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The site of blister formation in dermatitis herpetiformis is within the lamina lucida
The site of blister formation in dermatitis herpetiformis is within the lamina lucida CAPT Jeffrey B. Smith, USAF,a Ted B. Taylor, MS,b and John J. Zone, MDb,c
Salt Lake City, Utah BackgrQund: Because the initial neutrophilic inilltrate in dermatitis herpetiformis is within the dermal papillae, most investigators have assumed the vesicles occur in this same area. This was supported by electron microscopy studies. In 1983 Klein et al. refuted this concept, suggesting that vesicle formation was within the lamina lucida above the lamina densa. Despite this study, current literature continues to state that blister formation is below the lamina densa. Objective: Our purpose was to determine the ultrastructural site of blister formation in early and late vesicles of dermatitis herpetiformis. Methods: We evaluated eight biopsy specimens from four patients by immunomapping with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and epidermolysis bullosa acquisita antigen. Results: In both early and late vesicles blister formation was found to be above the lamina densa in the lamina lucida. Conclu$ion: These findings are contrary to the commonly held concept that the blister in dermatitis herpetiformis is below the lamina densa and confirm the findings of Klein et al. that the site of blister formation in dermatitis herpetiformis is above the lamina densa within the lamina lucida. (J AM ACAD DERMATOL 1992;27:209-13.)
Dermatitis herpetiformis (DR) is an intensely pruritic disease characterized by the occurrence of papulovesicles and occasionally bullae on extensor surfaces. Immunopathologically there is a pathognomonic deposition of granular IgA in dermal papillae. l ,2 The earliest histologic finding is neutrophilic infiltration with edema in dermal papillae. Subsequent vesicle formation occurs in the basement membrane zone (BMZ).3-5 Because the initial neutrophilic infiltrate and edema is within the papillary dermis, it could be assumed that the vesicles occur in the same location, below the lamina densa. In fact, many early electron microscopic (EM) studies supported this concept. 6-10 However, in 1983 Klein et al. ll suggested that vesicle formation in DH lesions was actually above the From the Emergency Department, Hill Hospital, Hill Air Force Basea ; the Division of Dermatology, Department of Internal Medicine, University of Utah School of Medicineb ; and the Dermatology Section, Veterans Affairs Medical Center.c Supported by Department ofVeterans Affairs Medical Research Funds and the Division of R~earch Resources MOI-RR-00064. Accepted for publication March 9, 1992. Reprint requests: John J. Zone, MD, Division of Dermatology, University of Utah Medical Center, 50 North Medical Dr., Salt Lake City, UT 84132,
16/1/37796
lamina densa in the lamina lucida. Other authors have also noted cases in which the DH blister is above the lamina densa. 12 Despite this information, dermatologic texts have continued to state that blister formation is below the lamina densa. 3,4, 13-19 Clinically, sub-lamina densa blister formation is characterized by scarring and milia formation in diseases such as epidermolysis bullosa acquisita (EBA) and epidermolysis bullosa dystrophica. However, D H produces neither scarring nor milia. On the basis of these observations, it seemed more likely to us that blister formation was above the lamina densa rather than below the lamina densa. The BMZ can be divided into four distinct and separate areas: (1) the plasma membrane of basal cells, (2) the lamina lucida, (3) the lamina densa, and (4) the sub-lamina densa. 2o The lamina lucida contains lamiriin and the bullous pemphigoid (BP) antigen, the majority of which is intracellular. The lamina densa contains type IV collagen as its principal component, and the sub-lamina densa contains the antigen of EBA. With the use of the indirect immunofluorescence technique with antibodies to the components of the various layers of the BMZ, it is possible to map precisely the level at which blister formation and separation take place. 21 Therefore, to test our hypothesis that the blister in DH was above
209
Journal of the American Academy of Dermatology
210 Smith et ai. Table I. Location of fluorescence in dermatitis herpetiformis blisters Erythematous skin Patient 1 Patient 2 Patient 3 Patient 4 Vesicular skin Patient 1 Patient 2 Patient 3 Patient 4
BP
Laminin
Collagen IV
E.BA
Roof+++ NB NB Roof+++
Floor + NB NB
Floor +++ NB NB Floor +++
Floor +++ NB NB Floor +
Floor +
Floor +++ Floor +++ Floor +++ Floor +++
Floor +++ Floor +++ Floor +++ Floor +
Roof++ Roof + Roof+++ Roof++
(-)
(-)
Both + Floor +
Fluorescence intensity; +++, strong; ++, moderate; +, weak. Both, part in floor and part in roof; BP, bullous pemphigoid; EBA, epidermolysis bullosa acquisita; NB, no blister.
