (Suppl) 88(60):1-24, 1976. 10. E1 Labban NG: Light and electron microscopicstudies of colloid bodies in lichen planus. Periodont Res 2:315-324, 1970.
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11. Ebner H, Gebhart W: Beitrag zur Histochemie und Ultrastruktur der sogenannten hyalinen bzw, kolloiden K6rperehen. Arch Dermatol Res 242:153-164, 1972. 12. Anton Lamprecht J, Tilgen W: Zur Entstehung und Tonofibrillennatur tier fibrillgxen K6rper. ArcJi Dermatol Res 246:317-327, 1973. 13. Ebner H, Gebhart W: Epidermal changes in lichen planus. J Cutan Pathol 3:167-174, 1976. 14. Eady RAJ, Cowen T: Half- and half-cells in lichen planus. Br J Dermatol 98:417-423, 1978. 15. Kerr JF, Wyllie AH, Currie AR: Apoptosis. A basic phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26:239-257, 1972.
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16. Sumegi J: Fibrinoid necrosis of liver parenchymal cells. Acta Pathol Microbiol Stand 83:109-119, 1975. 17. Sumegi J, Rajka G: Amyloldlike substance surrounding mammary cancer and basal cell carcinoma. Acta Pathol Microbiol Scand 80:185-192, 1972. 18. Sumegi J: Fibrinoid necrosis and downward motion of colloid bodies in lichen planus (apoptosis). Acta Derm Venereol (Stockh) 59:27-31, 1979. 19. Ebner H, Kraft D: Fibrinnachweis mit einem EnzymAntiserum-Konjugat beim Lichen tuber planus. Dermatologica 146:101-106, 1973. 20. Medenica M, Lorincz A: Lichen planus: An ultrastructural study. Acta Derm Venereol (Stockh) 57:55-62, 1977.
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Ultrastructural localization of immunoglobulin and fibrin in lichen planus K. Konrad, M.D., H. Pehamberger, M.D., and K. Holubar, M.D.
Vienna, Austria To demonstrate the exact localization of immunoglobulin and fibrin deposits in skin lesions of lichen planus, we employed the peroxidaseantiperoxidase technique, both light and electron microscopically. IgM is shown to be deposited preferentially in and on the cytoid bodies all over their cut surface in a typical stippled pattern, partly obscuring the characteristic fibrillar substructure. All other structures such as the basal lamina are devoid of IgM deposits. Fibrin (F) is also present in cytoid bodies but in a lesser amount; it is characteristically deposited on and along the basal lamina and its reduplications in the uppermost strata of the dermis. Our results indicate that IgM deposition in cytoid bodies occurs at a very early stage during the formation of these structures and that fibrinogen exhibits a specific affinity not only to cytoid bodies but also to basal lamina material. (J AM ACAODE•MATOL 1:233-239, 1979.)
In lichen planus, immunogtobulin (Ig) deposits can be demonstrated, by direct immunofluorescence, in so-called cytoid bodies (CB) located in small groups in the uppermost layers of the dermis and in the basal layer of the epidermis. The CB belong predominantly to the IgM class. IgG, IgA, and fibrin can, however, also be demonstrated in these structures. ''~ In addition, fibrin (F) deposits are observed in an almost bandlike pattern along the dermoepidermal junction and extending as irregular strands into the upper dermis. s5 Since the significance of these findings for the pathogenesis of lichen planus has not yet been established and an exact correlation of these deposits with preexisting structures is not possible by means of direct immunofluorescence, we decided to investigate the fine structural localization of Ig From the Department of Dermatology (I), University of Vienna. Supported, in part, by a grant fl'om Schering AG, Berlin. Reprint requests to: Doz. Klaus Konrad, M.D., Department of Dermatology (I), University of Vienna, Alserstrasse 4, A1090, Vienna, Austria. 0190-9622/79/030233+07500.70/0 9 1979 Am Acad Dermatol
and F deposits in lichen planus skin by employing the peroxidase-antiperoxidase technique, ~ both light and electron microscopically.
