5 88
0 1990
Case Reports
The Japanese Society of Pathology
Eosinophilic Gra nulo ma Showing Rapid Regression Report of a Mandibular Case with Application of a Modified PNA Staining Method for Demonstration of La ngerha ns-type Histiocytes
Motoo Kitano', Gabriel Landini', lchiro Semba', Atsushi Urago', Kazumasa Sugihara2, Hiroshi Mukai2, and Sukehide Yamashita2
A mandibular eosinophilic granuloma in a 16-year-old male is reported. This case showed rapid regression, which was clearly demonstrated by histopathological examinations of both preoperative biopsy and surgical materials. Transformation from an eosinophilic granuloma to a xanthomatous granuloma with multinucleated giant cells was observed after only 26 days. Special staining of paraffin sections with peanut agglutinin (PNA) and use of electron microscopy showed that the main component of the lesion in the biopsy material was Langerhans-type histiocytes. These cells had disappeared from the lesion by the time of the operation. At the same time, the number of infiltrating eosinophils was also markedly reduced. It seems appropriate to consider that the rapid regression of this disease was correlated with the rapid reduction in the number of Langerhans-type histiocytes appearlng in the granulomatous foci, as well as the number of infiltrating eosinophils. Acta Pathol Jpn 40 : 588-595, 1990.
Key words : Eosinophilic granuloma, Langerhans-type
histiocytes, Peanut agglutinin staining method
INTRODUCTION Eosinophilic granuloma has been considered as one of a group of disorders known as histiocytosis-X or Langerhans cell histiocytosis (1-3). In addition to eosinophilic granulomas, this group includes Hand-Schuller-Christian Received December 11, 1989. Accepted for publication May 17, 1990. 'Department of Oral Pathology and ZFirst Department of Oral and Maxillofacial Surgery, Kagoshima University Dental School, Kagoshima. Mailing address : Motoo Kitano (tBE+), Department of Oral Pathology, Kagoshima University, Usuki-cho 1208-1, Kagoshima 890, Japan.
disease (probably better described as multiple eosinophilic granuloma), and Letterer-Siwe disease (a malignant counterpart with a characteristically fulminant course occurring in infants), as well as many intermediate and unclassified forms (4). Eosinophilic granulomas are seen most commonly in children and young adults. They are localized destructive conditions which occur within the bone and occasionally involve the soft tissues (5-8). The most commonly affected bones are the proximal femoral metaphysis, skull, mandible, rib and vertebral column (2, 3). Usually the prognosis of this disease is good and patients with a unifocal lesion may show spontaneous regression (2-9). Histologically, eosinophilic granuloma is characterized by a sheet-like proliferation of specific large mononuclear cells, Langerhans-type histiocytes (LCs), and an admixture of eosinophils, lymphocytes, plasma cells, macrophages and multinucleated giant cells. Many of the LCs have indented or cleaved nuclei with abundant pale-staining cytoplasm (5, 10, 11). Electron microscopy of the LCs reveals special cytoplasmic inclusion bodies of unknown function known as Birbeck or Langerhans granules. Recent immunohistochemical methods have indicated that these LCs can be identified using anti-S-100, anti-T6 and anti-ATPase antibodies (1116). Ree and Kadin (1986) (17) described a peanut agglutinin (PNA) staining method that was useful and reliable for identification of the LCs in Langerhans cell histiocytosis. The present study was based upon a case of eosinophilic granuloma which developed in the mandible of a 16-year-old male, and showed very rapid spontaneous regression. This paper describes the clinical and pathological features of this case and discusses the relation-
589
Acta Pathologica Japonica 40 (8) : 1990
Figure 1. X-ray photograph of this case. A wide, abnormal translucent shadow (arrows) of the left mandibular ramus is apparent.
ship between the regression of this disease and the microscopical findings using a modified method of PNA staining and electron microscopy.
CASEREPORT A 16-year-old Japanese male high school student was hit in the left temporal region during a rugby game. Two days later, he noticed pain in the left temporomandibular joint and trismus. A swelling developed in the left anterior auricular region 17 days later and he then consulted a dentist. An X-ray examination revealed an abnormal translucent shadow in the mandibular ramus extending from the condyle to the coronoid process (Fig. 1). A primary malignant bone tumor was suspected and he was referred to the Kagoshima University Dental Hospital. On admission, a slight edematous swelling of the left temporomandibular joint region and slight trismus was observed. No abnormalities were found by routine laboratory tests, except for moderate elevation of the antistreptolysin-0 titer to 833 Todd units and slight elevation of serum alkaline phosphatase to 2 9 9 units/l. Results of blood analysis were within normal limits initially, but a slight elevation of eosinophils to 5% of the white cell profile was found two days after admission. On the fifth day, biopsies were performed at the site of the lesion in the mandible and an enlarged lymph node 1 2 m m in maximum diameter on the left side of the neck. The hist o pat ho log ica I d iag nos is was eosi no p hi I ic granuloma of the bone. Twenty-six days after the biopsy, a raminectomy of the left side of the mandible was performed with exarticulation of the left temporomandibular joint and sub-
sequent reconstruction of the joint with an iliac bone graft. The postoperative clinical course was good and there were no detectable recurrent foci in the mandible, other bones, or visceral organs two and a half years after the operation.
