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In situ characterization of cells in periapical granuloma by monoclonal antibodies
In situ characterization of cells in periapical granuloma by monoclonal antibodies P. Babbl,* P. Soler,** M. Brozman,*** J. Jakubovsky,*** M. Beyly,**** and F. Basset,** Bratislava, Czechoslovakia, and Paris, France
Monoclonal antibodies were used in histopathologic and immunohistologic studies of periapical granulomas. In cellular zones, plasma cells and lymphocytes predominated, with variable numbers of tfbroblasts, macrophages, and polymorphonuclear leukocytes. Labeling with monoclonal antibodies disclosed relatively infrequent, usually scattered macrophages. Plasma cells were numerous and frequently clumped. The vast majority of fymphocytes were T cells, scattered individually or in small groups of three or four cells and dispersed throughout the granulomas without any topographic predilection, with prevalence of T-suppressor Icytotoxic cells over T-helper/inducer cells. Our findings of numerous plasma cells, which were in agreement with descriptions of numerous immunoglobulin-producing cells by other authors, imply the participation of antibody-mediated immune reactions in periapical granulomas. The presence of cell-mediated immunity, in spite of numerous T cells, could not be confirmed. (ORAL
SIJRG. ORAL
MED.
ORAL
PATHOL.
1987;64348-52)
A lthough
periapical granuloma is usually regarded as a trivial lesion rather than a serious clinical complication, this process remains an object of interest for numerous investigators. Essentially, it is considered a reactive inflammatory process resulting from chronic irritation originating in the root canal system of the affected tooth.1.2 The precise pathogenesis of this lesion is still under discussion. The presence of different classes of immunoglobulins (IgG, IgM, IgA, and IgE) and the C3 fraction of complement and their localization have been already investigated.3.’ Skaug et al” demonstrated Fc receptor on the surface of the cells in granuloma. Farber’ and later Stern et al.* provided data about the presence of T-lymphocytes in the lesions. It has been proposed that antigen-antibody complexes and an IgE-mediated reaction could initiate preliminary changes in periapical tissues; also, it has been suggested that delayed hypersensitivity takes part in the perpetuation and progression of this process.‘, ‘. *. p However, these speculations have not been definitely
*Komenski University, Department of Pathology, Bratislava, Czechoslovakia. *‘INSERM U.82, Faculty X. Bichat. Paris, France. ***Medical Bionic Research Institute, Bratislava, Czechoslovakia. ****Department of Stomatology, Hospital X. Bichat, Paris, France.
348
confirmed as yet. Because of the uncertainties that remain about the pathogenesis of the periapical granuloma, it appeared desirable to obtain a more precise characterization of the cells involved in these lesions. As previous studies were performed on cell suspensions, analyzing the cell surface with the scanning electron microscope’ and by the rosetting technique,s it seemed useful to investigate tissue sections of granulomas with the use of monoclonal antibodies in order to show the cells in situ. MATERIALS
AND METHODS
Specimens were obtained from 31 patients, 16 to 67 years of age, by means of tooth extraction, with or without alveolar curettage. The patients had not been treated previously. Part of the tissue was fixed in 10% formalin and embedded in paraffin; sections were stained with hematoxylin and eosin, Giemsa stain, and Mallory’s phosphotungstic acid-hematoxylin. Another part was immediately frozen in liquid nitrogen, and 5 pm cryostat sections were made. After drying for 30 minutes at room temperature, they were stored at -40” C or immediately fixed in acetone for 10 minutes at room temperature for immunofluorescence and in acetone with 0.05% HzOz (to inhibit endogenous peroxidases) for the immunoperoxidase technique. After rehydration, appropriate antibodies were applied: OKT8 (Ortho) or Leu 2a (Becton-Dickinson) for T-suppressor/
Volume 64 Number 3
