Desquamative Gingivitis: Immunologic Findings*
nofluorescent testing. Tests on cryostat cut frozen sections from the gingival lesion revealed complement 4 (C4) deposition in linear pattern along the basement membrane zone. Tests on sections from the adjacent normal gingival tissue revealed a linear deposition of I g G , I g A , I g M and C 4 along the basement membrane zone (Fig. 2). Immunofluorescent tests on the serum were negative for epithelial basement membrane zone antibodies and epithelial intercellular tissue antibodies. The immunofluorescent examination of the gingival biopsies and serum were consistent with a diagnosis of cicatricial pemphigoid (benign mucous membrane pemphigoid). Hematoxylin and eosin stained frozen sections from the lesion and adjacent normal tissue revealed subepidermal bullae (Figs. 3 A & 3 B ) . 1
by RUSSELL J . NISENGARD, D.D.S., PH.D.f ANDREW M . ALPERT, D.M.D.X VICTOR KRESTOW, M.D.$
CASE
REPORT
O n the recommendation of Philip L . McCarthy, M . D . a dermatologist and Victor Krestow, M . D . the patient was started on systemic corticosteroids. This initially consisted of 10 mg t.i.d. of prednisone which was decreased after 1 week because of the development of increased blood glucose values. Because of this increased blood glucose, the steroid therapy had to be altered. The patient was then continued on prednisone, 20 mg on an alternate day basis. After 3 months the gingival tissues had responded partially to therapy with reduction in the extent of the lesions. Epithelial sloughing of the gingiva was still observed, however, from the maxillary right cuspid to the right central incisor. Six months later some gingival bullae and desquamation were still observed. Immunofluorescent tests on serum taken at this 6-month evaluation were still negative.
A 66 Y E A R O L D white female presented with sore, painful gingival lesions of over 5 years duration. E x amination revealed the lesions were limited to the gingiva and not seen elsewhere in the mouth, on the skin or involving the eyes. The gingival lesions were generalized but more severe on the buccal and lingual gingival aspects of the lower anterior teeth (Figs. 1 A , I B , 1C). The gingiva appeared erythematous, and edematous with ulcerated necrotic areas and evidence of intact and ruptured gingival bullae. Gingival manipulation caused epithelial desquamation. Further periodontal evaluation revealed generalized horizontal bone loss with periodontal pockets ranging from 5 to 7 mm in the posterior aspects of the mouth. The periodontal diagnosis was desquamative gingivitis and generalized moderate to advanced periodontitis. During the previous 2 years, the patient was under treatment for hypertension, obesity and occasional phlebitis. A t a subsequent complete physical examination requested by her periodontist, an abnormal 4hour glucose tolerance test led to a diagnosis of mild, adult onset diabetes. The patient was placed on a glucose restricted diet resulting in a normal 2 hour postprandial blood sugar. A gingival biopsy sent for routine histologic examination revealed inflamed granulation tissue partially covered by stratified squamous epithelium. Most of the epithelium had desquamated just beneath the basal cell layer. The granulation tissue consisting of loose collagen fiber bundles had an inflammatory infiltrate primarily of neutrophils but with some plasma cells and lymphocytes. The diagnosis made from this biopsy was chronic desquamative gingivitis and nonspecific ulceration. Biopsies from a gingival lesion and adjacent normal gingiva and serum were subsequently sent for immu-
2
COMMENT
Chronic desquamative gingivitis is a common clinical manifestation of a number of entities: dermatoses, hormonal factors, irritational factors and idiopathic factors. The dermatoses include cicatricial pemphigoid (benign mucous membrane pemphigoid), pemphigus and lichen planus. In a study of 40 patients with desquamative gingivitis, McCarthy and Shklar identified 17 cases of cicatricial pemphigoid, two cases of pemphigus and four cases of lichen planus. The remaining cases were hormonal changes, idiopathic disease and abnormal responses to local factors such as calculus. Because of the clinical course of pemphigus and cicatricial pemphigoid, early diagnosis and appropriate treatment is necessary. The diagnosis can be made frequently on the basis of histopathology. Sometimes however, a definite diagnosis is not possible by routine biopsy examination. This is exemplified by this particular case. Immunologic tests can provide valuable information in addition to clinical and histopathologic examination. Pemphigus and pemphigoid are thought to be autoimmune diseases while lichen planus has an 3
3
* Supported by U n i t e d States Public Health Service Grant DE03408. t State University of N e w Y o r k at Buffalo, Buffalo, New Y o r k 14214. t North M i a m i B e a c h , F l o r i d a .
27
28
J. Periodontol. January, 1978
Nisengard, Alpert, Krestow
FIGURE
1A
,
I B ,
1 C . Desquamative gingival lesions can be seen in areas throughout the mouth.
F I G U R E 2. Direct immunofluorescence test on biopsy from clinically normal gingiva. Section was stained with an antihuman IgG conjugate revealing IgG deposition along the basement membrane zone.
immunologic component. Characteristic and sometimes diagnostic findings are seen by immunofluorescent studies of these diseases. APPLICATION DIAGNOSIS
OF IMMUNOFLUORESCENCE FOR OF DESQUAMATIVE
GINGIVITIS
A . Immunofluorescent Methods Immunofluorescent (IF) studies are performed on both sera and biopsies: indirect IF tests on sera reveal circulating antibodies to tissue antigens and direct I F tests on biopsies reveal the histological location of in vivo bound immunoglobulins and complement. Figure 4
F I G U R E 3 A . Hematoxylin and eosin stained frozen section from clinically normal gingiva. These are small subepidermal bullae. B . Hematoxylin and eosin stained section from desquamative lesion. There is complete epithelial desquamation along the dermoepidermal junction.
