Dermatopathology
Direct immunofluorescence studies of sodium chloride-separated skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita W. R. Gammon, MD, a C. Kowalewski, MD,b T. P. Chorzelski, MD,b V. Kumar, PhD,c R. A. Briggaman, MD,a and E. H. Beutner, PhDc Chapel Hill, North Carolina, Warsaw, Poland, and Buffalo, New York Bullous pemphigoid and epidermolysis bullosa acquisita may have indistinguishable clinical, histologic, and routine immunohistologic features. In those cases these two diseases can be reliably distinguished in routine diagnostic studies only in seropositive cases by tests on lamina lucida-split skin and in research studies by direct immunoelectron microscopy or, in patients with circulating autoantibodies, by immunoblotting studies. The use of these methods is limited by the expense and unavailability of the methods, the requirement for circulating autoantibodies, or both. We describe a method to distinguish between the two diseases on the basis offindings of direct immunofluorescence of a biopsy specimen after separation through the lamina lucida with 1.0 mol/L sodium chloride. The IgG appeared in the dermal side of the split specimens in epidermolysis bullosa acquisita and predominantly or exclusively in the epidermal side in pemphigoid. The method was found to be relatively simple, inexpensive, applicable to specimens preserved in transport media, and 100% reliable in our group of 22 patients. (J AM ACAD DERMATOL 1990;22:664-70.)
Bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA) are chronic, inflammatory, subepidermal, blistering diseases characterized by circulating and tissue~bound autoantibodies to the cutaneous basement membrane zone (BMZ).1,2 Because anti~BMZ autoantibodies in both diseases belong predominantly to the IgG class and produce a linear pattern of IgG deposition at the BMZ, they cannot be reliably distinguished by routine direct and indirect immunofluorescence methods. However, they are readily distinguished by immunoelectron microscopy (IEM) and immunochemical
From the Department of Dermatology, The University of North Carolina, Chapel Hill,' the Department of Dermatology, Warsaw Academy of Medicine,b and the Departments of Microbiology. and Dermatology, The Ernest Witebsky Center for Immunology, University at Buffalo. SUNY.c SUPJXlrted in part by National Institu tes of Health grant Nos. AR30475 and ARI 0546, the Polish Academy ofScience, and Immco Diagnostics, Buffalo, N.Y. Reprint requests: W. R. Gammon, MD, Department of Dermatology. The University of North Carolina, Chapel Hill, NC 27514.
16/1/13567
664
methods. 3-9 Because of the expense and relative unavailability of IEM and immunochemical methods, differential diagnosis is usually made on the basis of differences in the epidemiologic, clinical, and histologic features of the diseases. Recent studies of patients with BP and EBA diagnosed on the basis of definitive IEM and/or immunochemical methods have provided evidence that attempts to distinguish between them on the basis of differences in epidemiologic, clinical, and histologic features can result in misdiagnosis. Patients with BP may have clinical and histologic features that resemble those classically associated with EBA, and patients with EBA may have clinical and histologic features that closely resemble those characteristic of BP. IO-12 Furthermore, patients have been reported with coexistent systemic lupus erythematosus (SLE) and BP who had clinical, histologic, and immunohistologic features that closely resembled those seen in bullous SLE (BSLE).13-19 BP and EBA also share similar, if not identical, routine direct and indirect immunofluorescent features, that is, linear deposits of predominantly IgG
Volume 22 Number 4
Direct immunofluorescence on NaCl-separated skin 665
April 1990
and C3 at the BMZ and IgG anti-BMZ autoantibodies, respectively.]], 12 In previous studies we2D described ~ method of indirect immunofluorescence on sodium chloride (NaCI)-separated normal human skin that could distinguish between circulating BP and EBA anti-BMZ autoantibodies. The method was shown to be a reliable means of distinguishing between patients with BP and those with EBA who had circulating anti-BMZ antibodies and offered the advantages of relative simplicity and economy over IEM and immunochemical methods. However, application of the method is limited because approximately 20% to 30% of patients with BP and 50% of those with EBA have been shown to lack those antibodies. To overcome that limitation, we investigated the use of direct immunofluorescence on 3 to 4 mm punch biopsy specimens of patients' clinically normal skin after separation of the epidermis from the dermis in 1.0 mol/L NaCl.
Table I. Diagnosis of patients entered in this study Method of diagnosis
Patient No.
10
BP BP BP BP EBA EBA EBA BSLE BP BP
11
BP
12 13
BP BP BP BP BP EBA EBA EBA EBA EBA EBA
1 2 3
4 5 6
7 8
9
14 15
16 17 18
MATERIAL AND METHODS Patients. Twenty-two patients (12 with BP, 9 with EBA, and 1 with BSLE) were studied. All had linear IgG deposits at the BMZ shown by routine direct immunofluorescence. The diagnosis of BP was made on the basis of clinical, histologic, and routine direct immunohistologic criteria. The diagnosis was confirmed in 10 patients with circulating IgG anti·BMZ autoantibodies by indirect immunofluorescence on NaCl-separated skin 20 (Table I). The diagnosis of EBA was made by clinical, histologic, and routine direct immunohistologic criteria. The diagnosis was confirmed in four patients by indirect immunofluorescence on NaCl-separated skin, IEM, or both20 (TableI). The diagnosis of BSLE was made in one patient on the basis of previously published clinical, histologic, routine direct immunohistologic, and IBM criteria. 2l Skin. Punch biopsy specimens (3 or 4 mm in diameter) of clinically normal perilesional skin were obtained with patients under local anesthesia. The specimens were rinsed brieflY with phosphate-buffered saline (PBS) and placed in 10 to 20 ml of 1.0 mol/L NaCl with or without 5 mmoljL EDTA. The specimens were incubated in NaCI with or without EDTA for 72 to 96 hours at 4 C and blotted on filter paper. With the use of fine forceps and a dissecting microscope, the epidermis was gently displaced laterally 1 mm across the dermis and was left adherent to the dermis. The specimens were then snapfrozen in liquid nitrogen, mounted in OCT compound (Miles Scientific, Naperville, IIl.) or PBS and sections 4 to 8 p,m thick were cut on a cryostat and mounted on glass slides. Specimens from four patients were placed in 0
Diagnosis
19 20 21 22
IIFon NaO-SS
I
!EM
+ +
+ +
+ +
+ + +
+ + +
+
+ + + + +
fEM, lmmunoelectron microscopy; IfF, indirect immunofluorescence; NaCl-8S, sodium chloride-separated skin; +, test performed; -, test not
performed.