cytotoxic lymphocytes; Leu 3a or OKT4 + OKT4a (1 : 1) for identification of T-helper/inducer lymphocytes; OKMl for mononuclear phagocytes, and OKIal for B cell populations. The second antibody was peroxidase-conjugated or fluorescein-conjugated goat anti-mouse IgG. All antibodies were diluted 1 : 20, except the fluorescein conjugate, which were diluted 1 : 30. The sections were incubated with each antibody for 30 minutes in a moist chamber at room temperature. Peroxidase was revealed by immersion of the sections for 4 minutes in a mixture of 10 mg 3-amino-9-ethyl carbazole dissolved in 2.5 ml of N-N-dimethylformamide and 40 ml of 0.05 M acetate buffer at pH 5.12 with three drops of 3% hydrogen peroxide. Counterstaining was carried out with Mayer’s hematoxylin for 30 seconds and the specimens were mounted with glycerin jelly. In addition, other sections were examined by indirect immunofluorescence after labeling with rabbit anticollagen type I antibody for the first step and sheep anti-rabbit IgG conjugated with fluorescein for the second step. For immunofluorescence examinations, all sections were mounted with 30% glycerol in phosphate-buffered saline solution. Only cells with typical membrane labeling were considered positive. Polymorphonuclear leukocytes, which were identified by their nuclei, were excluded from the counts of OKM 1-positive cells. Cryostat sections of sarcoid granulomas in lung and in lymph node were used as controls. As controls for labeling with antibodies, sections of all periapical granulomas were treated with the second antibody only. RESULTS
In sections stained with hematoxylin and eosin, plasma cells, lymphocytes, fibroblasts, and histiocytes appeared to be most numerous cell components of cellular areas of periapical lesions (Fig. 1). Neutrophilic leukocytes were predominant in zones of acute exudation, frequently accompanied by eosinophils. Multinucleated giant cells of the foreign body type were seen only rarely. Fibroblasts were sometimes disseminated among the inflammatory cells or formed swirllike parallel arrangements, bridging cellular and fibrous areas. In cellular areas small vessels, which frequently were lined with thick endothelium, were consistently observed, as were sometimes a few extravasated erythrocytes. These areas frequently took the appearance of granulation tissue. Foamy cells, usually in small groups, were seen occasionally. In fibrotic regions, fibrous acidophilic material prevailed and contained variable numbers of fibroblasts and small numbers of cells, such as lymphocytes and histiocytes. The blood vessel supply
Cell detection in periapical
granuloma
349
was poor. In about one half of the specimens squamous epithelium was found; this formed either small groups of three or four cells, clusters, or a continuous lining of cystic cavities, sometimes with a tendency to line the surface of the lesions. Mast cells in Giemsa stain most frequently were found in specimens with marked fibrosis, predominantly localized at the borders of cellular and fibrotic regions and usually close to blood vessels. Fibrin appeared in cellular areas as scarce, fine, blue fibers in Mallory’s phosphotungstic hematoxylin and in areas of acute exudation as large deposits. In this stain, Russell bodies were dark blue and appeared very numerous. Immunohistochemical techniques were performed on 17 samples. Leu 2a (OKT8)-positive T-suppressor/cytotoxic lymphocytes were found scattered throughout the cellular areas, either singly or in small groups of three or four cells (Fig. 2). Similarly, there were Leu 3a (OKT4)-labeled T-helper/inducer lymphocytes (Fig. 3). These cells usually were less numerous than were the Leu 2a-positive cells. The numbers of Leu l-positive cells (that is, the whole population of T cells) approximately corresponded to the sum of the two preceding groups of cells (Leu 2a and Leu 3a-positive cells) and formed up to one fourth of all the cells. OKIal-marked cells, representing predominantly the population of B cells, formed in cellular zones in up to one half (sometimes even more) of the cell total. They were usually in aggregates of relatively large cells; small cells corresponding to the size of lymphocytes were not numerous (Fig. 4). Small numbers of OKMl-positive cells, representing predominantly mononuclear phagocytes, were diffusely dispersed throughout the specimens; these cells were mostly isolated except in zones of acute exudation, where they were more numerous (Fig. 5). In general, all types of reactive cells were rare in fibrotic areas; some reactive cells were found in the epithelium and occasionally in cystic cavities. Immunohistologic studies with antibodies against type I collagen disclosed a number of irregularly arranged tiny fibrils and walls of small blood vessels in cellular areas (Fig. 6). In fibrotic areas large bundles of fibers were labeled. Control specimens treated only with the second antibody were all negative. The specimens of lung tissue and lymph node containing sarcoid granulomas showed the typical distribution of labeled cells. DISCUSSION
The purpose of this study was to identify precisly, with the use of monoclonal antibodies, the nature
350
Babirl et al.