4 summarizes the steps in both the direct and indirect IF tests. Cryostat cut biopsy sections, 4 p, or less in thickness are prepared for direct I F tests. Sections of the biopsy are incubated with monospecific fluorescein-labelled antihuman I g G , I g A , I g M , complement 3 or complement 4. After this, sections are washed in phosphate buffered saline and examined under a microscope equipped for fluorescence microscopy. The histologic localization of immunoglobulins and complement are revealed as an apple-green fluorescence. For indirect immunofluorescent tests, serum dilu-
Volume 49 Number 1
Desquamative Gingivitis 29
F I G U R E 4. Principles of direct immunofluorescence on biopsies for in vivo bound immunoglobulins cence on sera for circulating antibodies to tissue antigens.
tions are incubated on cryostat cut sections of monkey esophagus. The tissue sections are then washed in phosphate saline to remove free, nonbound serum constituents. To identify antibodies from the serum that are now bound to the tissue sections, fluoresceinlabelled antihuman I g G is incubated on the sections. The sections are again washed to remove any nonbound conjugate and are examined under the microscope for fluorescence of specific structures. The titers of the tissue antibodies are determined by examination of tissue sections incubated with doubling dilutions of the patient's serum. B . Specimens Sera. Serum from 8 to 10 ml of blood collected without anticoagulants is sufficient for testing. After the blood clots, the serum should be removed and sent by first class mail at ambient temperature to the testing laboratory. During transit the specimens do not have to be frozen or cooled. Biopsies. The sites of gingival biopsies must be carefully selected. The epithelium must be intact and not completely desquamated so the test can be properly evaluated. In addition, crevicular epithelium should be excluded since this tissue could give rise to false positive findings. Staining intercellularly could occur as a result of the crevicular fluid which contains immunoglobulins and complement. Two biopsies should be taken. One 3 to 5 mm diameter biopsy should be taken from the edge of a desquamative lesion, preferably a fresh lesion. A second 3 to 5 mm diameter biopsy should be taken from adjacent clinically normal gingival tissue. Biopsies for immunofluorescent tests must be specifically handled and not fixed in formalin. Two acceptable methods are currently in use: Biopsies can either be quick frozen and transported frozen on dry ice or secondly, transported at ambient temperature in holding solution described by M i c h e l which contains N ethylmaleimide and ammonium sulfate. Quick freezing 5
and indirect
immunofluores-
is more widely used and is still considered the method of choice even though it is more cumbersome, requiring that specimens be kept frozen during transport. Biopsies can be quick frozen by placing the tissue directly into liquid nitrogen or by placing a tube containing the tissue into a slush of dry ice mixed with acetone or ethanol. Once frozen, biopsies must be kept frozen until processed for testing. Such quick freezing of gingival biopsies would probably have to be done at a hospital which has liquid nitrogen and dry ice facilities. Transport of biopsies in Michel's holding solution is simple and best adapted for a clinical office. However, there has been only limited experience with this and the possibility of false negatives and false positives cannot be completely discounted. Biopsies are immediately placed in tubes of Michel's holding solution and mailed at ambient temperatures to the testing laboratory. Experience with this method have revealed that biopsies in transit for over 2 weeks from Warsaw, Poland to Buffalo, New Y o r k have yielded similar results as seen with quick frozen biopsies tested in Warsaw. C . Results For desquamative gingivitis, immunologic studies should be performed on both serum and biopsies. Immunologic findings for dermatoses manifesting as desquamative gingivitis are summarized in Table 1. Findings in cicatricial pemphigoid, bullous pemphigoid and pemphigus are diagnostic while those for lichen planus are found in several other dermatoses. Pemphigoid. Bullous pemphigoid appears clinically as bullous eruptions primarily of the skin and less commonly of the mucous membranes. Cicatricial pemphigoid, on the other hand, involves primarily the oral mucosa and conjunctiva and less commonly the skin. Skin lesions usually appear as subepidermal tense bullae. The mucosal lesions frequently rupture leading to ulcerated areas and subsequent residual scarring on healing. 4
30
J . Periodontol. January, 1978
Nisengard, Alpert, Krestow T A B L E 1. Immunologic
Findings of Importance in the Diagnosis of Desquamative
Gingivitis
Serum
Biopsies
Underlying Disease Types of antibodies Basement membrane antibodies Basement membrane antibodies Intercellular antibodies of epithelium
Cicatricial pemphigoid Bullous pemphigoid Pemphigus
Findings
Pattern of I F
Findings -or+*
Basement membrane zone
+
+ (80%)
Basement membrane zone
+ (100%)
+ (over 9 5 % )
Intercellular deposits
+ (100%)
Globular deposits in dermis and epidermis
+ (97%)
L i c h e n planus * Most cases are negative for serum antibodies.