Michel's transport medium immediately after being obtained. After several days in that medium, they were washed with PBS and incubated in NaCI as described. These specimens were found to be slightly more resistant to separation than ones placed directly into NaCl; however, all four separated without difficulty. Immunofluorescence mapping. The site of dermoepidermal separation in NaCl-separated specimens was determined with indirect immunofluorescence mapping,22 Reagents used to localize the site of separation included (1) a reference BP serum (1:40 dilution) from a patient with disease diagnosed by clinical, histologic, immunohistologic, and IBM criteria. The serum contained IgG anti-BMZ antibody (titer of 1:640) that consistently bound only to the epidermal side of N aCI-separated normal human skin by indirect immunofluorescence and to the lamina lucida-hemidesmosome complex of normal human skin by indirect IBM; (2) a previously characterized mouse monoclonal antibody (L3d) to human type VII collagen (1:100 dilution)]l; (3) polyc1onal rabbit antibodies to human Iaminin (l: 10 dilution; Gibeo #6803019, Gibeo, Madison, Wis.); and (4) polyclonal rabbit antibodies to human type IV collagen prepared by
Journal of the American Academy of Dermatology
666 Gammon et al.
Fig. 1. Immunofluorescence mapping of patient's biopsy specimen after separation in 1.0 mo1/L NaCl. Location of the split relative to lamina densa was examined by staining with monoclonal antibody to type VII coHagen. Type VII collagen staining is seen only on dermal side of separation, confirming separation above lamina densa. (X400.)
Fig. 2. Immunofluorescence mapping of biopsy specimen of patient with BP after separation in 1.0 mol/L NaCl. The biopsy specimen was stained for BP antigen with reference BP serum. Staining is observed only on epidermal side of separation, indicating separation in lamina lucida and confirming separation beneath keratinocyte plasma membrane. Direct IgG immunofluorescence of the biopsy specimen before incubation with reference BP serum showed the same pattern of staining. (X400.)
immunization with human type IV coHagen (1:16 dilution). Secondary antibodies were (1) fluorescein isothiocyanate (FITC)-conjugated anti-human IgG; (2) FITCconjugated goat IgG specific for mouse IgG (no cross-reactivity with human IgG); and (3) FITC-conjugatedgoat IgG specific for rabbit IgG. Direct immunofluorescence. Direct IgG, IgA, IgM, and C3 immunofluorescence of N aCI-separated biopsy specimens was performed with standard methods, that is, monospecific reagents with molar fluorescein to protein
ratios between 1.5 and 2.5 were diluted to contain 0.25 Vjml (approximately 50 p,g/ml of antibody protein).23
RESULTS
Immunofluorescence mapping Immunofluorescence mapping was performed on all 22 N aCI-separated biopsy specimens. When the specimens were examined with the use of antibodies to laminin or the lamina densa-sublamina densa
Volume 22 Number 4 April 1990
Direct immunofluorescence on NaCl-separated skin 667
Table II. Results of direct IgG immunofluorescence on patients' skin after separation in 1.0 moljL NaCl Location of IgG deposits No. of
Diagnosis
BP
EBA BSLE
patients
12 9 1
Epidermis
5
o o
I p~~d:::~ I Dermis 7
o
a
o 9 1
antigens, type VII collagen or type IV collagen, the results showed binding only to the dennal side of the separation (Fig. 1). We never observed binding of antibodies to lamina densa-sublamina densa antigens or laminin on the epidermal side of the separation as would occur if separation were beneath the lamina densa. These results showed that separation of the BMZ had occurred above the lamina densa in all specimens and was consistent with separation in the lamina lucida. To determine whether separation had occurred beneath the basal keratinocyte plasma membrane, mapping studies were performed with reference BP serum. Those results had to be interpreted in the context of direct immunofluorescent findings because all patients had human IgG deposited at the BMZ in vivo. In patients with a confirmed diagnosis of BP, there was no difference in the location of IgG deposits before and after indirect immunofluorescence with reference BP serum. In all cases, staining for IgG showed on the epidermal side of the separation consistent with separation beneath the keratinocyte plasma membrane. In some specimens, the intensity of immunofluorescence on the epidermal side of the separation was greater after treatment with the reference serum, which indicated that the site of separation was through the lamina lucida and below the BP antigen (Fig. 2). In patients with a previously confirmed diagnosis of EBA or BSLE, indirect immunofluorescence with reference BP serum also showed binding of IgG to the epidermal side of the separation, again indicative of separation beneath the keratinocyte plasma membrane (Fig. 3). Direct immunofluorescence of NaCl-separated biopsy specimens The results are shown in Table II. Direct IgG immunofluorescence of NaCl-separated biopsy specimens from the 12 patients with BP showed IgG staining either on only the epidermal side of the sep-
Fig. 3. Immunofluorescence mapping of biopsy specimen from patient with EBA after separation in 1.0 mol/L NaCl. a, IgG staining before incubation with reference BP serum shows in vivo-deposited IgG only on dermal side of separation. b, Same biopsy specimen after incubation with reference BP serum. IgG staining is observed on both epidermal and dermal sides of separation consistent with lamina lucida split beneath keratinocyte plasma membrane. (X400.)