Oral Surg. September 1987
Fig. 1. Cellular area with predominance of plasma cells. Note a strand of epitheliun section. Hematoxylin and eosin stain. Original magnification X250.)
at right. (Paraffin
Fig. 2. OKTI-reactive cells representing T-suppressor/cytotoxic lymphocytes scattered throughout (Frozen section, counterstained with hematoxylin. Original magnification, X200.) Fig. 3. Same sample as in Fig. 2. I-eu 3a-reactive X200.)
T-helper/inducer
lymphocytes.
(Original
tissue.
magnification
Fig. 4. Fluorescence micrograph of same case labeled with OKlal antibody. Positive cells are numerous and are frequently in clusters. Often only fragments of membranes are evident. (Original magnification x180.) Fig. 5. OKMI-positive magnification X200.)
cells disseminated
in tissue. Same specimen
Fig. 6. Frozen section from same sample labeled magnification X350.)
with rabbit
as in previous
anticollagen
figures. (Original
type I antibody.
(Original
Volume Number
64 3
and distribution of cell components of periapical granulomas. In our materials macrophages formed only a small portion of the inflammatory cells. Previous descriptions mentioned that macrophages were commonly present in periapical granulomas and were usually numerous.7q1o According to Stabholz and McArthur,’ the cellular composition of periapical lesions consisted of lymphocytes and macrophages; Weiner et al.” described a predominance of macrophages and monocytes forming granulomatous inflammatory reactions. Maybe these differences occurred because we studied only untreated cases. According to Paulus and Sitzmann’* and Yanagisawa,3 the treatment of teeth can basically influence the cell composition in these lesions. Surface Ia antigen is present on B cells, on some T-lymphocytes, and on the subset of mononuclear phagocytes.“s14 In zones with numerous OKIalpositive cells, OKMl-positive cells were rare. Thus the majority of OKIa l-positive cells represented plasma cells, which constituted the majority of inflammatory cells. Lymphocytes were regularly observed in periapical granulomas. As B-lymphocytes are plasma cell precursors, it is likely that some of small Ia-positive cells represented B-lymphocytes. Previously, their presence in periapical granulomas was indicated by demonstration of receptors for Fc fragment of IgG6 or their immunoglobulin contents.4*5 Farber, using scanning electron microscopic observations of cell surfaces, indicated that most lymphocytes in periapical granulomas appeared to be T cells. Recently, Stern et al8 produced data on the enumeration of T-lymphocytes by the rosetting technique. Their finding of 34.5% of T cells in suspensions of periapical granulomas was in agreement with our results. Delayed hypersensitivity reaction is characterized by perivascular accumulations of Tlymphocytes and macrophages; small groups of cells can also accumulate more distantly from blood vessels,‘5-L7and the mutual rate of T-helper to Tsuppressor lymphocytes in delayed hypersensitivity is about 2:l.” This implies that a reaction with prevalence of T-suppressor/cytotoxic lymphocytes and without any topographic accumulation of T cells, which took place in periapical granulomas, did not represent (or at least was different from) the typical delayed hypersensitivity reaction. Similarly, in human experimental gingivitis, even if large numbers of T cells (more than 70% of lymphocytes) were
Cell detection in periapical
granuloma
351
found, the presence of true cell-mediated immunity could not be stated. I* Since it is unlikely that cytotoxic activity took place in periapical granulomas, it could be supposed that OKTS (Leu 2a)positive cells represented T-suppressor cells acting on both T-helper lymphocytes and plasma cells. If we take into consideration that T-helper cells can modulate osteoclast and fibroblast activities,‘) the suppressant activity could be of great significance in the development of these lesions. The role of mast cells in cutaneous hypersensitivity 1v*20was intensively studied, and their participation in immediate immune reactions is known.21 These cells were abundant and present in all specimens, but their participation in immune processes in periapical granulomas remains to be confirmed. In the context of cell composition, the chronic inflammation that took place in periapical granulomas differed from that seen in other types of granuloma. According to McKinney,22 granulomatous lesions in periapical granulomas should be differentiated from granulation tissue. Weiner et al.” presented data that tended to exclude granulation tissue from periapical granulomas. Yanagisawa3 and others2sv*23described granulation tissue in periapical granulomas, and our findings in the present study are in agreement with this description. The histologic and immunohistologic findings of chronic inflammatory infiltrates with the predominance of plasma cells, lymphocytes, fibroblasts, and macrophages in a diffuse tangle of tiny collagen fibers corresponds to simple chronic inflammation as understood by others.1’s24The finding of granulation tissue probably corresponded with a decreasing number of inflammatory cells and the beginning of the healing process. It seems that use of the term granulomatous lesion in the case of periapical granuloma represents a mere question of terminology. It cannot be used in the sense of Sell’s25 granulomatous hypersensitivity reaction or the definitions by Adams26 or Williams and Williams,” which are limited to epithelioid granulomas. The periapical granuloma, in which organization and activation of mononuclear phagocytes cannot be demonstrated, could be included only in the broadest sense of the definition by Warren,28 who defined “granuloma” in general as a focal chronic inflammatory reaction characterized by accumulation and proliferation of leukocytes, principally of the mononuclear type. Granulation tissue represents a subsequent reparative process. Our findings of numerous plasma cells in periapi-
352
Babhl et al.