the lamina densa and not below it, we evaluated both early and mature vesicles from patients with DR by immunomapping with antibodies to BP antigen, laminin, EBA antigen, and collagen IV. MATERIAL AND METHODS
Eight skin biopsy specimens were taken from four patients with clinically typical DR not receiving treatment at that time. All patients demonstrated granular IgA deposition in dermal papillae of clinically normal-appearing perilesional skin. To evaluate both early and late lesions two specimens were obtained from each patient. One was taken from an area of erythema and induration without clinical vesiculation and one from a clinically apparent vesicle less than 24 hours old. Each biopsy specimen was evaluated for location of the cleavage plane of the blister by immunomapping. Mouse monoclonal antibody reactive with type IV collagen (Chemicon International Corp., Inc., EI Segundo, Calif.) and laminin (Calbiochem-Behring Corp., San Diego, Calif.), as well as serum from patients with bullous pemphigoid (BP) and EBA were used. Indirect immunofluorescence was performed on frozen sections of biopsy specimens with these primary antisera and appropriate secondary antibodies as described previously.22 Individual specimens were then evaluated for intensity of fluorescence (0 to 3+) and location (roofvs base) relative to the blister cavity with an epifiuorescence microscope. Control specimens were sodium chloride- and EDTA-split normal human skin treated with the same four antibodies used. RESULTS
The results are presented in Table 1. Hematoxylin-and-eosin stains showed mature blisters in six of eight specimens and neutrophilic infiltration in dermal papillae without blister formation in two ofeight (patients 2 and 3 listed under erythematous skin in Table 1). The remaining six biopsy specimens re-
vealed vesicles at various stages. In six of six specimens stained for type IV collagen, the fluorescence was clearly in the floor of the blister, indicating that the blister was above the lamina densa (Fig. 1). In six of six specimens stained with EBA serum the fluorescence was also in the floor of the blister (Fig. 2). In both Figs. 1 and 2 there is a small area oflucency beneath the fluorescence. This most likely represents crystallization artifact from the frozen sections but could represent dermal papillary edema. It is impossible to determine this with certainty. The important result is that the large vesicle containing cellular debris is clearly above the fluorescence in both of these figures. Sections stained with BP serum showed the fluorescence in the roof of the blister in six of six specimens (Fig. 3). The sections stained with antilaminin antibody stained weakly in the floor of blisters in three of six specimens, failed to stain at the blister in two of six, and in one of six stained weakly in the floor in parts and weakly in the roof in other parts. Despite the weak or absent staining at the blister proper, all six specimens stained with antilaminin antibody did stain well at the BMZ in adjacent nonblistered areas, thereby confirming the reactivity of the antibody. Therefore all specimens were consistent with blister formation above the lamina densa in the lower lamina lucida. We attribute the poor staining with antilaminin antibody to destruction of laminin by the blistering process. The control specimens done on normal human skin split in sodium chloride or EDTA showed the same results (i.e., the sections stained with collagen IV and EBA fluoresced brightly in the bases of blisters, the BP in the roof, and the laminin sections fluoresced weakly and variably as already stated.
Volume 27 Number 2, Part 1 August 1992
DH blister site 211
Fig. 1. DR blister stained with type IV collagen. Note fluorescence along floor of blister cavity. (X20.)
Fig. 2. DR blister stained using EBA serum. Note fluorescence along floor of blister cavities. (X20.)
DISCUSSION
Our findings indicate that mature vesicles in DR form above the layer of type IV collagen in the BMZ and therefore above the lamina densa and not below it as many current textbooks suggest.3, 13-19 Our most convincing piece of evidence to support this conclusion is that in all sections stained with type IV collagen (reactive with the lamina densa) the fluorescence was located in the floor of the blister and never in the roof. The fact that the EBA antigen was always in the base of the blister and the BP antigen was always in the roof is consistent with and
supportive of this conclusion. Indeed, if blister formation were in the papillary dermis below the lamina densa, we would have expected the EBA antigen to be found in the roof of the blister and not the base, as our results indicate. The fact that laminin stained well in perilesional areas but poorly if at all in the blisters suggests that the laminin within the lamina Lucida is being destroyed by inflammation and that the lamina lucida is the site of blister formation in DH. The BP antigen, the majority of which is intracellular, is also located in the lamina lucida but was not destroyed to the same extent as
Journal of the American Academy of Dermatology
212 Smith et al.
Fig. 3. DH blister stained using BP serum. Note fluorescence along roof of blister cavity. (X20.)