Similar immunologic findings of basement membrane zone antibodies are observed in both bullous pemphigoid and cicatricial pemphigoid, however, the incidence of serum antibodies is considerably lower in cicatricial pemphigoid. Antibodies to the basement membrane of stratified squamous epithelium are often found in the serum (Fig. 5) and bound in vivo to the basement membrane zone of biopsies (Fig. 2). Serum antibodies to basement membrane zone antigens of stratified zone antigens of stratified epithelium are specific and diagnostic for p e m p h i g o i d . In bullous pemphigoid, antibodies frequently of high titer (greater than 160) are found in more than 8 0 % of the cases. In cicatricial pemphigoid, a lower incidence of serum antibodies has been reported (10/43 or 23%) and these are of low t i t e r . ' Antibody titers to the basement membrane zone do not correlate well with the clinical severity of the disease. Sometimes they can't even be detected in serum during the disease or may persist long after the lesions disappear. 6,7
6
8 - 1 2
Biopsy studies are necessary when serum tests are negative or titers are 20 or less. Immunoglobulins and complement are localized along the basement membrane in virtually 1 0 0 % of bullous pemphigoid cases and approximately 8 0 % of cicatricial pemphigoid cases. In some cases, positive findings are only observed after examination of many serial sections from multiple biopsies. Pemphigus. Pemphigus, particularly pemphigus vulgaris which is the most common form, is a bullous disease of the mucous membranes and secondarily involving the skin. Skin lesions appear as flaccid bulla. These intraepithelial bullae contain free epithelial cells or acantholytic cells. Skin lesions and even more commonly mucosal lesions may rupture leaving an ulcerated surface. Intercellular epithelial antibodies are usually present in serum and/or fixed intercellularly to the oral mucosa and skin (Fig. 6 A & 6 B ) . Intercellular antibodies, sometimes termed pemphigus antibodies are both disease specific and diagnostic for pemphigus. Serum titers are found in over 9 5 % of active cases but may not be detectable during the early stages of the disease when there are only a few lesions or during remission. 7
6,7
Titers of 10 to 20 in the presence of typical clinical and histologic findings are significant. Atypical clinical or histologic findings and low titers of 10 or 20 may suggest "pemphigus-like antibodies" rather than true pemphigus antibodies. This can be confirmed by i m munofluorescence studies of biopsies since only true pemphigus antibodies bind intercellularly in vivo and will give positive biopsy findings. Pemphigus-like antibodies of low titer have been reported in patients with severe burns and in patients exhibiting penicillin reactions. Pemphigus antibody titers of 40 or more even with atypical clinical findings are significant. Confirmation by direct immunofluorescent studies is still desirable. 13-15
Titers of intercellular antibodies frequently correlate with disease activity. Titers rise during exacerbations and fall during remissions. Since these changes may sometimes precede clinical changes, monitoring titers by multiple serum samples can serve as guides to therapy. A rise in titer of two or more doubling dilutions may signal an impending relapse while a fall in titer of two or more doubling dilutions can occur during clinical control. Serum specimens should be tested every 2 to 3 weeks until remission and then at 1 to 6 month intervals depending on clinical control of the disease. Gingival biopsies should be tested by direct immunofluorescence when serum tests are negative or intercellular antibody titers are 10 to 20 or when there are atypical clinical and histologic findings. Intercellular deposits of I g G are seen in biopsies from almost all cases of true pemphigus. I g A , I g M and/or complement deposits are also frequently seen. Lichen Planus. Lichen planus appears as a papulosquamous eruption of the skin and mucous membrane. The ulcerative form on the gingiva can appear as desquamative gingivitis. Serum studies for tissue antibodies in lichen planus have been negative. Immunofluorescent studies of biopsies, however, reveal globular deposits of immunoglobulin and complement at the dermoepidermal junction (Fig. 7 ) . The deposits, sometimes called cytoid bodies or Civatte bodies, are usually seen in the papillary dermis at the dermoepidermal junction in clusters. These globules are more 16
7
17
Volume 49 Number 1
F I G U R E 5 . Indirect immunofluorescence membrane zone antibodies.
Desquamative
on monkey esophagus. Sera from
bullous pemphigoid
Gingivitis
31
patient reveals basement
F I G U R E 7. Direct immunofluorescence on biopsy from patient with lichen planus demonstrates staining of cytoid bodies.
common in lesions than in normal tissue and are more frequent in skin than in mucous membranes. Cytoid bodies are not disease specific but their identification can be helpful when the histopathology is inconclusive. The incidence of such deposits is: 9 7 % in lichen planus; 4 1 % in systemic lupus erythematosus; 7 0 % in discoid lupus erythematosus; 7 5 % in dermatomyositis; 5 0 % in erythema multiforme; 4 0 % in pemphigus; 5 0 % in pemphigoid and 4 0 % in normals. The globules seen in lichen planus sometimes can be differentiated from those in the other disease and normals on the basis of pattern and size. The globules tend to cluster in groups of 10 or more in lichen planus but have a 6
F I G U R E 6A. Indirect immunofluorescence on monkey esophagus. Sera from a pemphigus patient reveals epithelial intercellular antibodies. B . Direct immunofluorescence on biopsy from pemphigus patient reveals staining intercellularly in the epithelium and on intraepithelial bullae.
