Immunohistochemical identification of alpha-1-antitrypsin, alpha-1-antichymotrypsin, and lysozyme in focal hyperplastic gingivitis

Immunohistochemical identification of alpha- 1-antitrypsin, alph.a-1-antichymotrypsin, and lysozyme I in focal hyperplastic gingivitis Ruth Kivens, John J. Sauk, and Robert A. Vickers, Minneapolis, DEPARTMENT MINNESOT.4

OF ORAL

PATHOLOGY

AND

HUMAN

GENETICS,

SCHOOL

Minn.

OF DENTISTRY,

UNIVERSITY

OF

Tissues from twenty-four patients with focal hyperplastic gingival lesions containing calcification were stained for lysozyme (muramidase), alpha- 1-antitrypsin, and alpha- 1-antichymotrypsin. In eighteen of the twenty-four cases the tissues stained positively for lysozyme, and in all instances the tissues stained positively for alpha- 1-antitrypsin and alpha- 1-antichymotrypsin. These data suggest that the fibrous components of these lesions are derived from tissue histiocytes. (ORAL SURG. ORAL MED. ORAL PATHOL. 59:167-171, 1985)

S

oft-tissue masses composed predominantly of fibrous connective tissue constitute one of the most frequently encountered focal abnormalities of gingival mucosa. A variety of terms have been employed to describe these lesions and the moderate histologic differences they manifest. Peripheral ossifying$broand peripherma, peripheral cementifyingjbroma, al odontogenic jibroma are but some examples of this variation in nomenclature.’ Microscopically, these lesions are generally composed of an exceedingly cellular mass of connective tissue consisting of plump proliferating spindle cells that are dispersed in a delicate fibrillar stroma. In addition, several forms of calcification (bony trabeculae, dystrophic calcification, and globular calcified material resembling acellular cementurn) have been described in these lesions. The osteoid and cementoid patterns manifest by these lesions has suggested to some that these overgrowths are neoplasms derived from the periodontal ligament. In the present report we investigate the: possibility that these proliferative lesions are reactive inflammatory conditions and that the predominant cell type is derived from tissue histiocytes that function as facultative fibroblasts. For these investigators, immunohistochemical techniques were used to stain for alpha-1-antitrypsin (AlAT), alpha-1-antichymotrypsin (AlACT), and lysozyme, all of which have been shown to be

Table I. Staining of focal hyperplastic gingival lesions for lysozyme, Al AT, and Al ACT by indirect immunoperoxidase

Histology (type of lesion) Peripheral ossifying fibroma Peripheral cementifying fibroma Peripheral fibroma with dystrophic calcification

Lysozyme

AIAT

AIACT

3/a+

8/S+

8/S+

416-k

616-t

6/6+

10/10+

10/10+

10/10+

cytochemical markers for bone marrow and tissue histiocytes.2-5 MATERIALS

AND METHODS

Paraffin blocks were obtained from the files of the Department of Oral Pathology, University of Minnesota. All the tissue had been previously fixed in 10% buffered formalin and embedded in paraffin for diagnostic interpretation. In each case the tissues had been interpreted by standard light microscopy, after hematoxylin and eosin staining. For the present study, additional tissue sections were prepared and, subsequent to deparaffinization, were stained for Al AT, Al ACT, and lysozyme by a standard immunoperoxide technique.6 Samples were stained with 167

ivens, Sauk, and Vickers

Oral Surg. February, 1985

a, Fibrocellular components in peripheral odontogenic fibroma. b, Osteoid patterns observed in hyperplastic gingival lesion. c, Dystrophic deposits in peripheral odontogenic fibroma. d, Cementoid deposits in peripheral odontogenic fibroma. (Hematoxylin and eosinstain. Magnifications:a and b, x64; c, x80; d, x96.)

and without prior treatment with trypsin. Antisera to AlAT, AlACT, and lysozyme were obtained from AKOPATTS in Glostrup, Denmark. Negative controls were provided by a blank serum control and by absorption of the primary antisera with purified antigen. ESULTS

The soft-tissue growths examined are listed in Table I. The tissues used in the study included examples of focal hyperplastic gingivitis that contained bony trabeculae (Fig. 1, b), dystrophic calcifications (Fig. 1, c), and globular cementoid deposits (Fig. 1, d). The staining patterns obtained for AlAT, AlACT, and lysozyme are also summarized in Table I. Positive staining for AlAT and AlACT was resent in all of the casesexamined, regardless of the resence or type of calcification within the lesion (Figs. 2 to 4). In each case, intracytoplasmic granu-

lar staining depicted positivity for these cell markers. In general, the staining was relatively evenly distributed within the soft-tissue sections. In no instance was prior treatment with trypsin necessaryto elicit a positive stain; however, trypsin pretreatment was always noted to enhance the intensity of staining. Lysozyme was identified in seventeenof the twenty-four cases examined. When present, however, these stains were also evenly distributed within tumorlike proliferations (Fig. 5). The presence of occasional multinucleated giant cells was a recognized feature of some of these lesions. These cells, however, did not stain for lysozyme, although they did show reactivity when stained for AlAT and AlACT. Pretreatment of the tissue samples with trypsin was noted to enhance the staining intensity as noted above, but in no instance was a specimen positive after trypsin pretreatment if that specimen did not initially elicit a positive staining reaction.