aration in five patients (Fig. 2) or on both the epidermal and dermal sides of the separation in seven patients (Fig. 4). In the nine patients with EBA, direct immunofluorescence demonstrated IgG deposits only on the dermal side of the separation (Fig. 5). In the one patient with BSLE, direct immunofluorescence demonstrated IgG staining only on the dermal side of the separation. The IgA and IgM deposits in these specimens consistently codistributed with IgG deposits (results not shown). C3 deposits in patients with BP were consistently observed on both the epidermal and dermal sides of the separation even in patients whose IgG deposits were confined to the epidermal side. DISCUSSION
IEM and immunochemical studies have shown conclusively that BP and EBA are immunologically distinct diseases. Direct IEM studies have shown that the BMZ IgG deposits in BP are in the lamina
668 Gammon et aJ.
Journal of the American Academy of Dermatology
Fig. 4. Direct IgG immunofluorescence of biopsy specimen separated in 1.0 moljL NaCl from patient with BP. IgG staining is observed on both epidermal and dermal sides of separation. (X400.)
Fig. 5. Direct IgG immunofluorescence of biopsy specimen separated in 1.0 moljL NaCI from patient with EBA. IgG staining is observed on only dermal side of separation. (X400.)
lucida and are closely associated with the keratinocyte plasma membrane-hemidesrnosome complex whereas those in EBA are on and/or beneath the lamina densa. 3-5 Western immunoblots that use circulating BP and EBA anti-BMZ autoantibodies have shown that BP autoantibodies recognize a 240 or 180 kD noncollagenous glycoprotein in epidennal extracts whereas EBA autoantibodies recognize a 290 kD collagenous glycoprotein (type VII collagen) extracted from dermis-lamina densa. 6-9,24 Some patients with clinical, histologic, and immunohistologic features of BSLE also have anti-BMZ au-
toantibodies that are indistinguishable from EBA antibodies by IBM and immunoblotting methods. 19 Although directIEM and immunochemicalmethods can reliably distinguish between BP and EBA or BSLE, the methods are rarely used because of their unavailability and expense. In addition, immunochemical methods can only be used in patients with circulating anti-BMZ autoantibodies. Because routine direct and indirect immunofluorescence procedures cannot reliably distinguish the diseases, differential diagnosis is usually made on the basis of differences in epidemiologic, clinical, and histologic
Volume 22 Number 4 April 1990
Direct immunofluorescence on NaCl-separated skin 669
features. However, recent reports indicate that patients with BP may have some degree of skin fragility, trauma-induced lesions, lesions localized to extensor surfaces, an absence of inflammation, and healing with milia and sometimes scarring. I 1,12,25,26 They have also shown that patients with EBA may have a widespread inflammatory bullous eruption of flexural skin and an absence of clinically significant skin fragility, milia, or scarring early in the disease. 10, 11 The histologic features of lesions in patients with BP and EBA may also overlap. II It is apparent that there is no single and probably no combination of epidemiologic, clinical, and histologic features that can reliably distinguish all patients. Furthermore, evidence from some studies has shown that as many as 10% of patients with a diagnosis of BP may have EBA. lI In previous studies, we reported a method to distinguish BP and EBA antibodies by indirect immunofluorescence on 1.0 mol/L NaCl-separated normal human skin. In those studies, we found that circulating, anti-BMZ autoantibodies from patients with BP bound either to only the epidermal side of the separation (85% of cases) or to both the epidermal and dermal sides ofthe separation (15% of cases). In addition, we observed that anti-BMZ antibodies from patients with EBA and BSLE bound exclusively to the dermal side of the separation. I9,20 Although this method is relatively simple, inexpensive, and has become increasingly available, its application is limited to patients with circulating antiBMZ autoantibodies. This represents a significant limitation because as many as 20% to 30% of patients with BP and 50% of those with EBA may not have those antibodies. Incubation of normal human skin in 1.0 mol/L N aCl has been shown to separate the BMZ through the lamina lucida, leaving the keratinocyte plasma membrane and hemidesmosomes on the epidermal side of the separation and the lamina densa and sublamina densa zone on the dermal side of the separation. 2o,27,28 Because the BP antigen is in the high lamina lucida and closely associated with the keratinocyte plasma membrane-hemidesmosome complex and the EBA antigen is associated with the lamina densa-sublamina densa region, NaCl separation of the BMZ of patients' skin should allow separation of the two antigens as well as antibodies bound to those antigens in vivo. 2-5, 29,30 One of our concerns was that NaCI separation of patients' skin might result in atypical separation, es-