Oral Surg.
September 1987
cal granulomas are in agreement with the findings of others,3-5 wbo described immunoglobulin-producing cells in these lesions. This fact and the presence of C3 component of complemenP* 5 imply the presence of immune reactions type I and type III (Coombs and Gel1 classification). The participation of cell-mediated immunity, in spite of numerous T-lymphocytes, was not confirmed. We thank Dr. V.J. Ferransfor his helpful criticism, F. Mazin, Z. Hamidovi, and J. Jarland for technical assistance, and C. Ybert for typing the manuscript. REFERENCES
1. Torabinejad M, Bakland LK. Immunopathogenesis of chronic periapical lesions. ORAL SURG ORAL MED ORAL PATHOL 1978;46:658-99. 2. Okada H, Aono M, Yoshida M, Munemoto K, Nishida 0, Yokomizo J. Experimental study on focal infection in rabbits by prolonged sensitization through dental pulp canals. Arch Oral Biol 1967;12:1017-34. 3. Yanagisawa S. Pathologic study of periapical lesions. 1. Periapical granulomas: clinical, histologic and immunohistopathologic studies. J Oral Path01 1980;9:288-300. 4. Stern MH, Dreizen S, Mackler BF, Levy BM. Antibodyproducing cells in human periapical granulomas and cysts. J Endod 1981;7:447-452. 5. Johannessen AC, Nilsen R, Skaug N. Deposits of immunoglobulins and complement factor C3 in human dental periapical inflammatory lesions. Stand J Dent Res 1983;91:191-9. 6. Skaug N, Nilsen R, Matre R, Bernhoft CH, Johannessen AC. In situ characterization of cell infiltrates in human dental periapical granulomas. 1. Demonstration of receptors for the Fc region of IgG. J Oral Path01 1982;11:47-57. 7. Farber PA. Scanning electron microscopy of cells from periapical lesions. J Endod 1975;1:291-4. 8. Stern MH, Dreizen S, Mackler BF, Levy BM: Isolation and characterization of inflammatory cells from the human periapical granuloma. J Dent Res 1982;61:1408-12. 9. Stabholz A, McArthur WP. Cellular immune response of patients with periapical pathosis to necrotic dental pulp antigens determined by release of LIF. J Endod 1978;4: 282-7.
10. Stern MH, Dreizen S, Mackler BF, Selbst AG, Levy BM. Quantitative analysis of cellular composition of human periaoical aranuloma. J Endod 1981:7:117-22. 11. Weine;S, McKinney RV, Walton RE. Characterization of the periapical surgical specimen: a morphologic and histochemical study of the inflammatory patterns. ORAL SURG ORAL MED ORAL PATHOL 1982;53:293-302. 12. Paulus GW, Sitzmann F. Immunreaktion als Ursache einer nekrotisierenden epithelioidzelligen Endzilndung ? Dtsch Zahnlrztl Z 1979;36:38-41.