PATHOLOGICAL FINDINGS The mandibular biopsy material was composed of several small fragments of granulation tissue of high cell density including numerous eosinophils, lymphocytes, histiocytic cells and large irregular-shaped multinucleated giant cells. Most of the histiocytic cells were rather large, round or elongated in form with varying amounts of cytoplasm and round or cleaved nuclei (Fig. 2). These cells corresponded t o the LCs demonstrated by PNA staining. Other histiocytic cells showed apparent phagocytic activity for foreign bodies such as hemosiderin or erythrocyte debris, and were considered to be macrophages. Watanabe’s silver impregnation method for reticulin showed no proper stroma, but scattered fine streaks of collagen fibers with scanty, delicate blood vessels. Small abscess-like clusters of eosinophils as well as diffuse hemorrhage were seen. A small number of xanthomatous cells were interspersed within this tissue. The biopsied lymph node showed slight follicular hyperplasia and mild aggregation of eosinophils, histiocytic cells and multinucleated giant cells in the marginal sinuses, suggesting that the lymph node was also involved in the eosinophilic granuloma (6). The mandibular specimen which was removed surgically 2 6 days after the biopsy contained a large portion
5 90
Eosinophilic Granuloma Showing Rapid Regression (Kitano et a/.)
Figure 2. Photomicrograph of the granu loma of the biopsy specimen Prolifera tion of histiocytic cells intermingled with many intermingled with many eosinophilic leukocytes is evident (HE)
Figure 3. Photomicrograph of the surgical specimen. A wide area of the condylar process is replaced by a scar tissue with abundant new bone formation around its periphery. A small granuloma surrounded by a layer of lymphoid cells is present in the central portion of the scar (HE).
Figure 4. The granuloma in the surgical specimen, consisting of numerous foamy xanthomatous cells, multinucleated giant cells and lymphocytes (HE).
591
Acta Pathologica Japonica 40 (8) : 1990
consisting of cicatrized collagenous tissue in which extensive new bone formation was noted (Fig. 3). The cicatrized collagenous tissue was situated in the condyle, extending into the coronoid process. In the central area of the cicatrized tissue, an irregular-shaped granuloma about 1 5 m m in longest diameter was present. It had a two-layered structure with an outer layer of densely infiltrated lymphocytes and plasma cells and an inner layer of xanthomatous granuloma. In the latter, xanthomatous cells with abundant foamy lipid-laden cytoplasm predominated (Fig. 4). Huge multinucleated giant cells including Touton-type giant cells were scattered elsewhere. There was no marked hemorrhage in the granuloma, but phagocytosis of hemosiderin granules was marked in almost all of the foamy xanthomatous cells and also in the giant cells. Eosinophils infiltrated the lesion in small numbers.
lmmunohistochemical analysis lmmunohistochemical studies were done using polyclonal antibodies (Dakopatts) against bovine S-100 protein, human alpha-1-antichymotrypsin and human lysozyme, and an ABC kit (Vectastain, Vector) was used on paraffin sections of the biopsy materials and the granulation tissue obtained at surgery. Immunostaining of the biopsy material confirmed the presence of lysozyme and alpha-1 -antichymotrypsin reactivity in some of the histiocytic cells (Fig. 5) but reaction with anti-S-100 protein antibody was very weak. Immunohistochemical examination of the surgical specimen showed a large population of cells with alpha-1 -antichymotrypsin and lysozyme antigen expression, but no evidence of strong S-100 protein immunoreactivity (Table 1).
Figure 5. Photomicrograph of immuno histochemistry for alpha 1-antichymotryp sin in the biopsy specimen Positive his tiocytic cells (macrophages) are scattered throughout the field of view
Table 1. Summary of lmmunohistochemical Studies, PNA Staining Method, and Electron Microscopical Examinations of Various Types of Cells in the Present Case La ngerha ns-ty pe H istiocytes f LC) -,
s-100 Lysozyme U-l-ACT PNAa EM a
.-
---+
--* -A,-+
it
Langerhans granules (LG)
Macro phages
--' +-it *-it it
Phagolysosomes (PL) No LG
Mult inucleated Giant Cells
Xanthomatous Cells
---t_
---t_
f-+
-+-it
--*
---+.
--+
Poor in PL No LG
---+ Lipid particles
Touton-type Giant Cells
--' +-it
--+ ---+
Lipid particles
Both LCs and macrophages were strongly positive by the PNA staining method, but were clearly differentiated by their staining patterns.
5 92
Eosinophilic Granuloma Showing Rapid Regression (Kitano et a/.)
Figure 6. Photomicrographs of the biopsy specimen after modified PNA staining. Dense deposits of reaction products are apparent on the cell surface as well as in the perinuclear area in Langerhans-type histiocytic cells (a), whereas the cytoplasm of macrophages is stained diffusely (b).
Table 2. Peanut Agglutinin Staining Method (Modified from Ree and Kadin)
’
1.
2. 3. 4. 5.
6. 7.
8. 9.
10 11.
Cut Daraffin-embedded tissue in 5 - ~ msections and m o u i t on slides coated with 0.2% neoprene solution in toluene to prevent detachment. Deparaffinize the sections with xylene and rehydrate in a graded ethanol series. Incubate at 37°C for 3 0 rnin in an aqueous solution of 0.1% trypsin. Wash in two changes of PBS. Immerse sections in 3% hydrogen peroxide in distilled water for 10 min to block endogenous peroxidase activity. Wash in two changes of PBS. Cover the sections with biotinylated PNA (Vector; dilution 1 / 2 0 0 in PBS) for 30 min. Then sprinkle mouse liver powder over the solution and incubate for another 30 min. Wash in two changes of PBS. Incubate in avid in^ biotin complex (Vector; dilution 1/ 100 in PBS) for 20 rnin. Wash in two changes of PBS. Immerse the sections in diaminobenzidine solution (0.025%), pH 7.0, until the reaction in evident by light microscopy. Rinse in tap water, counterstain with methy l green and mount in Entellan New.
