Heat Shock Protein 27 Expression in the Epithelium of Periapical ...

JOURNAL OF ENDODONTICS Copyright © 2001 by The American Association of Endodontists

Printed in U.S.A. VOL. 27, NO. 2, FEBRUARY 2001

Heat Shock Protein 27 Expression in the Epithelium of Periapical Lesions R. Leonardi, DDS, L. Villari, DM, M. Caltabiano, DM, PhD, and S. Travali, DM, PhD

tion (4) and the migration of keratinocytes during wound healing (5); it blocks apoptosis and protects cells against the cytotoxicity of inflammatory mediators (6). The epithelial lining of periapical cysts is generally considered to be derived from the proliferation of epithelial rests of Malassez (7), but the factors responsible for the activation of these normally quiescent rests are poorly understood. Alterations in connective tissue environment, such as local changes in tissue pH or carbon dioxide tension are thought to be responsible for this activation (7). Moreover the local accumulation of various types of immune cells and inflammatory mediators is believed to be associated with epithelial proliferation (8). On the other hand cytokines are also related to cell death, and the reasons why epithelial proliferating rests are protected against a stressful environment are a matter of question. In view of the roles of HSP27 and the possible implication in the pathogenesis of periapical lesions, the expression of HSP27 was investigated immunohistochemically in periapical granulomas with proliferating epithelium and in radicular cysts focusing on the epidermal compartment.

Heat shock protein (HSP) 27 is a member of the small HSP family that plays a part in the regulation of epithelial cell growth and differentiation, wound healing, apoptosis and cell protection against inflammatory cytotoxicity mediators. Thus the expression of HSP27 was investigated immunohistochemically in periapical granulomas with epithelial rests of Malassez and in radicular cysts. AntiHSP27 mouse monoclonal antibody and peroxidase-labeled streptavidin-biotin standard technique were used to study the expression of HSP27. Proliferating epithelial cell rests, and islands of epithelium and epithelial lining of microcysts strongly reacted throughout all layers, whereas radicular cysts epithelial lining presented mainly a moderate suprabasal staining pattern. However both the proliferating epithelial cell rests and the radicular cysts shared an over-expression of HSP27 immunostaining intensity in coincidence with the presence of local infiltration of immune cells. HSP27 may play several roles in periapical lesions that include contributing to the migration of epithelial cell rests and an increased resistance both to necrotic and apoptotic cell deaths.

MATERIALS AND METHODS Tissue specimens were obtained from the archives of the Department of Dentistry, University of Catania. Diagnosis was based on histopathological criteria, clinical history, and radiographic appearance. The formalin-fixed paraffin-embedded specimens were 16 periapical lesions of endodontic origin obtained from periapical surgery or tooth extraction and consisted of 10 periapical granulomas and 6 periapical cysts. Specimens were sectioned at a thickness of 5 ␮m and processed for immunohistochemistry as previously described (9). Shortly, deparaffinized and rehydrated sections were incubated for 30 min in 0.3% H2O2/methanol to quench endogenous peroxidase activity, then rinsed for 20 min with phosphate-buffered saline (PBS) (Bio-Optica M107, Milan, Italy). Nonspecific protein binding was attenuated by incubation for 30 min with 5% horse serum. For localization of HSP27 a mouse anti-HSP27 monoclonal antibody (catalog no. NCLHSP27; Novocastra Laboratories Ltd., Newcastle, UK) was used at a dilution of 1:20. The antibody was applied directly to the section, and the slides were incubated overnight (4°C) in a “humified chamber.” The sections were washed three times with PBS at room temperature. Immune complexes were subsequently treated with the secondary antibody and then detected by means of streptavidin

Heat shock proteins (HSPs) are a highly conserved group of cellular proteins whose synthesis is increased in response to a variety of stresses including: elevated temperatures, heavy metals, toxins, oxidants, and bacterial and viral infections (1). Cell exposure to stresses causes an imbalance in protein metabolism, which challenges the cell to respond rapidly, yet precisely, to minimize the deleterious effects of environmental and physiological stresses (1). In general, the stress response represents a cellular defense mechanism that leads to a decreased production of some polypeptides, while upregulating the HSPs (2). The 27-kDa HSP (HSP27) is a member of the small HSP family (3). In addition to its putative function in thermotolerance, this protein plays a role in the regulation of epithelial cell differentia89