laminin perhaps because BP antigen is located in the upper lamina lucida. 2o These findings are consistent with a report by Klein et al. II in 1983 that mature vesicles found in five of five DH biopsy specimens occurred above the lamina densa. In one case in which only urticarial lesions and dermal papillary edema were noted, the immunomapping results showed multiple microvesicles mostly above the lamina densa but also a few were noted below the lamina densa. In addition, Horiguchi et al. I2 in 1987 in an EM study reported that in two of five oftheir DH patients the blister was unequivocally above the lamina densa; in two of five there was considerable fragmentation of the lamina densa that sometimes appeared in the roof and other times in the floor of the blisters. In a single patient the lamina densa was partially preserved in the roof of the blister. Why has the site of blister formation in DH been misstated for so many years? There are several possible explanations. Because one ofthe early events in DH is neutrophil accumulation and edema in the dermal papillae, many have assumed without experimental evidence that microvesicles coalesce in situ and form a mature vesicle in the dermal papillae and thus below the lamina densa. Another possibility is that early studies were done with EM, which evaluates very small sections at very high magnifications; thus a full and accurate picture is difficult to obtain. From our review of the EM liter-
ature on DH we find no evidence of mature vesicles located below the lamina densa. Because EM involves such high magnification, we believe that what many authors called vesicle formation was actually dermal papillary edema and not truly vesicles. Another possible explanation is that late blisters may have been evaluated when widespread tissue destruction had already taken place, making interpretation difficult. Many investigators who have used EM to study DH have stated that "the basal lamina (lamina densa) is disrupted or absent" in the early blister. 23 We propose that the inflammatory response induced by neutrophilic infiltration has instead obscured the electron density of the lamina densa on EM. Immunologic detection of antigens by immunomapping clearly shows that lamina densa components are present and intact in a continuous linear pattern in the floor of the vesicle. The newer technique of immunomapping has several additional advantages over EM. Because of the relatively low magnification it is possible to visualize large portions of blisters or even complete blisters and thus obtain an accurate picture of what has taken place. It also has the advantage that the location of a specific protein can be identified rather than an electron-dense or electron-lucent material, the morphology or density of which may be altered by the inflammatory process. We propose that the actual process in the development of a mature vesicle in DR is as follows. First, neutrophils infiltrate dermal papillae and release lysosomal enzymes that produce dermal papillary edema. Second, there is recruitment of many neutrophils that release more proteolytic enzymes that act locally in the dermal papillae as well as in the area of the BMZ. Third, the lower lamina lucida is destroyed by the proteolytic enzymes and a mature vesicle is formed in the lower lamina lucida above the lamina densa.
REFERENCES 1. Leonard JN, Haffenden GP, Fry L. Dermatitis herpetifarmis. In: Beutner EH, Chorzelski TP, Kumar V, eds. Immunopathology ofthe skin. New York: John Wiley & Sons, 1987:437-43. 2. Zone JJ. Dermatitis herpetiformis. Curr Probl Dermatol 1991:1-41. 3. Lever WF, Schaumburg-Lever G. Histopathology of the skin. Philadelphia: JB Lippincott, 1990: 103-4, 131-5. 4. Alexander JO. Dermatitis herpetiformis. London: WB Saunders, 1975:91-117, 145-160. 5. ParkerSC, Black MM. Subepidermal bullous disorders. In: Farmer ER, Hood AF, eds. Pathology afthe skin. Norwalk, Conn: Appleton & Lange, 1990:148-9.