Results of PNA staining The peanut agglutinin (PNA) staining method according to Ree and Kadin (1986)(17) with some modifications (shown in Table 2) was employed on the paraffin sections. A positive reaction was seen in two cell types, the LCs and macrophages, which occurred in a ratio of 3 : 2. LCs showed a unique and characteristic dense deposit of reaction products on their cell membranes as well as in the paranuclear cytoplasmic areas (Figs. 6, 7). The remaining cytoplasm stained only
LC
MP
Figure 7. Schematic drawing of a Langerhans-type histiocytic cell (LC) and a macrophage (Mp) stained with PNA. N : Nucleus.
weakly or not at all. The macrophages also showed a strong positive reaction with the PNA staining method, but the cytoplasmic staining pattern was diffuse (Figs. 6, 7). Multinucleated giant cells were essentially negative for PNA staining, but some of them stained weakly without any particular staining pattern. In the surgical specimen, there were no definitive cells with the same characteristic staining pattern as that of the LCs. The foamy xanthomatous cells and multinucleated giant cells showed variable, negative or positive reactivity, with no particular staining pattern.
Acta Pathologica Japonica 40 (8): 1990
Figure 8. Electron micrograph of a Langerhans-type histiocytic cell possessing Langerhans granules (arrows) from the biopsy specimen ( x 15,000).
Figure 9. High-magnification view of the Langerhans granules in Fig. 8 ( x 100,000).
593
5 94
Eosinophilic Granuloma Showing Rapid Regression (Kitano et a/.)
Electron microsco pica I findings In the biopsy material, typical LCs were recognizable by the presence of zipper cord-like Langerhans granules (Figs. 8, 9) (10). More than 40% of the observed cells possessed these granules. Sometimes the ends of the granules were directly connected to the cell membrane (11). Apart from their granules, LCs were characteristically large cells with an irregular outline and numerous peripheral villous processes. The cytoplasm of the LCs contained several dense bodies, many mitochondria and well developed Golgi apparatus. The nucleus often showed a thin rim of peripheral heterochromatin, a relatively electron-lucent nucleoplasm and one or two nucleoli. Intermingled with the LCs, macrophages and many phagolysosomes were present. Langerhans granules were never seen in these cells. In the multinucleated giant cells, Langerhans granules were absent and phagolysosomes were seldom present. These giant cells contained many small, sometimes bent mitochondria. In the surgical specimen there were high numbers of mononuclear and multinucleated cells possessing abundant lipid or lipoid particles. In these cells, phagocytosis of hemosiderin granules was marked, Langerhans granules were never seen and it was not possible to identify any of the characteristics of LCs.
DISCUSSION Eosinophilic granuloma, along with other diseases belonging to histiocytosis-X, is characterized by an a bnorma I proliferation of La ngerha ns- type histiocytes (LCS). LCs are normally present in areas of antigen penetration, such as the epidermis and mucosal epithelium of the digestive tract. LCs as well as the interdigitating reticulum cells of lymph nodes, which belong to the dendritic cell family, are potent stimulators of various lymphocyte reactions. These cells play a critical role in the development of delayed-type hypersensitivity responses and are required for the initial activation of Tlymphocytes. Therefore they are involved in the generation of the immune response, particularly during the afferent phase. Likewise, dendritic cells have been detected in various chronic inflammatory diseases (18) in which they seem to play an important role in disease initiation and/or propagat ion. Histopathologically, the present case showed the features of an active eosinophilic granuloma in the biopsy material, and then passed rapidly to an inactive phase at the end-stage of the disease, revealing a xanthomatous granuloma in the surgical specimen after only 2 6 days
(19). The PNA staining method(l7) and electron microscopical examinations (10) showed that the active phase was represented by an aggregation of numerous LCs, whereas these cells disappeared in the inactive phase. Since LCs are presumed to be able to trigger an effector inflammatory cascade, their relevance to the development and/or progression of histiocytosis-X seems to be crucial. Differences in the severity of a functional disorder of these cells may be responsible for differences in the clinical and histopathological presentation (11). LCs are widely known to express S-100 protein strongly (4, 12, 17), although we were unable to obtain any such evidence from the biopsy material. This may have been due to inappropriate fixation, causing unreliable detection of 5-100 protein in LCs in histiocytosis-X (20). Recently, Ree and Kadin(17) employed a PNA staining method for LCs in paraffin sections of histiocytosis-X. LCs were easily distinguished from other types of cells appearing in the lesions by their unique paranuclear and cell surface deposition of reaction products. This method was employed w i t h some modifications in the present study, and the results were confirmed by electron microscopy. This again emphasized that PNA staining for LCs in eosinophilic granuloma or other kinds of histiocytosis-X can be very useful. It is well known that solitary eosinophilic granuloma of the bone has an excellent prognosis in contrast to other types of histiocytosis-X. Spontaneous resolution may occur, and the responses to treatment including curettage, chemotherapy and radiation therapy are good. In general, if a second lesion does not appear within one year, the prognosis is believed to be especially good (2, 3, 9). Histopathological analysis of the present case revealed almost complete regression during a 26-day period from the initial biopsy to surgery on the mandible. There are no case reports in the literature describing such a rapid regression of this disease confirmed by histopathological analysis in the same location, as in the present case (21 -23). Therefore, it seems appropriate to consider that spontaneous regression may occur more rapidly and frequently than generally considered previously (21-23). In retrospect, it was inappropriate to employ surgical procedures (raminectomy and exarticulation of the mandible) in the present case as a first-choice treatment. According to Schajowicz (2), LCs in histiocytosis-X may show secondary transformation into lipid-, or lipoidladen foamy xanthomatous cells which may also possess hemosiderin granules and cellular debris in their cytoplasm. Conversely, Elema and Atomosoerodjo-Briggs
Acta Pathologica Japonica 40 (8) : 1990
(11) believed that transformation between the two cell types, i.e. LCs and macrophages including xanthomatous cells, never occurs. Unfortunately, the fate of LCs in the biopsy material in the present case could not be determined, since the surgical material showed no definitive LCs by PNA staining o r electron microscopy. Acknowledgements : We express our thanks to Prof. Munetomo Enjoji and Assoc. Prof. Masazumi Tsuneyoshi, Second Department of Pathology, Kyushu University Faculty of Medicine, Fukuoka, Japan, for their invaluable advice. We acknowledge the excellent assistance of Ms. Fusako Kataoka, Ms. Sayuri Kubota, and Ms. Shuko Toya. The manuscript was kindly reviewed by Dr. Richard M. Shelton, Birmingham University (U K).