pecially in patients with EBA. In some patients with EBA, especially those with severe skin fragility, the BMZ may separate spontaneously beneath the lamina densa. 2 That site of separation theoretically would place the IgG deposits in EBA on either the epidermal or both sides of the separation. To ensure that the site of NaCl-induced separation was inthe lamina lucida, we routinely performed immunofluorescence mapping with antibodies to the BP antigen and antibodies to antigens located in the lamina densa-sublamina densa region. These studies showed that in all specimens, antibodies to lamina densa-sublamina densa antigens consistently bound only to the dermal side of the separation. Furthermore, in those specimens with in vivodeposited Igs on and beneath the lamina densa, we consistently observed BP antigen on only the epidermal side of the separation. Although we did not observe atypical splitting of the BMZ, the possibility that it could occur requires that all specimens be checked by immunofluorescence mapping with antibodies to at least one lamina densa antigen. Another of our initial concerns was that separation of patients' skin in 1.0 mol jL N aCl might elute the in vivo-bound immune deposits but that proved not to be a problem. We also observed that Michel's medium did not significantly interfere with either separation or immunofluorescence results. In NaCl-separated patients' skin, we found that the IgG deposits were always located either on only the epidermal side of the separation or on both the epidermal and dermal sides of the separation in pa~ tients with BP and on only the dermal side of the separation in patients with EBA and the one patient with BSLE. These studies demonstrate the reliability of direct immunofluorescence on NaCI-separated patients' skin to differentiate BP from EBA and BSLE. REFERENCES 1. Lever WF. Pemphigus and pemphigoid. A review of the advances made since 1964. J AM ACAD DERMATOL 1979; I:2-31. 2. Briggaman RA, Gammon WR, Woodley DT. Epidermolysis bullosa acquisita of the immunopathological type (dermolytic pemphigoid). J Invest Dermatol I 985;85:798-84s. 3. Nieboer C, Boorsma DM, Woerdeman MJ, eta!. Epidermolysis bullosa acquisita. Immunofluorescence, electron microscopic and immunoelectron microscopic studies in four patients. Dr J Dermatol 1980;102:383-92. 4. Yaoita H, Briggaman RA, Lawley T J, et al. Epidermolysis bullosa acquisita: ultrastructural and immunological studies. J Invest Dermatol 1981 ;76:288-92. 5. Horiguchi Y, Imamura S. Discrepancy between the local-
670 Gammon et at.
6.
7. 8.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
ization of in vivo-bound immunoglobulins in the skin and in vitro binding sites of circulating anti-BMZ antibodies in bullous pemphigoid: immunoelectron microscopic studies. J Invest Dermatol 1986;87:715-9. Stanley JR, Hawley-Nelson P, Yaar M, et al. Laminin and bullous pemphigoid antigen are distinct basement membrane proteins synthesized by epidermal cells. J Invest Dermatol 1982;78:456-9. Labib RS, Anhalt GJ, Patel HP, et al. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J ImmunoI1986;136:1231-5. Woodley DT, Briggaman RA, O'Keefe EJ, et al. Identification of the skin basement-membrane autoantigen in epidermolysis bullosa acquisita. N EnglJ Med 1984;310:100713. Woodley DT, Burgeson RE, Lunstrum G, et al. The epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen. J Clin Invest 1988;81:683-7. Gammon WR, Briggaman RA, Wheeler CEo Epidermolysis bullosa acquisita presenting as an inflammatory bullous disease. J AM ACAD DERMATOL 1982;7:382-7. Gammon WR, Briggaman RA, Woodley DT, et al. Epidermolysis bullosa acquisita-a pemphigoid-like disease. J AM ACAD DERMATOL 1984;11:820-32. Liu HH, Su WPD, Rogers RS III. Clinical variants of pemphigoid. Int J DermatoI1986;25:17-27. Jordon RE, Muller SA, Hale WL, et al. Bullous pemphigoid associated with systemic lupus erythematosus. Arch DermatoI1969;99:17-25. Miller JF, Downham TFII, Chapel T A. Coexistent bullous pemphigoid and systemic lupus erythematosus. Cutis 1978;21:368-73. Kumar V, Binder WL, Schotland E, et al. Coexistence of bullous pemphigoid and systemic lupus erythematosus. Arch DermatoI1978;114:1187-90. Szabo E, Husz S, Kovacs L. Coexistent atypical bullous pemphigoid and systemic lupus erythematosus. Br J DermatoI1981;104:71-5. Clayton CA, Burnham TK. Systemic lupus erythematosus and coexisting bullous pemphigoid: immunofluorescent investigations. J AM ACAD DERMATOL 1982;7:236-45. Stoll DM, King LE Jr. Association of bullous pemphigoid with systemic lupus erythematosus. Arch Dermatol1984; 120:362-6.