J. Periodontal. January, 1978
32 N i s e n g a r d , A l p e r t , K r e s t o w more linear pattern in lupus erythematosus. Those seen in the skin of normal individuals are usually much smaller and fewer in numbers. SUMMARY
A case of desquamative gingivitis is presented that was a gingival manifestation of cicatricial pemphigoid. Immunologic studies of serum and biopsies were of diagnostic significance. The application of immuno fluorescence for the diagnosis of desquamative gingival lesions was discussed. If a definite diagnosis can not be made on the basis of light microscopy, immunofluores cence studies should be performed. ADDENDUM
The authors are continuing immunologic studies of desquamative gingivitis and would welcome specimens for study. REFERENCES
1. Beutner, E . H . , and Nisengard, R . J.: Defined immu nofluorescence in clinical immunology. Beutner, E . H . , Chorzelski, T. P., Bean, S. F . , and Jordan, R . E . (eds), I m m u n o p a t h o l o g y of t h e S k i n : L a b e l e d A n t i b o d y Studies. Stroudsburg, Pa., Dowden, Hutchinson and Ross, Inc., 1973. 2. Personal communication. 3. McCarthy, P. L . and Shklar, G . : Disease of t h e O r a l M u c o s a : D i a g n o s i s , M a n a g e m e n t a n d T h e r a p y , pp 183-191. New York, McGraw Book Co., 1964. 4. Jablonska, S., et al.: Uses for IF tests of skin and sera: Utilization of immunofluorescence in the diagnosis of bullous diseases, lupus erythematosus and certain other dermatoses.
A r c h D e r m a t o l 3: 371, 1975. 5. Michel, B., Milner, Y . , and David, K.: Preservation of tissue fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases: Preliminary report. J I n v e s t D e r m a t o l 59: 449, 1973. 6. Jablonska, D . , Chorzelski, T. P., Beutner, E . H . , and Holubar, K.: Indications for skin and serum immunofluorescent studies in dermatology. See 1 above, Chap. 1. 7. Beutner, E . H . , Chorzelski, T. P., and Jordan, R . E . : Autosensitization i n Pemphigus and Bullous Pemphigoid. Springfield, Illinois, Charles C. Thomas, 1970. 8. Bean, S. F . , and Michel, B . : Cicatricial pemphigoid. See 1 above, Chap. 4. 9. Holubar, K., Honigsmann, H . , and Wolff, K.: Cicatri cial pemphigoid immunofluorescent investigations. A r c h D e r m a t o l 108: 50, 1973. 10. Dantzig, P.: Circulating antibodies in cicatricial pem phigoid. A r c h D e r m a t o l 108: 264, 1973. 11. Dabelsteen, E . , Ullman, S., Thompson, K . , and Rygaard, J.: Demonstration of basement membrane auto antibodies in patients with benign mucous membrane pem phigoid. A eta D e r m V e n e r e o l 54: 189, 1974. 12. Rogers, R . S., Sheridan, P. J . , and Jordan, R . E . : Desquamative gingivitis. O r a l S u r g 42: 316, 1976. 13. Ablin, R. J . , Milgrom, F . , Kano, K . , Rapaport, F . , and Beutner, E . H . : Pemphigus-like antibodies in patients with skin burns. Vox Sang 16: 73, 1969. 14. Thivolet, J . , and Beyvin, A . J.: Recherche par im munofluorescence d'auto-anticorps seriques vis-a-vis des con stituants de l'epiderme chez les brules. E x p e r i e n t i a 24: 945, 1968. 15. Fellner, M . J . , and Prutkin, L . : Morbilliform erup tions caused by penicillin. J I n v e s t D e r m a t o l 55: 390, 1970. 16. Chorzelski, T. P., Jablonska, S., and Beutner, E . H . : Clinical significance of pemphigus antibodies. See 1 above, Chap. 2. 17. Michel, B., and Sy, E . K.: Tissue-fixed immunoglob ulins in lichen planus. See 1 above, Chap. 12.
Abstract MICROSCOPIC EVALUATION OF CLINICAL MEASUREMENTS OF CONNECTIVE TISSUE ATTACHMENT LEVELS
Armitage, Gary C , Svanberg, Gunnar K., and Löe, Harald J C l i n Periodontol 4: 173, 1977. The purpose of this study was to determine how accurately periodontal probes measure connective tissue attachment levels in beagle dogs with (1) clinically healthy gingivae, (2) experimental gingivitis, and (3) periodontitis. In the healthy and experimental gin givitis specimens the probes were inserted with a standardized force of 25 ponds. In periodontitis specimens the probes were inserted with a gentle, but nonstandardized force. After insertion, 120 plastic periodontal probes (40 in each group) were held in place by fusing them to the teeth. Blocks of periodontal tissue with the probes in situ were subsequently processed and serially sectioned. Histometric
measurements were made from the sections in order to compare the level of connective tissue attachment to the level of probe penetra tion. In healthy specimens the probes consistently failed to reach the apical termination of the junctional epithelium (x = -0.39 mm). In the experimental gingivitis group most probes came closer to the apical termination of the junctional epithelium, but on the average still fell short by x = —0.10 mm. In periodontitis specimens the probes consistently went past the most apical cells of the junctional epithe lium (x = +0.24 mm). A significant relationship between the degree of inflammation and level of probe penetration was found. No rela tionship was observed between histological and clinical sulcus depths. It is concluded that in beagle dogs (1) periodontal probes do not pre cisely measure connective tissue attachment levels, (2) inflammation has a significant influence on the degree of probe penetration, and (3) histological and clinical sulcus depths differ significantly.