REFERENCES 1. Lichtenstein L. Histiocytosis X. Integration of eosinophilic granuloma of bone, “Letterer-Siwe disease”, and ”Schuller-Christian disease” as related manifestations of a single nosologic entity. Arch Pathol 56 : 84-1 02, 1953. 2. Schajowicz F. Tumor and tumor-like lesions of bone and joints. Springer-Verlag, Heidelberg 1981. 3. Barnes L. Surgical pathology of the head and neck. Marcel Dekker, Inc., New York, 1985. 4. Watanabe S, Nakajima T, Shimosato Y, Sato Y, and Simizu K. Malignant histiocytosis and Letterer-Siwe disease: Neoplasms of T-zone histiocyte with SlOO protein. Cancer 51 : 1412-1424, 1983. 5. Kato T, Matsuda M, Ando H, and Sasaki H. A case of multifocal proliferations of histiocytic cells containing Langerhans’ cell granules. Am J Clin Pathol 7 6 : 480-485, 1981. 6. Sajjad SM and Osborne B. Lymph node involvement by histiocytosis-X. Arch Pathol Lab Med 1 0 6 : 9698, 1982. 7. Siegal GP, Dehner LP, and Rosai J. Histiocytosis X (Langerhans’ cell granulomatosis) of the thymus: A clinicopathologic study of four childhood cases. Am J Surg Pathol 9 : 117-124, 1985. 8. Goldberg NS, Bauer K, Rosen ST, et a/. Histiocytosis X : Flow cytometric DNA-content and immunohistochemical and ultrastructural analysis. Arch Dermatol 122 446-450, 1986. 9. RisdaII RJ, Dehner LP, Duray P, et a/. Histiocytosis X (Langerhans’ cell histiocytosis). Arch Pathol Lab Med
595
107 : 59-63, 1983. 10. Wolff K. The Langerhans cell. Curr Probl Dermatol 4 : 79-145, 1972. 11. Elema JD and Atomosoerodjo-Briggs JE. Langerhans‘ cells and macrophages i n eosinophilic granuloma : An enzyme-histochemical, enzyme-cytochemical, and ultrastructural study. Cancer 54 : 2174-2181, 1984. 12. Nakajima T, Sat0 Y, Watanabe S, et a/. Immunoelectron microscopical demonstration of S-100 protein in epidermal Langerhans cells. Biomed Res 3 : 226231, 1982. 13. Fox JL and Berman B. T6-antigen-bearing cells in eosinophilic granuloma of bone. JAMA 2 4 9 : 30713072, 1983. 14. Watanabe S, Sat0 Y, Kodama T, and Shimosato Y. lmmunohistochemical study with monoclonal antibodies on immune response in human lung cancers. Cancer Res 43 : 5883-5889, 1983. 15. Hall PA, ODoherty CJ, and Levison DA. Langerhans cell histiocytosis : An unusual case illustrating the value of immunohistochemistry in diagnosis. Histopathology 11: 1181-1191, 1987. 16. Wakuya J. lmmunohistochemical and ultrastructural studies on histiocytosis in children. Acta Pathol Jpn 3 7 : 901-913, 1987. 17. Ree HJ and Kadin ME. Peanut agglutinin: A useful marker for histiocytosis X and interdigitating reticulum cells. Cancer 57 : 282-287, 1986. 18. Demetris AJ, Sever C, Kakizoe S , et a / . SlOO protein positive dendritic cells in primary biliary cirrhosis and other chronic inflammatory liver diseases : Relevance to pathogenesis ? Am J Pathol 1 3 4 : 741-747, 1989. 19. Engelbreth-Holm J, Teilum G, and Christensen E. Eosinophilic granuloma of bone: Schuller-Christian’s disease. Acta Med Scand 9 8 : 292-312, 1944. 20. Finney DS, Rees TD, Wright JM, and Blanton PL. Solitary eosinophilic granuloma (histiocytosis X) of the gingiva : A report of two cases. J Periodontol 59: 457-460, 1988. 21. Mercer Sir W and Duthie RB: Histiocytic granulomatosis. J Bone Joint Surg (Br) 3 8 : 279-292, 1956. 22. Dumermuth G. Reticulogranulomatose : Zwei Falle von eosinophilem Granulom mit Ubergang in HandSchuller-Christiansche Krankheit. Helvet Paediatr Acta 1 3 : 15-39, 1958. 23. Ritter R A Jr. Histiocytosis X : A case report with electron microscopic observations. Cancer 19 : 1155-1 164, 1966.