Journal of the American Academy of Dermatology
19. Gammon WR, WoodleyDT,DoleKC, etal. Evidence that anti-basement membrane zone antibodies in bullous eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen. J Invest Dermatoll985; 84:472-6. 20. Gammon WR, Briggaman RA, Inman AO III, et al. Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin. J Invest Dermatol 1984;82:139-44. 21. Camisa C, Sharma HM. Vesiculobullous systemic lupus erythematosus. Report of two cases and a review of the literature. J AM ACAD DERMATOL 1983;9:924~33. 22. Fine JD, Hintner H, Katz S1. Immunofluorescence studies in epidermolysis· bullosa utilizing polyclonal and monoclonal antibodies. In: Beutner EH, Chorzelski TP, Kumar V, eds. Immunopathology of the skin. 3rd ed. NewYork: John Wiley & Sons, 1987:399-405. 23. Beutner EH, Kumar V, Krasny SA, et al. Defined immunofluorescence in immunodermatology. In: Beutner EH, Chorzelski TP, Kumar V, eds. Immunopathology of the skin. 3rd ed. New York: John Wiley & Sons, 1987:3-40. 24. Stanley JR, Hawley-Nelson P, Yuspa SH, et al. Characterization of bullous pemphigoid antigen-a unique basement membrane protein of stratified squamous epithelium. Cell 1981;24:897-904. 25. Comaish S, McVittic E. Section blisters in bullous pemphigoid and other dermatoses. Br J DermatoI1973;89:127-32. 26. Dahl MGC, Cook L1. Lesions induced by trauma in pemphigoid. Br J DermatoI1979;101:469-73. 27. Scaletta LJ, Occhino JC, MacCallum DK, et al. Isolation and immunologic identification of basement membrane zone antigens from human skin. Lab Invest 1978;39:1-9. 28. Woodley D, Sauder D, Talley MJ, et al. Localization of basement membrane components after dermal-epidermal junction separation. J Invest Dermatol 1983;81:149-53. 29. Mutasim DF, Takahashi Y, Labib RS, et al. A pool ofbullous pemphigoid antigen(s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex. J Invest Dermatol 1985;84:47-53. 30. Westgate GE, Weaver AC, Couchman JR. Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes. J Invest Dermatol 1985; 84:218-24.
Direct immunofluorescence studies of sodium chloride-separated skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita W. R. Gammon, MD, a C. Kowalewski, MD,b T. P. Chorzelski, MD,b V. Kumar, PhD,c R. A. Briggaman, MD,a and E. H. Beutner, PhDc Chapel Hill, North Carolina, Warsaw, Poland, and Buffalo, New York Bullous pemphigoid and epidermolysis bullosa acquisita may have indistinguishable clinical, histologic, and routine immunohistologic features. In those cases these two diseases can be reliably distinguished in routine diagnostic studies only in seropositive cases by tests on lamina lucida-split skin and in research studies by direct immunoelectron microscopy or, in patients with circulating autoantibodies, by immunoblotting studies. The use of these methods is limited by the expense and unavailability of the methods, the requirement for circulating autoantibodies, or both. We describe a method to distinguish between the two diseases on the basis offindings of direct immunofluorescence of a biopsy specimen after separation through the lamina lucida with 1.0 mol/L sodium chloride. The IgG appeared in the dermal side of the split specimens in epidermolysis bullosa acquisita and predominantly or exclusively in the epidermal side in pemphigoid. The method was found to be relatively simple, inexpensive, applicable to specimens preserved in transport media, and 100% reliable in our group of 22 patients. (J AM ACAD DERMATOL 1990;22:664-70.)
Bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA) are chronic, inflammatory, subepidermal, blistering diseases characterized by circulating and tissue~bound autoantibodies to the cutaneous basement membrane zone (BMZ).1,2 Because anti~BMZ autoantibodies in both diseases belong predominantly to the IgG class and produce a linear pattern of IgG deposition at the BMZ, they cannot be reliably distinguished by routine direct and indirect immunofluorescence methods. However, they are readily distinguished by immunoelectron microscopy (IEM) and immunochemical
From the Department of Dermatology, The University of North Carolina, Chapel Hill,' the Department of Dermatology, Warsaw Academy of Medicine,b and the Departments of Microbiology. and Dermatology, The Ernest Witebsky Center for Immunology, University at Buffalo. SUNY.c SUPJXlrted in part by National Institu tes of Health grant Nos. AR30475 and ARI 0546, the Polish Academy ofScience, and Immco Diagnostics, Buffalo, N.Y. Reprint requests: W. R. Gammon, MD, Department of Dermatology. The University of North Carolina, Chapel Hill, NC 27514.
16/1/13567
664
methods. 3-9 Because of the expense and relative unavailability of IEM and immunochemical methods, differential diagnosis is usually made on the basis of differences in the epidemiologic, clinical, and histologic features of the diseases. Recent studies of patients with BP and EBA diagnosed on the basis of definitive IEM and/or immunochemical methods have provided evidence that attempts to distinguish between them on the basis of differences in epidemiologic, clinical, and histologic features can result in misdiagnosis. Patients with BP may have clinical and histologic features that resemble those classically associated with EBA, and patients with EBA may have clinical and histologic features that closely resemble those characteristic of BP. IO-12 Furthermore, patients have been reported with coexistent systemic lupus erythematosus (SLE) and BP who had clinical, histologic, and immunohistologic features that closely resembled those seen in bullous SLE (BSLE).13-19 BP and EBA also share similar, if not identical, routine direct and indirect immunofluorescent features, that is, linear deposits of predominantly IgG
Volume 22 Number 4
Direct immunofluorescence on NaCl-separated skin 665
April 1990
and C3 at the BMZ and IgG anti-BMZ autoantibodies, respectively.]], 12 In previous studies we2D described ~ method of indirect immunofluorescence on sodium chloride (NaCI)-separated normal human skin that could distinguish between circulating BP and EBA anti-BMZ autoantibodies. The method was shown to be a reliable means of distinguishing between patients with BP and those with EBA who had circulating anti-BMZ antibodies and offered the advantages of relative simplicity and economy over IEM and immunochemical methods. However, application of the method is limited because approximately 20% to 30% of patients with BP and 50% of those with EBA have been shown to lack those antibodies. To overcome that limitation, we investigated the use of direct immunofluorescence on 3 to 4 mm punch biopsy specimens of patients' clinically normal skin after separation of the epidermis from the dermis in 1.0 mol/L NaCl.
Table I. Diagnosis of patients entered in this study Method of diagnosis
Patient No.