nofluorescent testing. Tests on cryostat cut frozen sections from the gingival lesion revealed complement 4 (C4) deposition in linear pattern along the basement membrane zone. Tests on sections from the adjacent normal gingival tissue revealed a linear deposition of I g G , I g A , I g M and C 4 along the basement membrane zone (Fig. 2). Immunofluorescent tests on the serum were negative for epithelial basement membrane zone antibodies and epithelial intercellular tissue antibodies. The immunofluorescent examination of the gingival biopsies and serum were consistent with a diagnosis of cicatricial pemphigoid (benign mucous membrane pemphigoid). Hematoxylin and eosin stained frozen sections from the lesion and adjacent normal tissue revealed subepidermal bullae (Figs. 3 A & 3 B ) . 1
by RUSSELL J . NISENGARD, D.D.S., PH.D.f ANDREW M . ALPERT, D.M.D.X VICTOR KRESTOW, M.D.$
CASE
REPORT
O n the recommendation of Philip L . McCarthy, M . D . a dermatologist and Victor Krestow, M . D . the patient was started on systemic corticosteroids. This initially consisted of 10 mg t.i.d. of prednisone which was decreased after 1 week because of the development of increased blood glucose values. Because of this increased blood glucose, the steroid therapy had to be altered. The patient was then continued on prednisone, 20 mg on an alternate day basis. After 3 months the gingival tissues had responded partially to therapy with reduction in the extent of the lesions. Epithelial sloughing of the gingiva was still observed, however, from the maxillary right cuspid to the right central incisor. Six months later some gingival bullae and desquamation were still observed. Immunofluorescent tests on serum taken at this 6-month evaluation were still negative.
A 66 Y E A R O L D white female presented with sore, painful gingival lesions of over 5 years duration. E x amination revealed the lesions were limited to the gingiva and not seen elsewhere in the mouth, on the skin or involving the eyes. The gingival lesions were generalized but more severe on the buccal and lingual gingival aspects of the lower anterior teeth (Figs. 1 A , I B , 1C). The gingiva appeared erythematous, and edematous with ulcerated necrotic areas and evidence of intact and ruptured gingival bullae. Gingival manipulation caused epithelial desquamation. Further periodontal evaluation revealed generalized horizontal bone loss with periodontal pockets ranging from 5 to 7 mm in the posterior aspects of the mouth. The periodontal diagnosis was desquamative gingivitis and generalized moderate to advanced periodontitis. During the previous 2 years, the patient was under treatment for hypertension, obesity and occasional phlebitis. A t a subsequent complete physical examination requested by her periodontist, an abnormal 4hour glucose tolerance test led to a diagnosis of mild, adult onset diabetes. The patient was placed on a glucose restricted diet resulting in a normal 2 hour postprandial blood sugar. A gingival biopsy sent for routine histologic examination revealed inflamed granulation tissue partially covered by stratified squamous epithelium. Most of the epithelium had desquamated just beneath the basal cell layer. The granulation tissue consisting of loose collagen fiber bundles had an inflammatory infiltrate primarily of neutrophils but with some plasma cells and lymphocytes. The diagnosis made from this biopsy was chronic desquamative gingivitis and nonspecific ulceration. Biopsies from a gingival lesion and adjacent normal gingiva and serum were subsequently sent for immu-
2
COMMENT
Chronic desquamative gingivitis is a common clinical manifestation of a number of entities: dermatoses, hormonal factors, irritational factors and idiopathic factors. The dermatoses include cicatricial pemphigoid (benign mucous membrane pemphigoid), pemphigus and lichen planus. In a study of 40 patients with desquamative gingivitis, McCarthy and Shklar identified 17 cases of cicatricial pemphigoid, two cases of pemphigus and four cases of lichen planus. The remaining cases were hormonal changes, idiopathic disease and abnormal responses to local factors such as calculus. Because of the clinical course of pemphigus and cicatricial pemphigoid, early diagnosis and appropriate treatment is necessary. The diagnosis can be made frequently on the basis of histopathology. Sometimes however, a definite diagnosis is not possible by routine biopsy examination. This is exemplified by this particular case. Immunologic tests can provide valuable information in addition to clinical and histopathologic examination. Pemphigus and pemphigoid are thought to be autoimmune diseases while lichen planus has an 3
3
* Supported by U n i t e d States Public Health Service Grant DE03408. t State University of N e w Y o r k at Buffalo, Buffalo, New Y o r k 14214. t North M i a m i B e a c h , F l o r i d a .
27
28
J. Periodontol. January, 1978
Nisengard, Alpert, Krestow
FIGURE
1A
,
I B ,
1 C . Desquamative gingival lesions can be seen in areas throughout the mouth.
F I G U R E 2. Direct immunofluorescence test on biopsy from clinically normal gingiva. Section was stained with an antihuman IgG conjugate revealing IgG deposition along the basement membrane zone.
immunologic component. Characteristic and sometimes diagnostic findings are seen by immunofluorescent studies of these diseases. APPLICATION DIAGNOSIS
OF IMMUNOFLUORESCENCE FOR OF DESQUAMATIVE
GINGIVITIS
A . Immunofluorescent Methods Immunofluorescent (IF) studies are performed on both sera and biopsies: indirect IF tests on sera reveal circulating antibodies to tissue antigens and direct I F tests on biopsies reveal the histological location of in vivo bound immunoglobulins and complement. Figure 4
F I G U R E 3 A . Hematoxylin and eosin stained frozen section from clinically normal gingiva. These are small subepidermal bullae. B . Hematoxylin and eosin stained section from desquamative lesion. There is complete epithelial desquamation along the dermoepidermal junction.