0 1990
Case Reports
The Japanese Society of Pathology
Eosinophilic Gra nulo ma Showing Rapid Regression Report of a Mandibular Case with Application of a Modified PNA Staining Method for Demonstration of La ngerha ns-type Histiocytes
Motoo Kitano', Gabriel Landini', lchiro Semba', Atsushi Urago', Kazumasa Sugihara2, Hiroshi Mukai2, and Sukehide Yamashita2
A mandibular eosinophilic granuloma in a 16-year-old male is reported. This case showed rapid regression, which was clearly demonstrated by histopathological examinations of both preoperative biopsy and surgical materials. Transformation from an eosinophilic granuloma to a xanthomatous granuloma with multinucleated giant cells was observed after only 26 days. Special staining of paraffin sections with peanut agglutinin (PNA) and use of electron microscopy showed that the main component of the lesion in the biopsy material was Langerhans-type histiocytes. These cells had disappeared from the lesion by the time of the operation. At the same time, the number of infiltrating eosinophils was also markedly reduced. It seems appropriate to consider that the rapid regression of this disease was correlated with the rapid reduction in the number of Langerhans-type histiocytes appearlng in the granulomatous foci, as well as the number of infiltrating eosinophils. Acta Pathol Jpn 40 : 588-595, 1990.
Key words : Eosinophilic granuloma, Langerhans-type
histiocytes, Peanut agglutinin staining method
INTRODUCTION Eosinophilic granuloma has been considered as one of a group of disorders known as histiocytosis-X or Langerhans cell histiocytosis (1-3). In addition to eosinophilic granulomas, this group includes Hand-Schuller-Christian Received December 11, 1989. Accepted for publication May 17, 1990. 'Department of Oral Pathology and ZFirst Department of Oral and Maxillofacial Surgery, Kagoshima University Dental School, Kagoshima. Mailing address : Motoo Kitano (tBE+), Department of Oral Pathology, Kagoshima University, Usuki-cho 1208-1, Kagoshima 890, Japan.
disease (probably better described as multiple eosinophilic granuloma), and Letterer-Siwe disease (a malignant counterpart with a characteristically fulminant course occurring in infants), as well as many intermediate and unclassified forms (4). Eosinophilic granulomas are seen most commonly in children and young adults. They are localized destructive conditions which occur within the bone and occasionally involve the soft tissues (5-8). The most commonly affected bones are the proximal femoral metaphysis, skull, mandible, rib and vertebral column (2, 3). Usually the prognosis of this disease is good and patients with a unifocal lesion may show spontaneous regression (2-9). Histologically, eosinophilic granuloma is characterized by a sheet-like proliferation of specific large mononuclear cells, Langerhans-type histiocytes (LCs), and an admixture of eosinophils, lymphocytes, plasma cells, macrophages and multinucleated giant cells. Many of the LCs have indented or cleaved nuclei with abundant pale-staining cytoplasm (5, 10, 11). Electron microscopy of the LCs reveals special cytoplasmic inclusion bodies of unknown function known as Birbeck or Langerhans granules. Recent immunohistochemical methods have indicated that these LCs can be identified using anti-S-100, anti-T6 and anti-ATPase antibodies (1116). Ree and Kadin (1986) (17) described a peanut agglutinin (PNA) staining method that was useful and reliable for identification of the LCs in Langerhans cell histiocytosis. The present study was based upon a case of eosinophilic granuloma which developed in the mandible of a 16-year-old male, and showed very rapid spontaneous regression. This paper describes the clinical and pathological features of this case and discusses the relation-
589
Acta Pathologica Japonica 40 (8) : 1990
Figure 1. X-ray photograph of this case. A wide, abnormal translucent shadow (arrows) of the left mandibular ramus is apparent.
ship between the regression of this disease and the microscopical findings using a modified method of PNA staining and electron microscopy.
CASEREPORT A 16-year-old Japanese male high school student was hit in the left temporal region during a rugby game. Two days later, he noticed pain in the left temporomandibular joint and trismus. A swelling developed in the left anterior auricular region 17 days later and he then consulted a dentist. An X-ray examination revealed an abnormal translucent shadow in the mandibular ramus extending from the condyle to the coronoid process (Fig. 1). A primary malignant bone tumor was suspected and he was referred to the Kagoshima University Dental Hospital. On admission, a slight edematous swelling of the left temporomandibular joint region and slight trismus was observed. No abnormalities were found by routine laboratory tests, except for moderate elevation of the antistreptolysin-0 titer to 833 Todd units and slight elevation of serum alkaline phosphatase to 2 9 9 units/l. Results of blood analysis were within normal limits initially, but a slight elevation of eosinophils to 5% of the white cell profile was found two days after admission. On the fifth day, biopsies were performed at the site of the lesion in the mandible and an enlarged lymph node 1 2 m m in maximum diameter on the left side of the neck. The hist o pat ho log ica I d iag nos is was eosi no p hi I ic granuloma of the bone. Twenty-six days after the biopsy, a raminectomy of the left side of the mandible was performed with exarticulation of the left temporomandibular joint and sub-
sequent reconstruction of the joint with an iliac bone graft. The postoperative clinical course was good and there were no detectable recurrent foci in the mandible, other bones, or visceral organs two and a half years after the operation.