10
BP BP BP BP EBA EBA EBA BSLE BP BP
11
BP
12 13
BP BP BP BP BP EBA EBA EBA EBA EBA EBA
1 2 3
4 5 6
7 8
9
14 15
16 17 18
MATERIAL AND METHODS Patients. Twenty-two patients (12 with BP, 9 with EBA, and 1 with BSLE) were studied. All had linear IgG deposits at the BMZ shown by routine direct immunofluorescence. The diagnosis of BP was made on the basis of clinical, histologic, and routine direct immunohistologic criteria. The diagnosis was confirmed in 10 patients with circulating IgG anti·BMZ autoantibodies by indirect immunofluorescence on NaCl-separated skin 20 (Table I). The diagnosis of EBA was made by clinical, histologic, and routine direct immunohistologic criteria. The diagnosis was confirmed in four patients by indirect immunofluorescence on NaCl-separated skin, IEM, or both20 (TableI). The diagnosis of BSLE was made in one patient on the basis of previously published clinical, histologic, routine direct immunohistologic, and IBM criteria. 2l Skin. Punch biopsy specimens (3 or 4 mm in diameter) of clinically normal perilesional skin were obtained with patients under local anesthesia. The specimens were rinsed brieflY with phosphate-buffered saline (PBS) and placed in 10 to 20 ml of 1.0 mol/L NaCl with or without 5 mmoljL EDTA. The specimens were incubated in NaCI with or without EDTA for 72 to 96 hours at 4 C and blotted on filter paper. With the use of fine forceps and a dissecting microscope, the epidermis was gently displaced laterally 1 mm across the dermis and was left adherent to the dermis. The specimens were then snapfrozen in liquid nitrogen, mounted in OCT compound (Miles Scientific, Naperville, IIl.) or PBS and sections 4 to 8 p,m thick were cut on a cryostat and mounted on glass slides. Specimens from four patients were placed in 0
Diagnosis
19 20 21 22
IIFon NaO-SS
I
!EM
+ +
+ +
+ +
+ + +
+ + +
+
+ + + + +
fEM, lmmunoelectron microscopy; IfF, indirect immunofluorescence; NaCl-8S, sodium chloride-separated skin; +, test performed; -, test not
performed.
Michel's transport medium immediately after being obtained. After several days in that medium, they were washed with PBS and incubated in NaCI as described. These specimens were found to be slightly more resistant to separation than ones placed directly into NaCl; however, all four separated without difficulty. Immunofluorescence mapping. The site of dermoepidermal separation in NaCl-separated specimens was determined with indirect immunofluorescence mapping,22 Reagents used to localize the site of separation included (1) a reference BP serum (1:40 dilution) from a patient with disease diagnosed by clinical, histologic, immunohistologic, and IBM criteria. The serum contained IgG anti-BMZ antibody (titer of 1:640) that consistently bound only to the epidermal side of N aCI-separated normal human skin by indirect immunofluorescence and to the lamina lucida-hemidesmosome complex of normal human skin by indirect IBM; (2) a previously characterized mouse monoclonal antibody (L3d) to human type VII collagen (1:100 dilution)]l; (3) polyc1onal rabbit antibodies to human Iaminin (l: 10 dilution; Gibeo #6803019, Gibeo, Madison, Wis.); and (4) polyclonal rabbit antibodies to human type IV collagen prepared by
Journal of the American Academy of Dermatology
666 Gammon et al.
Fig. 1. Immunofluorescence mapping of patient's biopsy specimen after separation in 1.0 mo1/L NaCl. Location of the split relative to lamina densa was examined by staining with monoclonal antibody to type VII coHagen. Type VII collagen staining is seen only on dermal side of separation, confirming separation above lamina densa. (X400.)
Fig. 2. Immunofluorescence mapping of biopsy specimen of patient with BP after separation in 1.0 mol/L NaCl. The biopsy specimen was stained for BP antigen with reference BP serum. Staining is observed only on epidermal side of separation, indicating separation in lamina lucida and confirming separation beneath keratinocyte plasma membrane. Direct IgG immunofluorescence of the biopsy specimen before incubation with reference BP serum showed the same pattern of staining. (X400.)