13. Reinherz EL, Schlossman SF. Regulation of the immune response-inducer and suppressor T-lymphocyte subsets in human beings. N Engl J Med 1980;301:1018-22. 14. Ballieux RE, Heijnen CJ. Immunoregulatory T cell subpopulations in man: dissection by monoclonal antibodies and Fc receptors. Immunol Rev 1983;74:5-28. 15. Turk JL. Delayed hypersensitivity. In: Neuberger A, Tatum EL, eds. Frontiers of biology. Vol. 4. Amsterdam: ElsevierNorth Holland Publishing Company, 1967;46. 16. Hay J.B.: Delayed Cellular Hypersensitivity. In: Movat HZ, ed. Inflammation, immunity and hypersensitivity: Cellular and Molecular Mechanisms. 2nd ed. New York; Medical Department, Harper & Row, 1979;271. 17. Poulter LW, Seymour GJ, Duke 0, Janossy G, Panayi G. Immunohistological analysis of delayed-type hypersensitivity in man. Cell Immunol 1982;74:358-69. 18. Seymour GJ, Powell RN, Aitken JF, et al. Experimental gingivitis in humans: a histochemical and immunological characterization of the lymphoid cell subpopulations. J Periodont Res 1983;18:375-85. 19. Dvorak AM, Mihm MC, Dvorak HF. Morphology of delayed-type hypersensitivity reaction in man. II. Ultrastrucrural alterations affecting the microvasculature and the tissue mast cells. Lab Invest 1976;34:179-9 1. 20. Askenase PW, Loveren H. Delayed-type hypersensitivity: activation of mast cells by antigen-specific T-cell factors initiates the cascade of cellular interactions. Immunol Today 1983;4:259-64. 21. Lagnuoff D, Chi EY. Cell biology of mast cells and basophils. In: Weissmann G, ed. Handbook of inflammation, Vol. 2. The cell biology of inflammation. Amsterdam: Elsevier/NorthHolland Biomedical Press, 1980:217. 22. McKinney RV. Clarification of the terms granulomatous and granulation tissue. J Oral Path01 1981;10:307-10. 23. Block RM, Bushel1 A, Rodrigues H, Langeland K. A histopathologic, histobacteriologic and radiographic study of periapical endodontic surgical specimens. ORAL SURG ORAL MED ORAL PATHOL 1979;42:656-78. 24. 25. 26. 27. 28.
Spector WG. Chronic inflammation. J Endod 1977;3: 218-21. Sell S. Immunopathology [Teaching monograph]. Am J Pathol 1978;90:215-79. Adams DO. The granulomatous inflammatory response: a review. Am J Path01 1976;84:164-92. Williams GT, Williams WJ. Granulomatous inflammationa review. J Clin Path01 1983;36:723-33. Warren KS. A functional classification of granulomatous inflammation. Ann NY Acad Sci 1976;278:7-18.
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requests
to:
Dr. Pave1 Babal Institute of Pathology Komensky University Sasinkova 4 8 11 08 Bratislava, Czechoslovakia
Monoclonal antibodies were used in histopathologic and immunohistologic studies of periapical granulomas. In cellular zones, plasma cells and lymphocytes predominated, with variable numbers of tfbroblasts, macrophages, and polymorphonuclear leukocytes. Labeling with monoclonal antibodies disclosed relatively infrequent, usually scattered macrophages. Plasma cells were numerous and frequently clumped. The vast majority of fymphocytes were T cells, scattered individually or in small groups of three or four cells and dispersed throughout the granulomas without any topographic predilection, with prevalence of T-suppressor Icytotoxic cells over T-helper/inducer cells. Our findings of numerous plasma cells, which were in agreement with descriptions of numerous immunoglobulin-producing cells by other authors, imply the participation of antibody-mediated immune reactions in periapical granulomas. The presence of cell-mediated immunity, in spite of numerous T cells, could not be confirmed. (ORAL
SIJRG. ORAL
MED.
ORAL
PATHOL.