PATHOLOGICAL FINDINGS The mandibular biopsy material was composed of several small fragments of granulation tissue of high cell density including numerous eosinophils, lymphocytes, histiocytic cells and large irregular-shaped multinucleated giant cells. Most of the histiocytic cells were rather large, round or elongated in form with varying amounts of cytoplasm and round or cleaved nuclei (Fig. 2). These cells corresponded t o the LCs demonstrated by PNA staining. Other histiocytic cells showed apparent phagocytic activity for foreign bodies such as hemosiderin or erythrocyte debris, and were considered to be macrophages. Watanabe’s silver impregnation method for reticulin showed no proper stroma, but scattered fine streaks of collagen fibers with scanty, delicate blood vessels. Small abscess-like clusters of eosinophils as well as diffuse hemorrhage were seen. A small number of xanthomatous cells were interspersed within this tissue. The biopsied lymph node showed slight follicular hyperplasia and mild aggregation of eosinophils, histiocytic cells and multinucleated giant cells in the marginal sinuses, suggesting that the lymph node was also involved in the eosinophilic granuloma (6). The mandibular specimen which was removed surgically 2 6 days after the biopsy contained a large portion
5 90
Eosinophilic Granuloma Showing Rapid Regression (Kitano et a/.)
Figure 2. Photomicrograph of the granu loma of the biopsy specimen Prolifera tion of histiocytic cells intermingled with many intermingled with many eosinophilic leukocytes is evident (HE)
Figure 3. Photomicrograph of the surgical specimen. A wide area of the condylar process is replaced by a scar tissue with abundant new bone formation around its periphery. A small granuloma surrounded by a layer of lymphoid cells is present in the central portion of the scar (HE).
Figure 4. The granuloma in the surgical specimen, consisting of numerous foamy xanthomatous cells, multinucleated giant cells and lymphocytes (HE).
591
Acta Pathologica Japonica 40 (8) : 1990
consisting of cicatrized collagenous tissue in which extensive new bone formation was noted (Fig. 3). The cicatrized collagenous tissue was situated in the condyle, extending into the coronoid process. In the central area of the cicatrized tissue, an irregular-shaped granuloma about 1 5 m m in longest diameter was present. It had a two-layered structure with an outer layer of densely infiltrated lymphocytes and plasma cells and an inner layer of xanthomatous granuloma. In the latter, xanthomatous cells with abundant foamy lipid-laden cytoplasm predominated (Fig. 4). Huge multinucleated giant cells including Touton-type giant cells were scattered elsewhere. There was no marked hemorrhage in the granuloma, but phagocytosis of hemosiderin granules was marked in almost all of the foamy xanthomatous cells and also in the giant cells. Eosinophils infiltrated the lesion in small numbers.
lmmunohistochemical analysis lmmunohistochemical studies were done using polyclonal antibodies (Dakopatts) against bovine S-100 protein, human alpha-1-antichymotrypsin and human lysozyme, and an ABC kit (Vectastain, Vector) was used on paraffin sections of the biopsy materials and the granulation tissue obtained at surgery. Immunostaining of the biopsy material confirmed the presence of lysozyme and alpha-1 -antichymotrypsin reactivity in some of the histiocytic cells (Fig. 5) but reaction with anti-S-100 protein antibody was very weak. Immunohistochemical examination of the surgical specimen showed a large population of cells with alpha-1 -antichymotrypsin and lysozyme antigen expression, but no evidence of strong S-100 protein immunoreactivity (Table 1).
Figure 5. Photomicrograph of immuno histochemistry for alpha 1-antichymotryp sin in the biopsy specimen Positive his tiocytic cells (macrophages) are scattered throughout the field of view
Table 1. Summary of lmmunohistochemical Studies, PNA Staining Method, and Electron Microscopical Examinations of Various Types of Cells in the Present Case La ngerha ns-ty pe H istiocytes f LC) -,
s-100 Lysozyme U-l-ACT PNAa EM a
.-
---+
--* -A,-+
it
Langerhans granules (LG)
Macro phages
--' +-it *-it it
Phagolysosomes (PL) No LG
Mult inucleated Giant Cells
Xanthomatous Cells
---t_
---t_
f-+
-+-it
--*
---+.
--+
Poor in PL No LG
---+ Lipid particles
Touton-type Giant Cells
--' +-it
--+ ---+
Lipid particles
Both LCs and macrophages were strongly positive by the PNA staining method, but were clearly differentiated by their staining patterns.
5 92
Eosinophilic Granuloma Showing Rapid Regression (Kitano et a/.)
Figure 6. Photomicrographs of the biopsy specimen after modified PNA staining. Dense deposits of reaction products are apparent on the cell surface as well as in the perinuclear area in Langerhans-type histiocytic cells (a), whereas the cytoplasm of macrophages is stained diffusely (b).
Table 2. Peanut Agglutinin Staining Method (Modified from Ree and Kadin)
’
1.
2. 3. 4. 5.
6. 7.
8. 9.
10 11.
Cut Daraffin-embedded tissue in 5 - ~ msections and m o u i t on slides coated with 0.2% neoprene solution in toluene to prevent detachment. Deparaffinize the sections with xylene and rehydrate in a graded ethanol series. Incubate at 37°C for 3 0 rnin in an aqueous solution of 0.1% trypsin. Wash in two changes of PBS. Immerse sections in 3% hydrogen peroxide in distilled water for 10 min to block endogenous peroxidase activity. Wash in two changes of PBS. Cover the sections with biotinylated PNA (Vector; dilution 1 / 2 0 0 in PBS) for 30 min. Then sprinkle mouse liver powder over the solution and incubate for another 30 min. Wash in two changes of PBS. Incubate in avid in^ biotin complex (Vector; dilution 1/ 100 in PBS) for 20 rnin. Wash in two changes of PBS. Immerse the sections in diaminobenzidine solution (0.025%), pH 7.0, until the reaction in evident by light microscopy. Rinse in tap water, counterstain with methy l green and mount in Entellan New.