immunization with human type IV coHagen (1:16 dilution). Secondary antibodies were (1) fluorescein isothiocyanate (FITC)-conjugated anti-human IgG; (2) FITCconjugated goat IgG specific for mouse IgG (no cross-reactivity with human IgG); and (3) FITC-conjugatedgoat IgG specific for rabbit IgG. Direct immunofluorescence. Direct IgG, IgA, IgM, and C3 immunofluorescence of N aCI-separated biopsy specimens was performed with standard methods, that is, monospecific reagents with molar fluorescein to protein
ratios between 1.5 and 2.5 were diluted to contain 0.25 Vjml (approximately 50 p,g/ml of antibody protein).23
RESULTS
Immunofluorescence mapping Immunofluorescence mapping was performed on all 22 N aCI-separated biopsy specimens. When the specimens were examined with the use of antibodies to laminin or the lamina densa-sublamina densa
Volume 22 Number 4 April 1990
Direct immunofluorescence on NaCl-separated skin 667
Table II. Results of direct IgG immunofluorescence on patients' skin after separation in 1.0 moljL NaCl Location of IgG deposits No. of
Diagnosis
BP
EBA BSLE
patients
12 9 1
Epidermis
5
o o
I p~~d:::~ I Dermis 7
o
a
o 9 1
antigens, type VII collagen or type IV collagen, the results showed binding only to the dennal side of the separation (Fig. 1). We never observed binding of antibodies to lamina densa-sublamina densa antigens or laminin on the epidermal side of the separation as would occur if separation were beneath the lamina densa. These results showed that separation of the BMZ had occurred above the lamina densa in all specimens and was consistent with separation in the lamina lucida. To determine whether separation had occurred beneath the basal keratinocyte plasma membrane, mapping studies were performed with reference BP serum. Those results had to be interpreted in the context of direct immunofluorescent findings because all patients had human IgG deposited at the BMZ in vivo. In patients with a confirmed diagnosis of BP, there was no difference in the location of IgG deposits before and after indirect immunofluorescence with reference BP serum. In all cases, staining for IgG showed on the epidermal side of the separation consistent with separation beneath the keratinocyte plasma membrane. In some specimens, the intensity of immunofluorescence on the epidermal side of the separation was greater after treatment with the reference serum, which indicated that the site of separation was through the lamina lucida and below the BP antigen (Fig. 2). In patients with a previously confirmed diagnosis of EBA or BSLE, indirect immunofluorescence with reference BP serum also showed binding of IgG to the epidermal side of the separation, again indicative of separation beneath the keratinocyte plasma membrane (Fig. 3). Direct immunofluorescence of NaCl-separated biopsy specimens The results are shown in Table II. Direct IgG immunofluorescence of NaCl-separated biopsy specimens from the 12 patients with BP showed IgG staining either on only the epidermal side of the sep-
Fig. 3. Immunofluorescence mapping of biopsy specimen from patient with EBA after separation in 1.0 mol/L NaCl. a, IgG staining before incubation with reference BP serum shows in vivo-deposited IgG only on dermal side of separation. b, Same biopsy specimen after incubation with reference BP serum. IgG staining is observed on both epidermal and dermal sides of separation consistent with lamina lucida split beneath keratinocyte plasma membrane. (X400.)
aration in five patients (Fig. 2) or on both the epidermal and dermal sides of the separation in seven patients (Fig. 4). In the nine patients with EBA, direct immunofluorescence demonstrated IgG deposits only on the dermal side of the separation (Fig. 5). In the one patient with BSLE, direct immunofluorescence demonstrated IgG staining only on the dermal side of the separation. The IgA and IgM deposits in these specimens consistently codistributed with IgG deposits (results not shown). C3 deposits in patients with BP were consistently observed on both the epidermal and dermal sides of the separation even in patients whose IgG deposits were confined to the epidermal side. DISCUSSION
IEM and immunochemical studies have shown conclusively that BP and EBA are immunologically distinct diseases. Direct IEM studies have shown that the BMZ IgG deposits in BP are in the lamina
668 Gammon et aJ.
Journal of the American Academy of Dermatology
Fig. 4. Direct IgG immunofluorescence of biopsy specimen separated in 1.0 moljL NaCl from patient with BP. IgG staining is observed on both epidermal and dermal sides of separation. (X400.)
Fig. 5. Direct IgG immunofluorescence of biopsy specimen separated in 1.0 moljL NaCI from patient with EBA. IgG staining is observed on only dermal side of separation. (X400.)
lucida and are closely associated with the keratinocyte plasma membrane-hemidesrnosome complex whereas those in EBA are on and/or beneath the lamina densa. 3-5 Western immunoblots that use circulating BP and EBA anti-BMZ autoantibodies have shown that BP autoantibodies recognize a 240 or 180 kD noncollagenous glycoprotein in epidennal extracts whereas EBA autoantibodies recognize a 290 kD collagenous glycoprotein (type VII collagen) extracted from dermis-lamina densa. 6-9,24 Some patients with clinical, histologic, and immunohistologic features of BSLE also have anti-BMZ au-
toantibodies that are indistinguishable from EBA antibodies by IBM and immunoblotting methods. 19 Although directIEM and immunochemicalmethods can reliably distinguish between BP and EBA or BSLE, the methods are rarely used because of their unavailability and expense. In addition, immunochemical methods can only be used in patients with circulating anti-BMZ autoantibodies. Because routine direct and indirect immunofluorescence procedures cannot reliably distinguish the diseases, differential diagnosis is usually made on the basis of differences in epidemiologic, clinical, and histologic
Volume 22 Number 4 April 1990
Direct immunofluorescence on NaCl-separated skin 669