Volume 27 Number 2, Part I August 1992
6. Bellone AG, Caputo R. Aspetti ultrastrutturali della dermatite erpetiforme di Duhring. G Ital Derm 1966;107: 173-89. 7. Pierard J, Kint A. Dermatite herpetiforme et pemphigoide bulleuse: etude comparative au microscope electronique du mecanisme de formation de la bulle. Ann Derm Syph 1968;95:391-404. 8. Fry L, Johnson FR. Electron microscopic study of dermatitis herpetiformis. Br J DermatoI1969;8I:44-50. 9. Jakubowicz K, Dabrowski J, Maciejewski W. Elektronenmikropische untersuchungen bei bullosem pemphigoid und dermatitis herpetiformis Duhring. Arch Klin Exp Derm 1970;238:272-84. 10. Rodrigo GF. Ultrastructural aspects of dermatitis herpetiformis (Duhring's disease). Br J DermatoI1972;86:348-55. 11. Klein GF, Hinter H, Schuler G, et al. Junctional blisters in acquired bullous disorders of the dermal-epidermal junction zone: role of the lamina lucida as the mechanical locus minoris resistente. Br J DermatoI1983;109:499-508. 12. Horiguchi Y, Danno K, Toda K. Ultrastructural sites of blister formation in dermatitis herpetiformis: report of a case and retrospective microscopy using routine histologic preparations. J DermatoI1987;14:462-70. 13. Zone JJ, Petersen MJ. Dermatitis herpetiform is. In: Thiers BH, Dobson R, eds. Pathogenesis of skin disease. New York: Churchill-Livingstone, 1985:159-83. 14. Zone JJ, Meyer LJ. Dermatitis herpetiformis. In: Norris DA, ed. Immune mechanisms in cutaneous disease. New York: Marcel Dekker, 1989:565-84. 15. Synkowski DR, Dore N, Provost IT. The dermoepidermal
DH blister site 213
16. 17.
18. 19.
20. 21.
22.
23.
junction: structure, function, and disorders. Clin Dermatol 1988;2:1-11. Pye RJ. Bullous eruptions. In: Rook A, Wilkinson OS, Ebling FJG, et aI, eds. Textbook of dermatology. Oxford: Blackwell Scientific, 1986:1651-4. Fritsch PO, Elias PM. Mechanisms of vesicle formation and classification. In: Fitzpatrick TB, Eisen AZ, Wolff K, et ai, eds. Dermatology in general medicine. New York: McGraw-Hili, 1986:546-54. Habif TP. Clinical dermatology. St Louis: CV Mosby, 1990:407-11. Hashimoto K, Takahashi S, Matsumoto M. Bullous dermatoses.In: Johannessen JV, Hashimoto K, eds. Electron microscopy in human medicine: the skin. New York: McGraw-Hill, 1985:180-6. Katz SI. The epidermal basement membrane zone-structure, ontogeny, and role in disease. J AM ACAD DERMATOL 1984;11:1025-37. Hinter H, Stingl G, Schuler G. Immunofluorescence mapping of antigenic determinants within the dermal-epidermal junction in mechanicobullous diseases. J Invest Dermatol 1981;76:113-8. ZoneJJ, TaylorTB, Kadunce DP, etal. Identification ofthe cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis. J Clin Invest 1990;85:812-20. Riches DJ, Martin BGH, Seah PP. The ultrastructural changes in the skin in dermatitis herpetiformis after withdrawal of dapsone. Hr J Dermatol 1976;94:31-7.
Salt Lake City, Utah BackgrQund: Because the initial neutrophilic inilltrate in dermatitis herpetiformis is within the dermal papillae, most investigators have assumed the vesicles occur in this same area. This was supported by electron microscopy studies. In 1983 Klein et al. refuted this concept, suggesting that vesicle formation was within the lamina lucida above the lamina densa. Despite this study, current literature continues to state that blister formation is below the lamina densa. Objective: Our purpose was to determine the ultrastructural site of blister formation in early and late vesicles of dermatitis herpetiformis. Methods: We evaluated eight biopsy specimens from four patients by immunomapping with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and epidermolysis bullosa acquisita antigen. Results: In both early and late vesicles blister formation was found to be above the lamina densa in the lamina lucida. Conclu$ion: These findings are contrary to the commonly held concept that the blister in dermatitis herpetiformis is below the lamina densa and confirm the findings of Klein et al. that the site of blister formation in dermatitis herpetiformis is above the lamina densa within the lamina lucida. (J AM ACAD DERMATOL 1992;27:209-13.)