MATERIAL AND METHODS Patients Three patients with typical clinical picture and histologic findings of lichen planus were investigated. Biopsies were obtained from lesional skin under local anesthesia by surgical excision for routine histopathology and immunofluorescence and by shave biopsy with a razor blade for immunoelectron microscopy. Direct immunofluorescence Tissue specimens were snap-frozen in liquid nitrogen and processed for the detection of in vivobound IgG, IgM, IgA, C3, and F with the use of standard methods. 7 Peroxidase-antiperoxidase technique The preparation and incubation procedure for the detection of Ig and C3 by light and electron 233
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Fig. 1. Direct immunofluorescence, a, IgM. Numerous cytoid bodies exhibiting bright fluorescence along the BMZ. (Original magnification, x400.) b, Fibrin. Irregular bandlike fluorescence in the BMZ and a few cytoid bodies intraepidermally. (Original magnification, x250.) c, Fibrin. Bandlike fluorescence pattern along the basement membrane zone with irregular strands extending into the upper papillary dermis. (Original magnification, x250.)
microscopy has been described previously. 6 For the demonstration of F depositions, a modification of the incubation chain was employed. Due to the rabbit origin of the F antiserum, a goat antirabbit serum was used as bridging antibody to bind the rabbit anti-F (first step) to the rabbit antigoat serum (third step). Controls. Appropriate controls as outlined in a previous paper s were done, including the use of normal goat or rabbit sera as a first step of the immunologic reaction chain.
Antisera All conjugates used for immunofluorescence and peroxidase-antiperoxidase technique were obtained commercially from Hyland Laboratories, Costa Mesa, C A (goat antihuman IgG, specific antibody concentration ( s A B c ) = 2.0 mg/ml, final concentration ( f c ) = a p p r o x i m a t e l y 100 /zg/ml; goat antihuman IgA, sABc = 2.0 mg/ml, f c = approximately 100 ~g/ml; goat antihuman lgM, sABc = 2.3 mg/ml, fc = approximately 130/zg/ml; goat antihuman C3, s A B c = 1.8 mg/ml, f c = a p proximately 90/~g/ml; rabbit antifibrin, total protein concentration, 13.3 mg/ml). The properties of the antisera and the cytochemical reagents have been described previously. ~
RESULTS Direct immunofluorescence Antihuman IgM antiserum, lgM deposits were found in the form of numerous CB with bright
fluorescence along the basement membrane zone (BMZ) (Fig. 1, a). The CB varied in number and size. Usually they appeared to be the size of basal cells, and only a small number of them were of larger size. The majority of the CB were located in the upper part of the dermis adjacent to the BMZ or in the dermal papillae distributed in small clusters or paralleling the course of the basal lamina. Occasionally, they were found singly in the basal layer of the epidermis. IgG and IgA were also present in the CB but in lesser amounts and exhibiting weak fluorescence only. Fibrin. F deposition occurred in a bandlike pattern along the BMZ with irregular strands extending from the bandlike deposition in the BMZ into the upper papillary dermis. In addition, F deposits were found in CB within the epidermis and in the uppermost dermis in the same distribution as IgM but in a smaller number and exhibiting weaker fluorescent intensity (Fig. 1, b and c).
Peroxidase-antiperoxidase technique, light microscopy IgM and F deposits could easily be identified by the presence of the dark brown reaction product. The localization and distribution of the reaction product were identical to results of direct immunofluorescence (Fig. 2). Due to the slight brownish background staining of the epidermal cells and of some dermal fibrillar components, the exact localization of the reaction product can be seen better than in direct immunofluoreseence.
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Fig. 2. Peroxidase-antiperoxidase technique, light microscopy, a, IgM. Reaction product on the cytoid bodies. Identical pattern as in Fig. 1, a. (Original magnification, x400.) b, Fibrin. Reaction product in bandlike arrangement along the dermoepidermal junction and in parallel lines in the uppermost dermis (arrows). (Original magnification, x400.)
The F deposits could be identified more clearly as irregular lines running parallel to the basal lamina (Fig. 2, b).