1987;64348-52)
A lthough
periapical granuloma is usually regarded as a trivial lesion rather than a serious clinical complication, this process remains an object of interest for numerous investigators. Essentially, it is considered a reactive inflammatory process resulting from chronic irritation originating in the root canal system of the affected tooth.1.2 The precise pathogenesis of this lesion is still under discussion. The presence of different classes of immunoglobulins (IgG, IgM, IgA, and IgE) and the C3 fraction of complement and their localization have been already investigated.3.’ Skaug et al” demonstrated Fc receptor on the surface of the cells in granuloma. Farber’ and later Stern et al.* provided data about the presence of T-lymphocytes in the lesions. It has been proposed that antigen-antibody complexes and an IgE-mediated reaction could initiate preliminary changes in periapical tissues; also, it has been suggested that delayed hypersensitivity takes part in the perpetuation and progression of this process.‘, ‘. *. p However, these speculations have not been definitely
*Komenski University, Department of Pathology, Bratislava, Czechoslovakia. *‘INSERM U.82, Faculty X. Bichat. Paris, France. ***Medical Bionic Research Institute, Bratislava, Czechoslovakia. ****Department of Stomatology, Hospital X. Bichat, Paris, France.
348
confirmed as yet. Because of the uncertainties that remain about the pathogenesis of the periapical granuloma, it appeared desirable to obtain a more precise characterization of the cells involved in these lesions. As previous studies were performed on cell suspensions, analyzing the cell surface with the scanning electron microscope’ and by the rosetting technique,s it seemed useful to investigate tissue sections of granulomas with the use of monoclonal antibodies in order to show the cells in situ. MATERIALS
AND METHODS
Specimens were obtained from 31 patients, 16 to 67 years of age, by means of tooth extraction, with or without alveolar curettage. The patients had not been treated previously. Part of the tissue was fixed in 10% formalin and embedded in paraffin; sections were stained with hematoxylin and eosin, Giemsa stain, and Mallory’s phosphotungstic acid-hematoxylin. Another part was immediately frozen in liquid nitrogen, and 5 pm cryostat sections were made. After drying for 30 minutes at room temperature, they were stored at -40” C or immediately fixed in acetone for 10 minutes at room temperature for immunofluorescence and in acetone with 0.05% HzOz (to inhibit endogenous peroxidases) for the immunoperoxidase technique. After rehydration, appropriate antibodies were applied: OKT8 (Ortho) or Leu 2a (Becton-Dickinson) for T-suppressor/
Volume 64 Number 3
cytotoxic lymphocytes; Leu 3a or OKT4 + OKT4a (1 : 1) for identification of T-helper/inducer lymphocytes; OKMl for mononuclear phagocytes, and OKIal for B cell populations. The second antibody was peroxidase-conjugated or fluorescein-conjugated goat anti-mouse IgG. All antibodies were diluted 1 : 20, except the fluorescein conjugate, which were diluted 1 : 30. The sections were incubated with each antibody for 30 minutes in a moist chamber at room temperature. Peroxidase was revealed by immersion of the sections for 4 minutes in a mixture of 10 mg 3-amino-9-ethyl carbazole dissolved in 2.5 ml of N-N-dimethylformamide and 40 ml of 0.05 M acetate buffer at pH 5.12 with three drops of 3% hydrogen peroxide. Counterstaining was carried out with Mayer’s hematoxylin for 30 seconds and the specimens were mounted with glycerin jelly. In addition, other sections were examined by indirect immunofluorescence after labeling with rabbit anticollagen type I antibody for the first step and sheep anti-rabbit IgG conjugated with fluorescein for the second step. For immunofluorescence examinations, all sections were mounted with 30% glycerol in phosphate-buffered saline solution. Only cells with typical membrane labeling were considered positive. Polymorphonuclear leukocytes, which were identified by their nuclei, were excluded from the counts of OKM 1-positive cells. Cryostat sections of sarcoid granulomas in lung and in lymph node were used as controls. As controls for labeling with antibodies, sections of all periapical granulomas were treated with the second antibody only. RESULTS