Dermatitis herpetiformis (DR) is an intensely pruritic disease characterized by the occurrence of papulovesicles and occasionally bullae on extensor surfaces. Immunopathologically there is a pathognomonic deposition of granular IgA in dermal papillae. l ,2 The earliest histologic finding is neutrophilic infiltration with edema in dermal papillae. Subsequent vesicle formation occurs in the basement membrane zone (BMZ).3-5 Because the initial neutrophilic infiltrate and edema is within the papillary dermis, it could be assumed that the vesicles occur in the same location, below the lamina densa. In fact, many early electron microscopic (EM) studies supported this concept. 6-10 However, in 1983 Klein et al. ll suggested that vesicle formation in DH lesions was actually above the From the Emergency Department, Hill Hospital, Hill Air Force Basea ; the Division of Dermatology, Department of Internal Medicine, University of Utah School of Medicineb ; and the Dermatology Section, Veterans Affairs Medical Center.c Supported by Department ofVeterans Affairs Medical Research Funds and the Division of R~earch Resources MOI-RR-00064. Accepted for publication March 9, 1992. Reprint requests: John J. Zone, MD, Division of Dermatology, University of Utah Medical Center, 50 North Medical Dr., Salt Lake City, UT 84132,
16/1/37796
lamina densa in the lamina lucida. Other authors have also noted cases in which the DH blister is above the lamina densa. 12 Despite this information, dermatologic texts have continued to state that blister formation is below the lamina densa. 3,4, 13-19 Clinically, sub-lamina densa blister formation is characterized by scarring and milia formation in diseases such as epidermolysis bullosa acquisita (EBA) and epidermolysis bullosa dystrophica. However, D H produces neither scarring nor milia. On the basis of these observations, it seemed more likely to us that blister formation was above the lamina densa rather than below the lamina densa. The BMZ can be divided into four distinct and separate areas: (1) the plasma membrane of basal cells, (2) the lamina lucida, (3) the lamina densa, and (4) the sub-lamina densa. 2o The lamina lucida contains lamiriin and the bullous pemphigoid (BP) antigen, the majority of which is intracellular. The lamina densa contains type IV collagen as its principal component, and the sub-lamina densa contains the antigen of EBA. With the use of the indirect immunofluorescence technique with antibodies to the components of the various layers of the BMZ, it is possible to map precisely the level at which blister formation and separation take place. 21 Therefore, to test our hypothesis that the blister in DH was above
209
Journal of the American Academy of Dermatology
210 Smith et ai. Table I. Location of fluorescence in dermatitis herpetiformis blisters Erythematous skin Patient 1 Patient 2 Patient 3 Patient 4 Vesicular skin Patient 1 Patient 2 Patient 3 Patient 4
BP
Laminin
Collagen IV
E.BA
Roof+++ NB NB Roof+++
Floor + NB NB
Floor +++ NB NB Floor +++
Floor +++ NB NB Floor +
Floor +
Floor +++ Floor +++ Floor +++ Floor +++
Floor +++ Floor +++ Floor +++ Floor +
Roof++ Roof + Roof+++ Roof++
(-)
(-)
Both + Floor +
Fluorescence intensity; +++, strong; ++, moderate; +, weak. Both, part in floor and part in roof; BP, bullous pemphigoid; EBA, epidermolysis bullosa acquisita; NB, no blister.