Peroxidase-antiperoxidase technique, electron microscopy The reaction product indicating in vivo deposition of IgM or F appeared as dense precipitate and therefore could easily be identified. Due to the many incubation procedures, tissue fixation was delayed, and, thus, the preservation of the tissue was below the standard for routine electron microscopy; nevertheless, the subcellular details were still well preserved and easily identifiable. IgM. The CB in the papillary dermis, in the BMZ, and also in the epidermis preferentially exhibited the reaction product. All other structures, such as the basal lamina, were devoid of electrondense deposits (Fig. 3, a). The reaction product was uniformly distributed all over the CB areas; it formed small, round dots of almost equal size between and upon the characteristic fibrillar structures of the CB that were partly obscured but still recognizable (Fig. 3, b). The CB therefore exhibited a typical stippled appearance (Fig. 3, a). There were no definite differences in the amount or in the distribution of the reaction product in CB in the epidermis or in the dermis, although the amount of deposits varied from one CB to another. Fibrin, The reaction product was found in CB and along the basal lamina and its reduplications. Only part of the CB exhibited F depositions. Some
of them were completely free. The amount of the reaction product which was seen in one single CB varied considerably (Fig. 4, a). Again, as described for the IgM deposits, the reaction product was almost uniformly distributed all over the cut surface of the CB. The fine-fibrillar structural details of the CB were easily identifiable in all instances (Fig. 4, b). The reaction product could be seen as small dots and also as thin, short strands which appeared partly interconnected (Fig. 4, b). A more intensive staining by the reaction product could be seen in an almost bandlike pattern in the BMZ. Fibrin was located selectively on and below the basal lamina and also on the multiple reduplications of the basal lamina that were present in the uppermost strata of the papillary dermis, running parallel to each other with reaction product densely aggregated along the undulating course of the basal lamina (Fig. 4, a). In some instances, the newly formed basal lamina below regenerating keratinocytes was free of F deposits, the reaction product being present only along the reduplications of the basal lamina further down in the dermis. All other structures of the BMZ were fi'ee of reaction product. Controls. Control specimens (Fig. 3, c) incubated with phosphate-buffered saline instead of the anti-IgM or antifibrin serum in the first step of the reaction chain were completely devoid of peroxidase reaction product. The CB exhibited their characteristic fibrillar substructure as shown in Fig. 3, c.
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Fig. 3. Peroxidase-antiperoxidase technique, electron microscopy. IgM. a, The reaction product is present in a stippled pattern all over the cut surface of the cytoid body in the uppermost dermis. The basal lamina(arrows) and a reduplication of the basal lamina below the CB (double arrow) are free of reaction product. K, Keratinocyte. (Original magnification, x 12,000.) b, Higher magnification of CB shown in a. The reaction product forms small dots (arrows) between and upon the characteristic fibrillar substructures of the CB. (Original magnification, x75,000.) c, Control specimens. Incubation in antMgM conjugate was omitted. Note the typical fine filaments of a CB without the reaction product. Compare with b. (Original magnification, x75,000.) DISCUSSION Eosinophilic, periodic acid-Schiff (PAS)-positire, round structures, approximately the size of epidermal keratinocytes, can be found quite frequently at the dermoepidermal junction in skin lesions of lichen planus. T h e y are synonymously
called CB, colloid bodies, hyaline bodies, or Civatte bodies. 'a It has been shown by electron microscopy that these CB consist of a dense meshwork of 70 A filaments that most likely correspond to the tonofilaments in epidermal keratinocytes. 1~ It is, therefore, the general belief