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FIG 1. Immunolocalization of HSP27 in proliferating epithelium by peroxidase-labeled streptavidin-biotin technique. Staining with monoclonal antibody against HSP27 is strong for all the epithelial cells. (⫻280.)

peroxidase, both incubated for 30 min at room temperature (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). After rinsing with three changes of PBS the immunoreactivity was visualized by development for 2 min with 0.1% 3,3⬘-diaminobenzidine and 0.02% hydrogen peroxide (DAB substrate kit, Vector Laboratories). Sections were counterstained with Mayer-hematoxylin, mounted with Permount, and examined by light microscopy. Positive controls consisted of tissue specimens with known antigenic positivity and included sections of breast carcinoma tissue. Negative control consisted of periapical lesion sections that were incubated with normal rabbit serum, omitting the primary antibody. The staining intensity was assigned on a subjective scale as follows: 0 for no reactivity, 1 for weak reactivity, 2 for moderate, 3 for strong, and grade 4 for a very strong reactivity. Three observers independently assessed the immunohistochemical reaction; and where the findings were divergent an agreement was reached after discussion.

FIG 2. Photomicrograph showing HSP27-positive epithelial cells in a periapical lesion in which epithelial rests appeared to have proliferated to form trabeculae by a peroxidase-labeled streptavidin-biotin technique. (⫻120.)

RESULTS At light microscopic examination all the specimens showed the presence of epithelium, fibrous connective tissue with varying degrees of leukocytic infiltration, and newly formed blood vessels. Three of the 10 periapical granulomas contained rests of Malassez. Seven of periapical granulomas contained epithelium rests that appeared to have proliferated to form strands and trabeculae of epithelium. The six periapical cysts lumen was lined by nonkeratinized stratified squamous epithelium, with an average thickness of 4 to 6 layers. Cytoplasmic staining for HSP27 antibody was detected in both the epithelium of radicular cysts lining and the proliferating epithelial rests. Although cysts lining and epithelial proliferating rests were immunostained by anti-HSP27, there was a different trend of labeling intensity for this antibody. Proliferating cell rests, islands, and lining of microcysts (Figs. 1 and 2) reacted strongly (grade 3), whereas radicular cyst epithelial lining (Fig. 3) presented a moderate immunoreaction (grade 2). Thus the intensity of HSP27 staining within the epithelium varied along with pathological findings.

FIG 3. Immunolocalization of HSP27 in a radicular cyst epithelial lining, by peroxidase-labeled streptavidin-biotin technique, revealing a suprabasal staining pattern and a patchy staining of the basal layer. (⫻280.)

Also the distribution of HSP27 immunoreactive epithelial cells was different in proliferating rests of periapical granulomas and radicular cysts. The proliferating epithelial cell rests (islands, strands, and trabeculae) were characterized, always, by a uniform pattern and intensity of staining throughout the layers (Figs. 1 and 2). On the other hand the epithelium of radicular cysts showed HSP27 immunolabeled cells mainly in the suprabasal region, but patchy and discontinuous immunolabeling of the basal layer could also be seen. However both proliferating epithelial cell rests and radicular cysts shared an overexpression of HSP27 immunostaining intensity coincident with the presence of local intraepithelial or subepithelial infiltration of immune cells (Figs. 4 and 5).

Vol. 27, No. 2, February 2001

FIG 4. HSP27 strongly positive epithelial cells in a periapical granuloma in the presence of intraepithelial and subepithelial infiltration of immune cells. (⫻280.)

FIG 5. Photomicrograph showing an upregulation of HSP27 expression in the epithelium of a radicular cyst in the presence of inflammatory infiltrate (arrow). (⫻280.)