the lamina densa and not below it, we evaluated both early and mature vesicles from patients with DR by immunomapping with antibodies to BP antigen, laminin, EBA antigen, and collagen IV. MATERIAL AND METHODS
Eight skin biopsy specimens were taken from four patients with clinically typical DR not receiving treatment at that time. All patients demonstrated granular IgA deposition in dermal papillae of clinically normal-appearing perilesional skin. To evaluate both early and late lesions two specimens were obtained from each patient. One was taken from an area of erythema and induration without clinical vesiculation and one from a clinically apparent vesicle less than 24 hours old. Each biopsy specimen was evaluated for location of the cleavage plane of the blister by immunomapping. Mouse monoclonal antibody reactive with type IV collagen (Chemicon International Corp., Inc., EI Segundo, Calif.) and laminin (Calbiochem-Behring Corp., San Diego, Calif.), as well as serum from patients with bullous pemphigoid (BP) and EBA were used. Indirect immunofluorescence was performed on frozen sections of biopsy specimens with these primary antisera and appropriate secondary antibodies as described previously.22 Individual specimens were then evaluated for intensity of fluorescence (0 to 3+) and location (roofvs base) relative to the blister cavity with an epifiuorescence microscope. Control specimens were sodium chloride- and EDTA-split normal human skin treated with the same four antibodies used. RESULTS
The results are presented in Table 1. Hematoxylin-and-eosin stains showed mature blisters in six of eight specimens and neutrophilic infiltration in dermal papillae without blister formation in two ofeight (patients 2 and 3 listed under erythematous skin in Table 1). The remaining six biopsy specimens re-
vealed vesicles at various stages. In six of six specimens stained for type IV collagen, the fluorescence was clearly in the floor of the blister, indicating that the blister was above the lamina densa (Fig. 1). In six of six specimens stained with EBA serum the fluorescence was also in the floor of the blister (Fig. 2). In both Figs. 1 and 2 there is a small area oflucency beneath the fluorescence. This most likely represents crystallization artifact from the frozen sections but could represent dermal papillary edema. It is impossible to determine this with certainty. The important result is that the large vesicle containing cellular debris is clearly above the fluorescence in both of these figures. Sections stained with BP serum showed the fluorescence in the roof of the blister in six of six specimens (Fig. 3). The sections stained with antilaminin antibody stained weakly in the floor of blisters in three of six specimens, failed to stain at the blister in two of six, and in one of six stained weakly in the floor in parts and weakly in the roof in other parts. Despite the weak or absent staining at the blister proper, all six specimens stained with antilaminin antibody did stain well at the BMZ in adjacent nonblistered areas, thereby confirming the reactivity of the antibody. Therefore all specimens were consistent with blister formation above the lamina densa in the lower lamina lucida. We attribute the poor staining with antilaminin antibody to destruction of laminin by the blistering process. The control specimens done on normal human skin split in sodium chloride or EDTA showed the same results (i.e., the sections stained with collagen IV and EBA fluoresced brightly in the bases of blisters, the BP in the roof, and the laminin sections fluoresced weakly and variably as already stated.
Volume 27 Number 2, Part 1 August 1992
DH blister site 211
Fig. 1. DR blister stained with type IV collagen. Note fluorescence along floor of blister cavity. (X20.)
Fig. 2. DR blister stained using EBA serum. Note fluorescence along floor of blister cavities. (X20.)
DISCUSSION
Our findings indicate that mature vesicles in DR form above the layer of type IV collagen in the BMZ and therefore above the lamina densa and not below it as many current textbooks suggest.3, 13-19 Our most convincing piece of evidence to support this conclusion is that in all sections stained with type IV collagen (reactive with the lamina densa) the fluorescence was located in the floor of the blister and never in the roof. The fact that the EBA antigen was always in the base of the blister and the BP antigen was always in the roof is consistent with and
supportive of this conclusion. Indeed, if blister formation were in the papillary dermis below the lamina densa, we would have expected the EBA antigen to be found in the roof of the blister and not the base, as our results indicate. The fact that laminin stained well in perilesional areas but poorly if at all in the blisters suggests that the laminin within the lamina Lucida is being destroyed by inflammation and that the lamina lucida is the site of blister formation in DH. The BP antigen, the majority of which is intracellular, is also located in the lamina lucida but was not destroyed to the same extent as
Journal of the American Academy of Dermatology
212 Smith et al.
Fig. 3. DH blister stained using BP serum. Note fluorescence along roof of blister cavity. (X20.)