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Fig. 4. Peroxidase-antiperoxidase technique. Fibrin. a, Lower magnification of the dermoepidermal junction. Reaction product is present in variable amounts on the CB (l, 2, 3) and also in an undiluting bandlike pattern in the basal lamina and its reduplications in the uppermost dermis (arrows); K, keratinocytes; A, artifacts due to the delayed fixation of the tissue after completion of the immunologic incubation procedures; D, dermis. (Original magnification, x5,400.) b, Higher magnification of CB 2 in a. Reaction product appearing as small dots and as interconnected thin, short strands (arrows). (Original magnification, x23,800.) that they represent degenerated, dyskeratotic basal keratinocytes that are eliminated into the uppermost strata of the papillary dermis. 1~a4 This downward movement and extrusion of damaged keratinocytes from the epidermis into the dermis is also called "apoptosis" according to a suggestion by Kerr, Wyllie, and Currie in 1972. m CB are characteristic but not pathognomonic for lichen planus; they can be seen also in many other skin diseases in which damage to the basal cells occurs? ,'~ It has been shown by immunofluorescence that Ig, especially IgM and to a lesser degree also IgG and IgA,
are deposited in these CB.1,2,'~ lmmunoelectron microscopic investigations disclose that not only are the IgM deposits present on the surface of the CB but also that the entire structure, even the centrally located parts in cross sections of CB, exhibits evenly distributed deposits of IgM. Since a penetration of the Ig molecules into a fully developed CB with its densely interwoven masses of fine fibrils seems to be very unlikely, we have to assume that the deposition of the IgM in CB occurs at a very early stage during the formation of these structures. Recently, SumegP ~ demonstrated, by
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Journal of the American Academy of Detanatology
K o n r a d et a l
histochemical methods, various paraproteins in necrotic epithelial cells of the liver, around malignant tumors o f various organs, 17 and also in the skin of lichen p l a n u s ? s He found fibrinoid necrosis and keratinization in necrotic keratinocytes and suggested the term "keratofibriuoid" for the pathologic substance that is present in C B ? 8 This mixture o f proteins and keratin in the CB is welt in accordance with our findings. It should be pointed out that we were unable to detect IgM deposits in association with the basal lamina, as has been described b y Baart de la Faille-Kuyper. z This further supports the findings of Abbel et al. z In addition to the IgM deposits, fibrinogen and F can regularly be detected b y immunofluorescence in lichen planus skin lesions, both in association with CB and also in a more bandlike pattern along the BMZ. 3,5 The presence of F could further be substantiated by an immunohistochemical technique using a peroxidase anti-F conjugate? 9 By routine electron microscopy, these F depositions have been shown to be localized on the surface and within the CB, bet w e e n collagen bundles and also following the BMZ. 4 Our results confirm these previous observations and, in addition, provide further information on the exact localization of F in lichen planus skin lesions. O f special interest seems to be the fact that we could regularly observe a selective deposition of F in a linear arrangement below the basal lamina and its reduplications in the u p p e r m o s t strata of the dermis. This indicates that there is some affinity of the fibrinogen to basal lamina material, which presents evidence against the assumption that the polymerization of fibrinogen takes place unspecifically after contact with any preexisting structures such as collagen bundles, elastic fibers, or cell surface. The number of CB with F deposition is definitely smaller than with IgM deposits. This difference can be o b s e r v e d by immunofluorescence and by the peroxidase-ant[peroxidase technique, both at the light microscopic and at the ultrastructural levels. Th e am ount of reaction product changes very much from one CB to another. The pattern of F deposition in the CB is almost identical as compared to deposition of IgM. The reaction product again appears both on the surface and within the central parts o f these structures. Our findings support the pathogenetic concept that a damage to the basal layer of the epidermis, resulting both in the formation of CB and in fragmentation and reduplication of the basal lamina, is