4 summarizes the steps in both the direct and indirect IF tests. Cryostat cut biopsy sections, 4 p, or less in thickness are prepared for direct I F tests. Sections of the biopsy are incubated with monospecific fluorescein-labelled antihuman I g G , I g A , I g M , complement 3 or complement 4. After this, sections are washed in phosphate buffered saline and examined under a microscope equipped for fluorescence microscopy. The histologic localization of immunoglobulins and complement are revealed as an apple-green fluorescence. For indirect immunofluorescent tests, serum dilu-
Volume 49 Number 1
Desquamative Gingivitis 29
F I G U R E 4. Principles of direct immunofluorescence on biopsies for in vivo bound immunoglobulins cence on sera for circulating antibodies to tissue antigens.
tions are incubated on cryostat cut sections of monkey esophagus. The tissue sections are then washed in phosphate saline to remove free, nonbound serum constituents. To identify antibodies from the serum that are now bound to the tissue sections, fluoresceinlabelled antihuman I g G is incubated on the sections. The sections are again washed to remove any nonbound conjugate and are examined under the microscope for fluorescence of specific structures. The titers of the tissue antibodies are determined by examination of tissue sections incubated with doubling dilutions of the patient's serum. B . Specimens Sera. Serum from 8 to 10 ml of blood collected without anticoagulants is sufficient for testing. After the blood clots, the serum should be removed and sent by first class mail at ambient temperature to the testing laboratory. During transit the specimens do not have to be frozen or cooled. Biopsies. The sites of gingival biopsies must be carefully selected. The epithelium must be intact and not completely desquamated so the test can be properly evaluated. In addition, crevicular epithelium should be excluded since this tissue could give rise to false positive findings. Staining intercellularly could occur as a result of the crevicular fluid which contains immunoglobulins and complement. Two biopsies should be taken. One 3 to 5 mm diameter biopsy should be taken from the edge of a desquamative lesion, preferably a fresh lesion. A second 3 to 5 mm diameter biopsy should be taken from adjacent clinically normal gingival tissue. Biopsies for immunofluorescent tests must be specifically handled and not fixed in formalin. Two acceptable methods are currently in use: Biopsies can either be quick frozen and transported frozen on dry ice or secondly, transported at ambient temperature in holding solution described by M i c h e l which contains N ethylmaleimide and ammonium sulfate. Quick freezing 5
and indirect
immunofluores-
is more widely used and is still considered the method of choice even though it is more cumbersome, requiring that specimens be kept frozen during transport. Biopsies can be quick frozen by placing the tissue directly into liquid nitrogen or by placing a tube containing the tissue into a slush of dry ice mixed with acetone or ethanol. Once frozen, biopsies must be kept frozen until processed for testing. Such quick freezing of gingival biopsies would probably have to be done at a hospital which has liquid nitrogen and dry ice facilities. Transport of biopsies in Michel's holding solution is simple and best adapted for a clinical office. However, there has been only limited experience with this and the possibility of false negatives and false positives cannot be completely discounted. Biopsies are immediately placed in tubes of Michel's holding solution and mailed at ambient temperatures to the testing laboratory. Experience with this method have revealed that biopsies in transit for over 2 weeks from Warsaw, Poland to Buffalo, New Y o r k have yielded similar results as seen with quick frozen biopsies tested in Warsaw. C . Results For desquamative gingivitis, immunologic studies should be performed on both serum and biopsies. Immunologic findings for dermatoses manifesting as desquamative gingivitis are summarized in Table 1. Findings in cicatricial pemphigoid, bullous pemphigoid and pemphigus are diagnostic while those for lichen planus are found in several other dermatoses. Pemphigoid. Bullous pemphigoid appears clinically as bullous eruptions primarily of the skin and less commonly of the mucous membranes. Cicatricial pemphigoid, on the other hand, involves primarily the oral mucosa and conjunctiva and less commonly the skin. Skin lesions usually appear as subepidermal tense bullae. The mucosal lesions frequently rupture leading to ulcerated areas and subsequent residual scarring on healing. 4
30
J . Periodontol. January, 1978
Nisengard, Alpert, Krestow T A B L E 1. Immunologic
Findings of Importance in the Diagnosis of Desquamative
Gingivitis
Serum
Biopsies
Underlying Disease Types of antibodies Basement membrane antibodies Basement membrane antibodies Intercellular antibodies of epithelium
Cicatricial pemphigoid Bullous pemphigoid Pemphigus
Findings
Pattern of I F
Findings -or+*
Basement membrane zone
+
+ (80%)
Basement membrane zone
+ (100%)
+ (over 9 5 % )
Intercellular deposits
+ (100%)
Globular deposits in dermis and epidermis
+ (97%)
L i c h e n planus * Most cases are negative for serum antibodies.