In sections stained with hematoxylin and eosin, plasma cells, lymphocytes, fibroblasts, and histiocytes appeared to be most numerous cell components of cellular areas of periapical lesions (Fig. 1). Neutrophilic leukocytes were predominant in zones of acute exudation, frequently accompanied by eosinophils. Multinucleated giant cells of the foreign body type were seen only rarely. Fibroblasts were sometimes disseminated among the inflammatory cells or formed swirllike parallel arrangements, bridging cellular and fibrous areas. In cellular areas small vessels, which frequently were lined with thick endothelium, were consistently observed, as were sometimes a few extravasated erythrocytes. These areas frequently took the appearance of granulation tissue. Foamy cells, usually in small groups, were seen occasionally. In fibrotic regions, fibrous acidophilic material prevailed and contained variable numbers of fibroblasts and small numbers of cells, such as lymphocytes and histiocytes. The blood vessel supply
Cell detection in periapical
granuloma
349
was poor. In about one half of the specimens squamous epithelium was found; this formed either small groups of three or four cells, clusters, or a continuous lining of cystic cavities, sometimes with a tendency to line the surface of the lesions. Mast cells in Giemsa stain most frequently were found in specimens with marked fibrosis, predominantly localized at the borders of cellular and fibrotic regions and usually close to blood vessels. Fibrin appeared in cellular areas as scarce, fine, blue fibers in Mallory’s phosphotungstic hematoxylin and in areas of acute exudation as large deposits. In this stain, Russell bodies were dark blue and appeared very numerous. Immunohistochemical techniques were performed on 17 samples. Leu 2a (OKT8)-positive T-suppressor/cytotoxic lymphocytes were found scattered throughout the cellular areas, either singly or in small groups of three or four cells (Fig. 2). Similarly, there were Leu 3a (OKT4)-labeled T-helper/inducer lymphocytes (Fig. 3). These cells usually were less numerous than were the Leu 2a-positive cells. The numbers of Leu l-positive cells (that is, the whole population of T cells) approximately corresponded to the sum of the two preceding groups of cells (Leu 2a and Leu 3a-positive cells) and formed up to one fourth of all the cells. OKIal-marked cells, representing predominantly the population of B cells, formed in cellular zones in up to one half (sometimes even more) of the cell total. They were usually in aggregates of relatively large cells; small cells corresponding to the size of lymphocytes were not numerous (Fig. 4). Small numbers of OKMl-positive cells, representing predominantly mononuclear phagocytes, were diffusely dispersed throughout the specimens; these cells were mostly isolated except in zones of acute exudation, where they were more numerous (Fig. 5). In general, all types of reactive cells were rare in fibrotic areas; some reactive cells were found in the epithelium and occasionally in cystic cavities. Immunohistologic studies with antibodies against type I collagen disclosed a number of irregularly arranged tiny fibrils and walls of small blood vessels in cellular areas (Fig. 6). In fibrotic areas large bundles of fibers were labeled. Control specimens treated only with the second antibody were all negative. The specimens of lung tissue and lymph node containing sarcoid granulomas showed the typical distribution of labeled cells. DISCUSSION
The purpose of this study was to identify precisly, with the use of monoclonal antibodies, the nature
350
Babirl et al.
Oral Surg. September 1987
Fig. 1. Cellular area with predominance of plasma cells. Note a strand of epitheliun section. Hematoxylin and eosin stain. Original magnification X250.)
at right. (Paraffin
Fig. 2. OKTI-reactive cells representing T-suppressor/cytotoxic lymphocytes scattered throughout (Frozen section, counterstained with hematoxylin. Original magnification, X200.) Fig. 3. Same sample as in Fig. 2. I-eu 3a-reactive X200.)
T-helper/inducer
lymphocytes.
(Original
tissue.
magnification
Fig. 4. Fluorescence micrograph of same case labeled with OKlal antibody. Positive cells are numerous and are frequently in clusters. Often only fragments of membranes are evident. (Original magnification x180.) Fig. 5. OKMI-positive magnification X200.)
cells disseminated
in tissue. Same specimen
Fig. 6. Frozen section from same sample labeled magnification X350.)
with rabbit
as in previous
anticollagen
figures. (Original
type I antibody.