the basic underlying process in lichen planus. 1~,14 What really initiates this process, the deposition of IgM and/or of F onto or into the basal cells (as a consequence of either circulatory disturbances leading to hypoxia-anoxia of the basal epidermal cells is or a primary injury in the nuclei of the keratinocytes with liberation of an antigen and thus initiation of a cell-mediated delayed type of hypersensitivity reaction), 2~ cannot be concluded on the basis of our results in fully developed lichen ptanus lesions. It will be interesting to investigate experimentally induced lichen planus lesions (for example, by K6bner's phenomenon) to study in detail the sequence of both the morphologic changes and the IgM and F depositions in very early lesions. It will also be interesting to investigate lichenoid eruptions in graft-vs-host disease. The technical assistance of Mrs. Steffi Pogantsch, Mrs. Lotte Polasek, Mrs. Susan Csegezi, and Mr. GfJnther Raffesberg is gratefully acknowledged. REFERENCES
1. UekiH: Hyalinebodies in subepidermalpapillae: Immunohistochcmical studies in several dermatoses. Arch Dermatol 100:610-617, 1969. 2. Baartde la Faille-KuyperEH, Baart de la Fai|le-KuyperH: An immunofluorescentstudy of lichenplanus. Br J Dermatol 90:365-371, 1974. 3. Abbel E, Presbury DGC, Marks R, Ramnarain D: The diagnostic significanceof immunoglobulinand fibrindeposition in lichen planus. Br J Dermatol 93:17-24, 1975. 4. EbnerH, Kraft D: Fibrinablagerungenbeim Lichen ruber planus. Arch Dermatol Res 243:305-317, 1972. 5. Michel B, Erlinda KS: Tissue fixed immunoglobulins in lichen planus, in Beutner EH, Chorzelski TP, Bean SF, Jordon RE, editors: Immunopathology of the skin. Stroudsburg, PA, 1973, Dowden, Hutchinson and Ross, Inc., pp. 182-193. 6. Ho|ubar K, WolffK, Konrad K, Beutner EH: Ultrastructural localization of immunogiobulins in buUous pemphigold skin. J Invest Dermatol 64:220-227, 1975. 7. Beutner EH, Nisengard RJ, Hale MA, Chorzelski TP, Holubar K: Defined immunofluorescencein clinical immunopathology,in BeutnerEH, ChorzelskiTP, Bean SF, Jordon RE, editors: Immunopathology of the skin. Stroudsburg, PA, 1973, Dowden, Hutchinson and Ross, Inc., pp. 197-247. 8. PehambergerH, Konrad K, Holubar K: Immunoelectron microscopyof skin in lupus erythematosus.J Cutan Pathol 5:319-328, I978. 9. Gebhart W: Zytoide K6rperchen in der menschlichen Haut. Wien Klin Wochenschr (Suppl) 88(60):1-24, 1976. 10. E1 Labban NG: Light and electron microscopicstudies of colloid bodies in lichen planus. Periodont Res 2:315-324, 1970.
Volume 1 Number 3 September, 1979
11. Ebner H, Gebhart W: Beitrag zur Histochemie und Ultrastruktur der sogenannten hyalinen bzw, kolloiden K6rperehen. Arch Dermatol Res 242:153-164, 1972. 12. Anton Lamprecht J, Tilgen W: Zur Entstehung und Tonofibrillennatur tier fibrillgxen K6rper. ArcJi Dermatol Res 246:317-327, 1973. 13. Ebner H, Gebhart W: Epidermal changes in lichen planus. J Cutan Pathol 3:167-174, 1976. 14. Eady RAJ, Cowen T: Half- and half-cells in lichen planus. Br J Dermatol 98:417-423, 1978. 15. Kerr JF, Wyllie AH, Currie AR: Apoptosis. A basic phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26:239-257, 1972.
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16. Sumegi J: Fibrinoid necrosis of liver parenchymal cells. Acta Pathol Microbiol Stand 83:109-119, 1975. 17. Sumegi J, Rajka G: Amyloldlike substance surrounding mammary cancer and basal cell carcinoma. Acta Pathol Microbiol Scand 80:185-192, 1972. 18. Sumegi J: Fibrinoid necrosis and downward motion of colloid bodies in lichen planus (apoptosis). Acta Derm Venereol (Stockh) 59:27-31, 1979. 19. Ebner H, Kraft D: Fibrinnachweis mit einem EnzymAntiserum-Konjugat beim Lichen tuber planus. Dermatologica 146:101-106, 1973. 20. Medenica M, Lorincz A: Lichen planus: An ultrastructural study. Acta Derm Venereol (Stockh) 57:55-62, 1977.