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Fig. 2. Focal hypoplastic gingivitis containing osteoid. The section is stained for alpha-1-antitrypsin. The dark deposits among the spindle-shaped cells are stain deposits from an immunoperoxidase reaction. The blue hematoxylin stain was absorbed by the use of a Wratten 45 filter during the photographic procedure. (Magnification, X290.)

Fig. 3. Focal hyperplastic gingivitis stained for alpha-1-antichymotrypsin. Many of the fibrocellular components of the lesion display a positive reaction for the enzyme marker. (Indirect immunoperoxidase, hematoxylin counter stain; photographed through a Wratten 45 filter. Magnification, X200.)

DISCUSSION

The present study demonstrates that the focal gingival lesions designated as peripheral ossifying fibroma, peripheral cementifying fibroma, and peripheral odontogenic fibroma all share the cell markers alpha- 1-antitrypsin and alpha- 1-antichymotrypsin. In addition, seventeen of the twenty-four lesions examined also demonstrated the presence of lysozyme (muramidase). In contrast, control sections of normal gingiva and periodontal ligament fibroblasts were unreactive to staining for these proteins, except that within control normal gingiva microvascular pericytes showed reactivity to both Al AT and

AlACT. This latter finding, however, is not surprising since these cells have been regarded as having histiocytic properties.7 Although lysozyme has been regarded as a sensitive marker for reactive histiocytes, it is yet not clear whether this enzyme is a suitable indicator for tissue histiocytes, since many tumors that have been regarded as fibrohistiocytic lesions,*,3 especially of the skin, have not always manifested this enzyme. Conversely, Al AT and Al ACT have been shown to be more useful indicators of both reactive histiocytes and neoplastic histiocytes of bone marrow origin.3-5 Meister and Nathrath4 have shown that it is easier to

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Focal hyperplastic gingivitis containing numerous unstained osteoid islands (arrows). Many of the surrounding stroma cells contain Al ACT after immunoperoxidase stain photographed through a Wratten 45 filter. (Magnification, X200.)

5. Focal hyperplastic gingivitis stained for lysozyme. The light gray-staining regions are portions of the tissue stained with hematoxyiin and photographed through a Wratten 45 filter. The black deposits (arrows) are precipitates resuiting from an immunoperoxidase reaction. (Magnification, x300.)

Fig.

identify tumor cells in malignant histiocytosis and malignant fibrous histiocytomas using AlACT since lysozyme-containing cells are infrequent and scattered; also, AIACT will not stain granulocytes, in contrast to their positive reaction with lysozyme.5z8It should, however, be acknowledged that AlAT and AlACT are not exclusively histiocyte markers, since these proteins have been demonstrated in a variety of neoplasms and cells that have included renal cell carcinoma, mesothelioma, and Wilms tumors.6 The specificity and distribution of AlAT and AlACT does not present a dilemma in the present discussion, since the histologic content of these lesions is clearly fibrous in nature and a high

proportion of these lesions also demonstrated the presence of lysozyme. Thus, it would appear that these peripheral gingival lesions represent fibrohistiocytic proliferations that probably are sequelaeto a local inflammatory response. Although the patterns of calcification within these lesions remain a curiosity, there appears to be no basis for further delineation of these tissue responses. In conclusion, the use of cell markers in the field of soft-tissue pathology has made possible a more detailed examination of fibrous lesions. Specifically, AlAT and AlACT appear to be reliable markers for proliferations involving fibrous histiocytes. However, Rosai has noted that the current available criteria

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for tissue histiocytes fall short of those developed for other cells (that is lymphocytes). Thus, the use of cell markers described herein cannot yet be regarded as definitive, in that further probes are necessary to more completely define the cell population designated as tissue histiocytes or facultative fibroblasts. REFERENCES 1. Vickers RA: Mesenchymlal tumors of the oral region. In Gorlin RJ, Goldman HM (editors): Thoma’s oral pathology, St. Louis, 1981, The C. V. Mosby Company, p. E61. 2. Burgdorf WHC, Duray P, Rosai J: Immunohistochernical identification of lysozym’e in cutaneous lesions of alleged histiocytic nature. Am J Clin Path01 75: 162-167, 1981. 3. Isaacson P, Jones DB, Millward-Sadler GH, Judd MA, Payne S: Alpha-1-antitrypsin in human macrophages. J Clin Pathol 34: 982-990, 1981. 4. Meister P, Nathrath W: Immunohistochemical markers of histiocytic tumors. Hum Path01 11: 300-301, 1980.

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Meister P, Huhn D, Nathrath W: Malignant histiocytosisimmunochemical characterization on paraffin-embedded tissue. Virchow’s Arch[A] 385: 233-246, 1980. du Boulay CEH: Demonstration of alpha-1-antitrypsin in fibrous histiocytomas using the immunoperoxide technique. Am J Surg Path01 6: 559-564, 1982. Haidu SI: Patholoav of soft tissue tumors. Philadelnhia, L,. 1979. Lea & Febiger, pi.* 77-121. Mann R, Mendelsohn G, Eggleston J: Relationship of lysozyme to histiocytic differentiation in malignant histiocytosis. Lab Invest 40: 270, 1979. 9. Rosai J: Ackerman’s surgical pathology, St. Louis, 198 1, The C. V. Mosby Company, p. 1420. rZnn

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Dr. Ruth Kivens Department of Oral Pathology School of Dentistry University of Minnesota 515 Delaware St., S.E. Minneapolis, MN 55455