laminin perhaps because BP antigen is located in the upper lamina lucida. 2o These findings are consistent with a report by Klein et al. II in 1983 that mature vesicles found in five of five DH biopsy specimens occurred above the lamina densa. In one case in which only urticarial lesions and dermal papillary edema were noted, the immunomapping results showed multiple microvesicles mostly above the lamina densa but also a few were noted below the lamina densa. In addition, Horiguchi et al. I2 in 1987 in an EM study reported that in two of five oftheir DH patients the blister was unequivocally above the lamina densa; in two of five there was considerable fragmentation of the lamina densa that sometimes appeared in the roof and other times in the floor of the blisters. In a single patient the lamina densa was partially preserved in the roof of the blister. Why has the site of blister formation in DH been misstated for so many years? There are several possible explanations. Because one ofthe early events in DH is neutrophil accumulation and edema in the dermal papillae, many have assumed without experimental evidence that microvesicles coalesce in situ and form a mature vesicle in the dermal papillae and thus below the lamina densa. Another possibility is that early studies were done with EM, which evaluates very small sections at very high magnifications; thus a full and accurate picture is difficult to obtain. From our review of the EM liter-
ature on DH we find no evidence of mature vesicles located below the lamina densa. Because EM involves such high magnification, we believe that what many authors called vesicle formation was actually dermal papillary edema and not truly vesicles. Another possible explanation is that late blisters may have been evaluated when widespread tissue destruction had already taken place, making interpretation difficult. Many investigators who have used EM to study DH have stated that "the basal lamina (lamina densa) is disrupted or absent" in the early blister. 23 We propose that the inflammatory response induced by neutrophilic infiltration has instead obscured the electron density of the lamina densa on EM. Immunologic detection of antigens by immunomapping clearly shows that lamina densa components are present and intact in a continuous linear pattern in the floor of the vesicle. The newer technique of immunomapping has several additional advantages over EM. Because of the relatively low magnification it is possible to visualize large portions of blisters or even complete blisters and thus obtain an accurate picture of what has taken place. It also has the advantage that the location of a specific protein can be identified rather than an electron-dense or electron-lucent material, the morphology or density of which may be altered by the inflammatory process. We propose that the actual process in the development of a mature vesicle in DR is as follows. First, neutrophils infiltrate dermal papillae and release lysosomal enzymes that produce dermal papillary edema. Second, there is recruitment of many neutrophils that release more proteolytic enzymes that act locally in the dermal papillae as well as in the area of the BMZ. Third, the lower lamina lucida is destroyed by the proteolytic enzymes and a mature vesicle is formed in the lower lamina lucida above the lamina densa.
REFERENCES 1. Leonard JN, Haffenden GP, Fry L. Dermatitis herpetifarmis. In: Beutner EH, Chorzelski TP, Kumar V, eds. Immunopathology ofthe skin. New York: John Wiley & Sons, 1987:437-43. 2. Zone JJ. Dermatitis herpetiformis. Curr Probl Dermatol 1991:1-41. 3. Lever WF, Schaumburg-Lever G. Histopathology of the skin. Philadelphia: JB Lippincott, 1990: 103-4, 131-5. 4. Alexander JO. Dermatitis herpetiformis. London: WB Saunders, 1975:91-117, 145-160. 5. ParkerSC, Black MM. Subepidermal bullous disorders. In: Farmer ER, Hood AF, eds. Pathology afthe skin. Norwalk, Conn: Appleton & Lange, 1990:148-9.
Volume 27 Number 2, Part I August 1992
6. Bellone AG, Caputo R. Aspetti ultrastrutturali della dermatite erpetiforme di Duhring. G Ital Derm 1966;107: 173-89. 7. Pierard J, Kint A. Dermatite herpetiforme et pemphigoide bulleuse: etude comparative au microscope electronique du mecanisme de formation de la bulle. Ann Derm Syph 1968;95:391-404. 8. Fry L, Johnson FR. Electron microscopic study of dermatitis herpetiformis. Br J DermatoI1969;8I:44-50. 9. Jakubowicz K, Dabrowski J, Maciejewski W. Elektronenmikropische untersuchungen bei bullosem pemphigoid und dermatitis herpetiformis Duhring. Arch Klin Exp Derm 1970;238:272-84. 10. Rodrigo GF. Ultrastructural aspects of dermatitis herpetiformis (Duhring's disease). Br J DermatoI1972;86:348-55. 11. Klein GF, Hinter H, Schuler G, et al. Junctional blisters in acquired bullous disorders of the dermal-epidermal junction zone: role of the lamina lucida as the mechanical locus minoris resistente. Br J DermatoI1983;109:499-508. 12. Horiguchi Y, Danno K, Toda K. Ultrastructural sites of blister formation in dermatitis herpetiformis: report of a case and retrospective microscopy using routine histologic preparations. J DermatoI1987;14:462-70. 13. Zone JJ, Petersen MJ. Dermatitis herpetiform is. In: Thiers BH, Dobson R, eds. Pathogenesis of skin disease. New York: Churchill-Livingstone, 1985:159-83. 14. Zone JJ, Meyer LJ. Dermatitis herpetiformis. In: Norris DA, ed. Immune mechanisms in cutaneous disease. New York: Marcel Dekker, 1989:565-84. 15. Synkowski DR, Dore N, Provost IT. The dermoepidermal
DH blister site 213
16. 17.
18. 19.
20. 21.
22.
23.
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