DISCUSSION In man epithelial rests of Malassez are not proliferative, but previous studies indicate that proliferating epithelium is commonly found in inflammatory periapical lesions. Although the reasons for proliferation of epithelium in this disease are not clear, it is always connected with local accumulation of various types of immune cells (8). The fact that periapical granulomas with altered proliferating epithelium have larger number of immune cells than those of periapical granulomas containing only the rests of Malassez correlates with this hypothesis (8). Some studies on cytokine and cell adhesion molecules expression in periapical lesions have been conducted to elucidate the mechanism of epithelial proliferation (9 –12). On the other hand no information is available on the mechanisms that allow epithelial cell survival in periapical lesion’s stressful environment. Recent results showing that HSPs play crucial roles in a wide variety of normal and pathological cellular processes have made them an objective of broad interest to specialists in various field of

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medicine, including infection diseases, immunology, oncology, and autoimmunity (3). Important roles of HSP27 in regulation of keratinocytes differentiation (4, 13), protection against the cytotoxicity of inflammatory mediators (6), and blocking apoptosis (14, 15) have led us to conduct this immunohistochemical investigation in periapical lesions. Our study has demonstrated HSP27 immunoreactivity in epithelial cells of periapical lesions, although with differences related to the nature of the lesion and the presence of local inflammatory infiltrates. HSP27 was mainly expressed by suprabasal epithelial cells in radicular cyst linings (i.e. cells in a low proliferative and highly differentiated state). This pattern of immunostaining is consistent with data published by others on keratinocytes (4, 13). In proliferating epithelial cell rests, islands, strands, and trabecule, all epithelial cell layers were strongly immunoreactive for HSP27. This uniform pattern of expression should not be correlated with the presence of well-differentiated epithelial cells as in proliferating epithelium. In fact keratin expression is different from the cyst’s well-differentiated stratified epithelium (16). The strong immunolabeling associates better with an activated and migrating state of epithelial cells (5). In fact the only study reported for epithelial tissue describes a basal and suprabasal HSP27 immunostaining of migrating keratinocytes in the wound healing process (5). It seems plausible, therefore, that in proliferating epithelial cell rests HSP27 expression may account both for a regulatory function mediating cell migration (17) and a protective function (18) against the periapical lesion stressful environment. Moreover HSP27 upregulation was seen in some areas of epithelial islands, strands, trabeculae, microcysts, and epithelial lining of radicular cysts, and it seemed to be correlated with the presence of local inflammatory infiltrates. This direct relationship was clearly evident in heavily stained epithelial tissues that were associated with inflammatory infiltrates in comparison with the adjacent not inflamed epithelium. HSP27 upregulation in these areas correlates well with the already described multiprotective effects of this molecule on epithelial cells in the presence of inflammation (6). Cells, in fact, protect themselves against the cytotoxicity of inflammatory mediators, including reactive oxygen species and cytokines, upregulating HSP27 (6). HSP27 overexpression in epithelial cells increases their resistance to cytotoxic agents and impairs their susceptibility to necrotic cell death. In combination with cell protection against inflammatory agents, evidence has demonstrated that HSP27 increases cell resistance to apoptosis by blocking Fas/APO-1 pathways (14). In view of the importance of apoptosis in the morphogenesis of human fetal skin and maintenance of adult epidermal homeostasis, it seems conceivable to presume also a role for HSP27 in the epithelial tissue of periapical lesions. It may be that cytotoxic agents, such as in a periapical granuloma, induce HSP27 expression in epithelial cell rests. This upregulation could allow epithelium to protect itself from necrotic death, as well as programmed cell death. The removal of a stressful environment (i.e. endodontic therapy) downregulates HSP27 expression and facilitates epithelial cell death and healing processes. In conclusion HSP27 expression may play several roles in periapical lesions that include inducing epithelial cell rests migration and increasing resistance both to necrotic and apoptotic cell deaths. Due to the possible important clinical significance of HSP27 in pathogenesis, as well as novel implications in therapy of

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periapical lesions, further studies are required to clarify roles of this HSP. Drs. Leonardi and Caltabiano are affiliated with Cattedra di Ortognatodonzia, Dr. Villari is affiliated with the Istituto di Anatomia Patologica, and Dr. Travali is affiliated with the Istituto di Patologia Generale, University of Catania, Catania, Italy. Address requests for reprints to Dr. Rosalia Leonardi, Istituto di II Clinica Odontoiatrica, Policlinico, Citta´ Universitaria, Universita` di Catania, Viale A. Doria no. 6, Catania, Italy.

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