Results of PNA staining The peanut agglutinin (PNA) staining method according to Ree and Kadin (1986)(17) with some modifications (shown in Table 2) was employed on the paraffin sections. A positive reaction was seen in two cell types, the LCs and macrophages, which occurred in a ratio of 3 : 2. LCs showed a unique and characteristic dense deposit of reaction products on their cell membranes as well as in the paranuclear cytoplasmic areas (Figs. 6, 7). The remaining cytoplasm stained only
LC
MP
Figure 7. Schematic drawing of a Langerhans-type histiocytic cell (LC) and a macrophage (Mp) stained with PNA. N : Nucleus.
weakly or not at all. The macrophages also showed a strong positive reaction with the PNA staining method, but the cytoplasmic staining pattern was diffuse (Figs. 6, 7). Multinucleated giant cells were essentially negative for PNA staining, but some of them stained weakly without any particular staining pattern. In the surgical specimen, there were no definitive cells with the same characteristic staining pattern as that of the LCs. The foamy xanthomatous cells and multinucleated giant cells showed variable, negative or positive reactivity, with no particular staining pattern.
Acta Pathologica Japonica 40 (8): 1990
Figure 8. Electron micrograph of a Langerhans-type histiocytic cell possessing Langerhans granules (arrows) from the biopsy specimen ( x 15,000).
Figure 9. High-magnification view of the Langerhans granules in Fig. 8 ( x 100,000).
593
5 94
Eosinophilic Granuloma Showing Rapid Regression (Kitano et a/.)
Electron microsco pica I findings In the biopsy material, typical LCs were recognizable by the presence of zipper cord-like Langerhans granules (Figs. 8, 9) (10). More than 40% of the observed cells possessed these granules. Sometimes the ends of the granules were directly connected to the cell membrane (11). Apart from their granules, LCs were characteristically large cells with an irregular outline and numerous peripheral villous processes. The cytoplasm of the LCs contained several dense bodies, many mitochondria and well developed Golgi apparatus. The nucleus often showed a thin rim of peripheral heterochromatin, a relatively electron-lucent nucleoplasm and one or two nucleoli. Intermingled with the LCs, macrophages and many phagolysosomes were present. Langerhans granules were never seen in these cells. In the multinucleated giant cells, Langerhans granules were absent and phagolysosomes were seldom present. These giant cells contained many small, sometimes bent mitochondria. In the surgical specimen there were high numbers of mononuclear and multinucleated cells possessing abundant lipid or lipoid particles. In these cells, phagocytosis of hemosiderin granules was marked, Langerhans granules were never seen and it was not possible to identify any of the characteristics of LCs.
DISCUSSION Eosinophilic granuloma, along with other diseases belonging to histiocytosis-X, is characterized by an a bnorma I proliferation of La ngerha ns- type histiocytes (LCS). LCs are normally present in areas of antigen penetration, such as the epidermis and mucosal epithelium of the digestive tract. LCs as well as the interdigitating reticulum cells of lymph nodes, which belong to the dendritic cell family, are potent stimulators of various lymphocyte reactions. These cells play a critical role in the development of delayed-type hypersensitivity responses and are required for the initial activation of Tlymphocytes. Therefore they are involved in the generation of the immune response, particularly during the afferent phase. Likewise, dendritic cells have been detected in various chronic inflammatory diseases (18) in which they seem to play an important role in disease initiation and/or propagat ion. Histopathologically, the present case showed the features of an active eosinophilic granuloma in the biopsy material, and then passed rapidly to an inactive phase at the end-stage of the disease, revealing a xanthomatous granuloma in the surgical specimen after only 2 6 days
(19). The PNA staining method(l7) and electron microscopical examinations (10) showed that the active phase was represented by an aggregation of numerous LCs, whereas these cells disappeared in the inactive phase. Since LCs are presumed to be able to trigger an effector inflammatory cascade, their relevance to the development and/or progression of histiocytosis-X seems to be crucial. Differences in the severity of a functional disorder of these cells may be responsible for differences in the clinical and histopathological presentation (11). LCs are widely known to express S-100 protein strongly (4, 12, 17), although we were unable to obtain any such evidence from the biopsy material. This may have been due to inappropriate fixation, causing unreliable detection of 5-100 protein in LCs in histiocytosis-X (20). Recently, Ree and Kadin(17) employed a PNA staining method for LCs in paraffin sections of histiocytosis-X. LCs were easily distinguished from other types of cells appearing in the lesions by their unique paranuclear and cell surface deposition of reaction products. This method was employed w i t h some modifications in the present study, and the results were confirmed by electron microscopy. This again emphasized that PNA staining for LCs in eosinophilic granuloma or other kinds of histiocytosis-X can be very useful. It is well known that solitary eosinophilic granuloma of the bone has an excellent prognosis in contrast to other types of histiocytosis-X. Spontaneous resolution may occur, and the responses to treatment including curettage, chemotherapy and radiation therapy are good. In general, if a second lesion does not appear within one year, the prognosis is believed to be especially good (2, 3, 9). Histopathological analysis of the present case revealed almost complete regression during a 26-day period from the initial biopsy to surgery on the mandible. There are no case reports in the literature describing such a rapid regression of this disease confirmed by histopathological analysis in the same location, as in the present case (21 -23). Therefore, it seems appropriate to consider that spontaneous regression may occur more rapidly and frequently than generally considered previously (21-23). In retrospect, it was inappropriate to employ surgical procedures (raminectomy and exarticulation of the mandible) in the present case as a first-choice treatment. According to Schajowicz (2), LCs in histiocytosis-X may show secondary transformation into lipid-, or lipoidladen foamy xanthomatous cells which may also possess hemosiderin granules and cellular debris in their cytoplasm. Conversely, Elema and Atomosoerodjo-Briggs
Acta Pathologica Japonica 40 (8) : 1990
(11) believed that transformation between the two cell types, i.e. LCs and macrophages including xanthomatous cells, never occurs. Unfortunately, the fate of LCs in the biopsy material in the present case could not be determined, since the surgical material showed no definitive LCs by PNA staining o r electron microscopy. Acknowledgements : We express our thanks to Prof. Munetomo Enjoji and Assoc. Prof. Masazumi Tsuneyoshi, Second Department of Pathology, Kyushu University Faculty of Medicine, Fukuoka, Japan, for their invaluable advice. We acknowledge the excellent assistance of Ms. Fusako Kataoka, Ms. Sayuri Kubota, and Ms. Shuko Toya. The manuscript was kindly reviewed by Dr. Richard M. Shelton, Birmingham University (U K).