Similar immunologic findings of basement membrane zone antibodies are observed in both bullous pemphigoid and cicatricial pemphigoid, however, the incidence of serum antibodies is considerably lower in cicatricial pemphigoid. Antibodies to the basement membrane of stratified squamous epithelium are often found in the serum (Fig. 5) and bound in vivo to the basement membrane zone of biopsies (Fig. 2). Serum antibodies to basement membrane zone antigens of stratified zone antigens of stratified epithelium are specific and diagnostic for p e m p h i g o i d . In bullous pemphigoid, antibodies frequently of high titer (greater than 160) are found in more than 8 0 % of the cases. In cicatricial pemphigoid, a lower incidence of serum antibodies has been reported (10/43 or 23%) and these are of low t i t e r . ' Antibody titers to the basement membrane zone do not correlate well with the clinical severity of the disease. Sometimes they can't even be detected in serum during the disease or may persist long after the lesions disappear. 6,7
6
8 - 1 2
Biopsy studies are necessary when serum tests are negative or titers are 20 or less. Immunoglobulins and complement are localized along the basement membrane in virtually 1 0 0 % of bullous pemphigoid cases and approximately 8 0 % of cicatricial pemphigoid cases. In some cases, positive findings are only observed after examination of many serial sections from multiple biopsies. Pemphigus. Pemphigus, particularly pemphigus vulgaris which is the most common form, is a bullous disease of the mucous membranes and secondarily involving the skin. Skin lesions appear as flaccid bulla. These intraepithelial bullae contain free epithelial cells or acantholytic cells. Skin lesions and even more commonly mucosal lesions may rupture leaving an ulcerated surface. Intercellular epithelial antibodies are usually present in serum and/or fixed intercellularly to the oral mucosa and skin (Fig. 6 A & 6 B ) . Intercellular antibodies, sometimes termed pemphigus antibodies are both disease specific and diagnostic for pemphigus. Serum titers are found in over 9 5 % of active cases but may not be detectable during the early stages of the disease when there are only a few lesions or during remission. 7
6,7
Titers of 10 to 20 in the presence of typical clinical and histologic findings are significant. Atypical clinical or histologic findings and low titers of 10 or 20 may suggest "pemphigus-like antibodies" rather than true pemphigus antibodies. This can be confirmed by i m munofluorescence studies of biopsies since only true pemphigus antibodies bind intercellularly in vivo and will give positive biopsy findings. Pemphigus-like antibodies of low titer have been reported in patients with severe burns and in patients exhibiting penicillin reactions. Pemphigus antibody titers of 40 or more even with atypical clinical findings are significant. Confirmation by direct immunofluorescent studies is still desirable. 13-15
Titers of intercellular antibodies frequently correlate with disease activity. Titers rise during exacerbations and fall during remissions. Since these changes may sometimes precede clinical changes, monitoring titers by multiple serum samples can serve as guides to therapy. A rise in titer of two or more doubling dilutions may signal an impending relapse while a fall in titer of two or more doubling dilutions can occur during clinical control. Serum specimens should be tested every 2 to 3 weeks until remission and then at 1 to 6 month intervals depending on clinical control of the disease. Gingival biopsies should be tested by direct immunofluorescence when serum tests are negative or intercellular antibody titers are 10 to 20 or when there are atypical clinical and histologic findings. Intercellular deposits of I g G are seen in biopsies from almost all cases of true pemphigus. I g A , I g M and/or complement deposits are also frequently seen. Lichen Planus. Lichen planus appears as a papulosquamous eruption of the skin and mucous membrane. The ulcerative form on the gingiva can appear as desquamative gingivitis. Serum studies for tissue antibodies in lichen planus have been negative. Immunofluorescent studies of biopsies, however, reveal globular deposits of immunoglobulin and complement at the dermoepidermal junction (Fig. 7 ) . The deposits, sometimes called cytoid bodies or Civatte bodies, are usually seen in the papillary dermis at the dermoepidermal junction in clusters. These globules are more 16
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Volume 49 Number 1
F I G U R E 5 . Indirect immunofluorescence membrane zone antibodies.
Desquamative
on monkey esophagus. Sera from
bullous pemphigoid
Gingivitis
31
patient reveals basement
F I G U R E 7. Direct immunofluorescence on biopsy from patient with lichen planus demonstrates staining of cytoid bodies.
common in lesions than in normal tissue and are more frequent in skin than in mucous membranes. Cytoid bodies are not disease specific but their identification can be helpful when the histopathology is inconclusive. The incidence of such deposits is: 9 7 % in lichen planus; 4 1 % in systemic lupus erythematosus; 7 0 % in discoid lupus erythematosus; 7 5 % in dermatomyositis; 5 0 % in erythema multiforme; 4 0 % in pemphigus; 5 0 % in pemphigoid and 4 0 % in normals. The globules seen in lichen planus sometimes can be differentiated from those in the other disease and normals on the basis of pattern and size. The globules tend to cluster in groups of 10 or more in lichen planus but have a 6
F I G U R E 6A. Indirect immunofluorescence on monkey esophagus. Sera from a pemphigus patient reveals epithelial intercellular antibodies. B . Direct immunofluorescence on biopsy from pemphigus patient reveals staining intercellularly in the epithelium and on intraepithelial bullae.