(Original
Volume Number
64 3
and distribution of cell components of periapical granulomas. In our materials macrophages formed only a small portion of the inflammatory cells. Previous descriptions mentioned that macrophages were commonly present in periapical granulomas and were usually numerous.7q1o According to Stabholz and McArthur,’ the cellular composition of periapical lesions consisted of lymphocytes and macrophages; Weiner et al.” described a predominance of macrophages and monocytes forming granulomatous inflammatory reactions. Maybe these differences occurred because we studied only untreated cases. According to Paulus and Sitzmann’* and Yanagisawa,3 the treatment of teeth can basically influence the cell composition in these lesions. Surface Ia antigen is present on B cells, on some T-lymphocytes, and on the subset of mononuclear phagocytes.“s14 In zones with numerous OKIalpositive cells, OKMl-positive cells were rare. Thus the majority of OKIa l-positive cells represented plasma cells, which constituted the majority of inflammatory cells. Lymphocytes were regularly observed in periapical granulomas. As B-lymphocytes are plasma cell precursors, it is likely that some of small Ia-positive cells represented B-lymphocytes. Previously, their presence in periapical granulomas was indicated by demonstration of receptors for Fc fragment of IgG6 or their immunoglobulin contents.4*5 Farber, using scanning electron microscopic observations of cell surfaces, indicated that most lymphocytes in periapical granulomas appeared to be T cells. Recently, Stern et al8 produced data on the enumeration of T-lymphocytes by the rosetting technique. Their finding of 34.5% of T cells in suspensions of periapical granulomas was in agreement with our results. Delayed hypersensitivity reaction is characterized by perivascular accumulations of Tlymphocytes and macrophages; small groups of cells can also accumulate more distantly from blood vessels,‘5-L7and the mutual rate of T-helper to Tsuppressor lymphocytes in delayed hypersensitivity is about 2:l.” This implies that a reaction with prevalence of T-suppressor/cytotoxic lymphocytes and without any topographic accumulation of T cells, which took place in periapical granulomas, did not represent (or at least was different from) the typical delayed hypersensitivity reaction. Similarly, in human experimental gingivitis, even if large numbers of T cells (more than 70% of lymphocytes) were
Cell detection in periapical
granuloma
351
found, the presence of true cell-mediated immunity could not be stated. I* Since it is unlikely that cytotoxic activity took place in periapical granulomas, it could be supposed that OKTS (Leu 2a)positive cells represented T-suppressor cells acting on both T-helper lymphocytes and plasma cells. If we take into consideration that T-helper cells can modulate osteoclast and fibroblast activities,‘) the suppressant activity could be of great significance in the development of these lesions. The role of mast cells in cutaneous hypersensitivity 1v*20was intensively studied, and their participation in immediate immune reactions is known.21 These cells were abundant and present in all specimens, but their participation in immune processes in periapical granulomas remains to be confirmed. In the context of cell composition, the chronic inflammation that took place in periapical granulomas differed from that seen in other types of granuloma. According to McKinney,22 granulomatous lesions in periapical granulomas should be differentiated from granulation tissue. Weiner et al.” presented data that tended to exclude granulation tissue from periapical granulomas. Yanagisawa3 and others2sv*23described granulation tissue in periapical granulomas, and our findings in the present study are in agreement with this description. The histologic and immunohistologic findings of chronic inflammatory infiltrates with the predominance of plasma cells, lymphocytes, fibroblasts, and macrophages in a diffuse tangle of tiny collagen fibers corresponds to simple chronic inflammation as understood by others.1’s24The finding of granulation tissue probably corresponded with a decreasing number of inflammatory cells and the beginning of the healing process. It seems that use of the term granulomatous lesion in the case of periapical granuloma represents a mere question of terminology. It cannot be used in the sense of Sell’s25 granulomatous hypersensitivity reaction or the definitions by Adams26 or Williams and Williams,” which are limited to epithelioid granulomas. The periapical granuloma, in which organization and activation of mononuclear phagocytes cannot be demonstrated, could be included only in the broadest sense of the definition by Warren,28 who defined “granuloma” in general as a focal chronic inflammatory reaction characterized by accumulation and proliferation of leukocytes, principally of the mononuclear type. Granulation tissue represents a subsequent reparative process. Our findings of numerous plasma cells in periapi-
352
Babhl et al.
Oral Surg.
September 1987
cal granulomas are in agreement with the findings of others,3-5 wbo described immunoglobulin-producing cells in these lesions. This fact and the presence of C3 component of complemenP* 5 imply the presence of immune reactions type I and type III (Coombs and Gel1 classification). The participation of cell-mediated immunity, in spite of numerous T-lymphocytes, was not confirmed. We thank Dr. V.J. Ferransfor his helpful criticism, F. Mazin, Z. Hamidovi, and J. Jarland for technical assistance, and C. Ybert for typing the manuscript. REFERENCES
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