features. However, recent reports indicate that patients with BP may have some degree of skin fragility, trauma-induced lesions, lesions localized to extensor surfaces, an absence of inflammation, and healing with milia and sometimes scarring. I 1,12,25,26 They have also shown that patients with EBA may have a widespread inflammatory bullous eruption of flexural skin and an absence of clinically significant skin fragility, milia, or scarring early in the disease. 10, 11 The histologic features of lesions in patients with BP and EBA may also overlap. II It is apparent that there is no single and probably no combination of epidemiologic, clinical, and histologic features that can reliably distinguish all patients. Furthermore, evidence from some studies has shown that as many as 10% of patients with a diagnosis of BP may have EBA. lI In previous studies, we reported a method to distinguish BP and EBA antibodies by indirect immunofluorescence on 1.0 mol/L NaCl-separated normal human skin. In those studies, we found that circulating, anti-BMZ autoantibodies from patients with BP bound either to only the epidermal side of the separation (85% of cases) or to both the epidermal and dermal sides ofthe separation (15% of cases). In addition, we observed that anti-BMZ antibodies from patients with EBA and BSLE bound exclusively to the dermal side of the separation. I9,20 Although this method is relatively simple, inexpensive, and has become increasingly available, its application is limited to patients with circulating antiBMZ autoantibodies. This represents a significant limitation because as many as 20% to 30% of patients with BP and 50% of those with EBA may not have those antibodies. Incubation of normal human skin in 1.0 mol/L N aCl has been shown to separate the BMZ through the lamina lucida, leaving the keratinocyte plasma membrane and hemidesmosomes on the epidermal side of the separation and the lamina densa and sublamina densa zone on the dermal side of the separation. 2o,27,28 Because the BP antigen is in the high lamina lucida and closely associated with the keratinocyte plasma membrane-hemidesmosome complex and the EBA antigen is associated with the lamina densa-sublamina densa region, NaCl separation of the BMZ of patients' skin should allow separation of the two antigens as well as antibodies bound to those antigens in vivo. 2-5, 29,30 One of our concerns was that NaCI separation of patients' skin might result in atypical separation, es-
pecially in patients with EBA. In some patients with EBA, especially those with severe skin fragility, the BMZ may separate spontaneously beneath the lamina densa. 2 That site of separation theoretically would place the IgG deposits in EBA on either the epidermal or both sides of the separation. To ensure that the site of NaCl-induced separation was inthe lamina lucida, we routinely performed immunofluorescence mapping with antibodies to the BP antigen and antibodies to antigens located in the lamina densa-sublamina densa region. These studies showed that in all specimens, antibodies to lamina densa-sublamina densa antigens consistently bound only to the dermal side of the separation. Furthermore, in those specimens with in vivodeposited Igs on and beneath the lamina densa, we consistently observed BP antigen on only the epidermal side of the separation. Although we did not observe atypical splitting of the BMZ, the possibility that it could occur requires that all specimens be checked by immunofluorescence mapping with antibodies to at least one lamina densa antigen. Another of our initial concerns was that separation of patients' skin in 1.0 mol jL N aCl might elute the in vivo-bound immune deposits but that proved not to be a problem. We also observed that Michel's medium did not significantly interfere with either separation or immunofluorescence results. In NaCl-separated patients' skin, we found that the IgG deposits were always located either on only the epidermal side of the separation or on both the epidermal and dermal sides of the separation in pa~ tients with BP and on only the dermal side of the separation in patients with EBA and the one patient with BSLE. These studies demonstrate the reliability of direct immunofluorescence on NaCI-separated patients' skin to differentiate BP from EBA and BSLE. REFERENCES 1. Lever WF. Pemphigus and pemphigoid. A review of the advances made since 1964. J AM ACAD DERMATOL 1979; I:2-31. 2. Briggaman RA, Gammon WR, Woodley DT. Epidermolysis bullosa acquisita of the immunopathological type (dermolytic pemphigoid). J Invest Dermatol I 985;85:798-84s. 3. Nieboer C, Boorsma DM, Woerdeman MJ, eta!. Epidermolysis bullosa acquisita. Immunofluorescence, electron microscopic and immunoelectron microscopic studies in four patients. Dr J Dermatol 1980;102:383-92. 4. Yaoita H, Briggaman RA, Lawley T J, et al. Epidermolysis bullosa acquisita: ultrastructural and immunological studies. J Invest Dermatol 1981 ;76:288-92. 5. Horiguchi Y, Imamura S. Discrepancy between the local-
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19. Gammon WR, WoodleyDT,DoleKC, etal. Evidence that anti-basement membrane zone antibodies in bullous eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen. J Invest Dermatoll985; 84:472-6. 20. Gammon WR, Briggaman RA, Inman AO III, et al. Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin. J Invest Dermatol 1984;82:139-44. 21. Camisa C, Sharma HM. Vesiculobullous systemic lupus erythematosus. Report of two cases and a review of the literature. J AM ACAD DERMATOL 1983;9:924~33. 22. Fine JD, Hintner H, Katz S1. Immunofluorescence studies in epidermolysis· bullosa utilizing polyclonal and monoclonal antibodies. In: Beutner EH, Chorzelski TP, Kumar V, eds. Immunopathology of the skin. 3rd ed. NewYork: John Wiley & Sons, 1987:399-405. 23. Beutner EH, Kumar V, Krasny SA, et al. Defined immunofluorescence in immunodermatology. In: Beutner EH, Chorzelski TP, Kumar V, eds. Immunopathology of the skin. 3rd ed. New York: John Wiley & Sons, 1987:3-40. 24. Stanley JR, Hawley-Nelson P, Yuspa SH, et al. Characterization of bullous pemphigoid antigen-a unique basement membrane protein of stratified squamous epithelium. Cell 1981;24:897-904. 25. Comaish S, McVittic E. Section blisters in bullous pemphigoid and other dermatoses. Br J DermatoI1973;89:127-32. 26. Dahl MGC, Cook L1. Lesions induced by trauma in pemphigoid. Br J DermatoI1979;101:469-73. 27. Scaletta LJ, Occhino JC, MacCallum DK, et al. Isolation and immunologic identification of basement membrane zone antigens from human skin. Lab Invest 1978;39:1-9. 28. Woodley D, Sauder D, Talley MJ, et al. Localization of basement membrane components after dermal-epidermal junction separation. J Invest Dermatol 1983;81:149-53. 29. Mutasim DF, Takahashi Y, Labib RS, et al. A pool ofbullous pemphigoid antigen(s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex. J Invest Dermatol 1985;84:47-53. 30. Westgate GE, Weaver AC, Couchman JR. Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes. J Invest Dermatol 1985; 84:218-24.