J. Periodontal. January, 1978
32 N i s e n g a r d , A l p e r t , K r e s t o w more linear pattern in lupus erythematosus. Those seen in the skin of normal individuals are usually much smaller and fewer in numbers. SUMMARY
A case of desquamative gingivitis is presented that was a gingival manifestation of cicatricial pemphigoid. Immunologic studies of serum and biopsies were of diagnostic significance. The application of immuno fluorescence for the diagnosis of desquamative gingival lesions was discussed. If a definite diagnosis can not be made on the basis of light microscopy, immunofluores cence studies should be performed. ADDENDUM
The authors are continuing immunologic studies of desquamative gingivitis and would welcome specimens for study. REFERENCES
1. Beutner, E . H . , and Nisengard, R . J.: Defined immu nofluorescence in clinical immunology. Beutner, E . H . , Chorzelski, T. P., Bean, S. F . , and Jordan, R . E . (eds), I m m u n o p a t h o l o g y of t h e S k i n : L a b e l e d A n t i b o d y Studies. Stroudsburg, Pa., Dowden, Hutchinson and Ross, Inc., 1973. 2. Personal communication. 3. McCarthy, P. L . and Shklar, G . : Disease of t h e O r a l M u c o s a : D i a g n o s i s , M a n a g e m e n t a n d T h e r a p y , pp 183-191. New York, McGraw Book Co., 1964. 4. Jablonska, S., et al.: Uses for IF tests of skin and sera: Utilization of immunofluorescence in the diagnosis of bullous diseases, lupus erythematosus and certain other dermatoses.
A r c h D e r m a t o l 3: 371, 1975. 5. Michel, B., Milner, Y . , and David, K.: Preservation of tissue fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases: Preliminary report. J I n v e s t D e r m a t o l 59: 449, 1973. 6. Jablonska, D . , Chorzelski, T. P., Beutner, E . H . , and Holubar, K.: Indications for skin and serum immunofluorescent studies in dermatology. See 1 above, Chap. 1. 7. Beutner, E . H . , Chorzelski, T. P., and Jordan, R . E . : Autosensitization i n Pemphigus and Bullous Pemphigoid. Springfield, Illinois, Charles C. Thomas, 1970. 8. Bean, S. F . , and Michel, B . : Cicatricial pemphigoid. See 1 above, Chap. 4. 9. Holubar, K., Honigsmann, H . , and Wolff, K.: Cicatri cial pemphigoid immunofluorescent investigations. A r c h D e r m a t o l 108: 50, 1973. 10. Dantzig, P.: Circulating antibodies in cicatricial pem phigoid. A r c h D e r m a t o l 108: 264, 1973. 11. Dabelsteen, E . , Ullman, S., Thompson, K . , and Rygaard, J.: Demonstration of basement membrane auto antibodies in patients with benign mucous membrane pem phigoid. A eta D e r m V e n e r e o l 54: 189, 1974. 12. Rogers, R . S., Sheridan, P. J . , and Jordan, R . E . : Desquamative gingivitis. O r a l S u r g 42: 316, 1976. 13. Ablin, R. J . , Milgrom, F . , Kano, K . , Rapaport, F . , and Beutner, E . H . : Pemphigus-like antibodies in patients with skin burns. Vox Sang 16: 73, 1969. 14. Thivolet, J . , and Beyvin, A . J.: Recherche par im munofluorescence d'auto-anticorps seriques vis-a-vis des con stituants de l'epiderme chez les brules. E x p e r i e n t i a 24: 945, 1968. 15. Fellner, M . J . , and Prutkin, L . : Morbilliform erup tions caused by penicillin. J I n v e s t D e r m a t o l 55: 390, 1970. 16. Chorzelski, T. P., Jablonska, S., and Beutner, E . H . : Clinical significance of pemphigus antibodies. See 1 above, Chap. 2. 17. Michel, B., and Sy, E . K.: Tissue-fixed immunoglob ulins in lichen planus. See 1 above, Chap. 12.
Abstract MICROSCOPIC EVALUATION OF CLINICAL MEASUREMENTS OF CONNECTIVE TISSUE ATTACHMENT LEVELS
Armitage, Gary C , Svanberg, Gunnar K., and Löe, Harald J C l i n Periodontol 4: 173, 1977. The purpose of this study was to determine how accurately periodontal probes measure connective tissue attachment levels in beagle dogs with (1) clinically healthy gingivae, (2) experimental gingivitis, and (3) periodontitis. In the healthy and experimental gin givitis specimens the probes were inserted with a standardized force of 25 ponds. In periodontitis specimens the probes were inserted with a gentle, but nonstandardized force. After insertion, 120 plastic periodontal probes (40 in each group) were held in place by fusing them to the teeth. Blocks of periodontal tissue with the probes in situ were subsequently processed and serially sectioned. Histometric
measurements were made from the sections in order to compare the level of connective tissue attachment to the level of probe penetra tion. In healthy specimens the probes consistently failed to reach the apical termination of the junctional epithelium (x = -0.39 mm). In the experimental gingivitis group most probes came closer to the apical termination of the junctional epithelium, but on the average still fell short by x = —0.10 mm. In periodontitis specimens the probes consistently went past the most apical cells of the junctional epithe lium (x = +0.24 mm). A significant relationship between the degree of inflammation and level of probe penetration was found. No rela tionship was observed between histological and clinical sulcus depths. It is concluded that in beagle dogs (1) periodontal probes do not pre cisely measure connective tissue attachment levels, (2) inflammation has a significant influence on the degree of probe penetration, and (3) histological and clinical sulcus depths differ significantly.