REFERENCES 1. Lichtenstein L. Histiocytosis X. Integration of eosinophilic granuloma of bone, “Letterer-Siwe disease”, and ”Schuller-Christian disease” as related manifestations of a single nosologic entity. Arch Pathol 56 : 84-1 02, 1953. 2. Schajowicz F. Tumor and tumor-like lesions of bone and joints. Springer-Verlag, Heidelberg 1981. 3. Barnes L. Surgical pathology of the head and neck. Marcel Dekker, Inc., New York, 1985. 4. Watanabe S, Nakajima T, Shimosato Y, Sato Y, and Simizu K. Malignant histiocytosis and Letterer-Siwe disease: Neoplasms of T-zone histiocyte with SlOO protein. Cancer 51 : 1412-1424, 1983. 5. Kato T, Matsuda M, Ando H, and Sasaki H. A case of multifocal proliferations of histiocytic cells containing Langerhans’ cell granules. Am J Clin Pathol 7 6 : 480-485, 1981. 6. Sajjad SM and Osborne B. Lymph node involvement by histiocytosis-X. Arch Pathol Lab Med 1 0 6 : 9698, 1982. 7. Siegal GP, Dehner LP, and Rosai J. Histiocytosis X (Langerhans’ cell granulomatosis) of the thymus: A clinicopathologic study of four childhood cases. Am J Surg Pathol 9 : 117-124, 1985. 8. Goldberg NS, Bauer K, Rosen ST, et a/. Histiocytosis X : Flow cytometric DNA-content and immunohistochemical and ultrastructural analysis. Arch Dermatol 122 446-450, 1986. 9. RisdaII RJ, Dehner LP, Duray P, et a/. Histiocytosis X (Langerhans’ cell histiocytosis). Arch Pathol Lab Med
595
107 : 59-63, 1983. 10. Wolff K. The Langerhans cell. Curr Probl Dermatol 4 : 79-145, 1972. 11. Elema JD and Atomosoerodjo-Briggs JE. Langerhans‘ cells and macrophages i n eosinophilic granuloma : An enzyme-histochemical, enzyme-cytochemical, and ultrastructural study. Cancer 54 : 2174-2181, 1984. 12. Nakajima T, Sat0 Y, Watanabe S, et a/. Immunoelectron microscopical demonstration of S-100 protein in epidermal Langerhans cells. Biomed Res 3 : 226231, 1982. 13. Fox JL and Berman B. T6-antigen-bearing cells in eosinophilic granuloma of bone. JAMA 2 4 9 : 30713072, 1983. 14. Watanabe S, Sat0 Y, Kodama T, and Shimosato Y. lmmunohistochemical study with monoclonal antibodies on immune response in human lung cancers. Cancer Res 43 : 5883-5889, 1983. 15. Hall PA, ODoherty CJ, and Levison DA. Langerhans cell histiocytosis : An unusual case illustrating the value of immunohistochemistry in diagnosis. Histopathology 11: 1181-1191, 1987. 16. Wakuya J. lmmunohistochemical and ultrastructural studies on histiocytosis in children. Acta Pathol Jpn 3 7 : 901-913, 1987. 17. Ree HJ and Kadin ME. Peanut agglutinin: A useful marker for histiocytosis X and interdigitating reticulum cells. Cancer 57 : 282-287, 1986. 18. Demetris AJ, Sever C, Kakizoe S , et a / . SlOO protein positive dendritic cells in primary biliary cirrhosis and other chronic inflammatory liver diseases : Relevance to pathogenesis ? Am J Pathol 1 3 4 : 741-747, 1989. 19. Engelbreth-Holm J, Teilum G, and Christensen E. Eosinophilic granuloma of bone: Schuller-Christian’s disease. Acta Med Scand 9 8 : 292-312, 1944. 20. Finney DS, Rees TD, Wright JM, and Blanton PL. Solitary eosinophilic granuloma (histiocytosis X) of the gingiva : A report of two cases. J Periodontol 59: 457-460, 1988. 21. Mercer Sir W and Duthie RB: Histiocytic granulomatosis. J Bone Joint Surg (Br) 3 8 : 279-292, 1956. 22. Dumermuth G. Reticulogranulomatose : Zwei Falle von eosinophilem Granulom mit Ubergang in HandSchuller-Christiansche Krankheit. Helvet Paediatr Acta 1 3 : 15-39, 1958. 23. Ritter R A Jr. Histiocytosis X : A case report with electron microscopic observations. Cancer 19 : 1155-1 164, 1966.
