Cheng-Chun Huang

Expressions of c-jun and p53 proteins in human middle ear cholesteatoma: Relationship to keratinocyte proliferation, differentiation, and programmed cell death

The c‐jun protein functions as a transcription factor for many genes, and the p53 protein functions as a negative regulator of cellular proliferation, which is related to the apoptosis pathway that induces DNA damage. It has recently been shown that c‐jun promotes keratinocyte proliferation and p53 induces apoptosis of various cells.

Localization of interleukin-1 in human cholesteatoma

Recent studies by other investigators have shown that interleukin-1 (IL-1) promotes bone resorption by stimulating various cells. Interleukin-1 not only stimulates collagenase production by fibroblasts and macrophages, but also acts as an osteoclast-activating factor. In this study, IL-1 was localized in human cholesteatoma tissues using both immunoperoxidase and immunofluorescent-staining methods with specific monoclonal antibodies. Highly concentrated IL-1 was found in the epithelial layer and granulation tissue. More specifically, intense staining was seen in basal and spinous cells of the epithelial layer, and in fibroblasts and macrophages of the granulation layer. We also located IL-1 in the normal external ear canal skin; however, the intensity of the staining in the cholesteatoma epithelium was found to be stronger. The presence of IL-1 in the epithelial layer and granulation tissue of the cholesteatoma suggests that IL-1 from the stimulated keratinocytes of the cholesteatoma could be one factor responsible for the markedly increased bone resorption observed in cholesteatoma patients.

The role of tumor necrosis factor-alpha in bone resorption of cholesteatoma

Recent investigations have indicated that cytokines such as tumor necrosis factor-alpha (TNF-alpha) play a potential role in the bone resorption associated with inflammatory diseases. In this immunoperoxidase study, TNF-alpha was localized in mononuclear cells, macrophages, fibroblasts, osteoblasts, and osteoclasts adjacent to bone resorption areas in both human and experimental middle ear cholesteatomas. In vitro, TNF-alpha stimulated monocytes to form multinucleated cells that demonstrate tartrate-resistant acid phosphatase activity, a marker enzyme for osteoclasts. These multinucleated osteoclast-like cells induce resorption of devitalized bone. The extent of bone resorption was increased by the co-cultures of osteoblasts and osteoclasts in the presence of TNF-alpha, suggesting that cell to cell interaction plays a significant role in bone resorption. Moreover, TNF-alpha was capable of stimulating macrophages to produce acid phosphatase and collagenase, and osteoblasts to produce prostaglandin E2 and collagenase. These chemical mediators have been known to lead to bone resorption. Our findings suggest that TNF-alpha may play an important clinical role in the destructive process of cholesteatoma.

Colony-stimulating factor in middle ear cholesteatoma.

Granulocyte-macrophage colony stimulating factor (GM-CSF) was found in human middle ear cholesteatoma tissues by immunohistochemical technique using mouse monoclonal anti-(human) GM-CSF IgG. Immunofluorescent staining showed the presence of GM-CSF in the peribasal area, in some suprabasal cells of the epithelium, and in the inflammatory connective tissue, especially in the monocytes and fibroblasts. These findings were confirmed by the immunoperoxidase method. Staining of the external ear canal epithelium, however, was significantly weaker than that of the cholesteatoma epithelium. The presence of GM-CSF in cholesteatoma appears to be a response to inflammation occurring in the middle ear cavity. Moreover, our in vitro study showed that GM-CSF induced the proliferation and protein synthesis of basal keratinocytes. This study suggests that GM-CSF is involved in the development and destructive effects of middle ear cholesteatoma.

Expression of cytokeratins 13 and 16 in middle ear cholesteatoma.

The accumulation of keratinizing epithelium in the middle ear cavity is a crucial factor in the pathogenesis of cholesteatoma. We hypothesize that keratinocytes from the skin of the ear canal migrate and hyperprollferate in response to Inflammation in the middle ear cavity to cause accumulation of keratin debris. In the present study, we Investigated the expression of specific cytokeratins (CKs) in the cholesteatoma matrix to determine whether cholesteatoma is a hyperproliferative disease. Cytokeratin expression was examined in cholesteatoma, meatal skin, and tympanic membrane with two monoclonal antibodies, one for both cytokeratins 13 and 16 (antibody K8.12), and another for cytokeratin 13 only (antibody K5–1A3). CK 13 (MW 51 KD) Is a marker of differentiation and CK 16 (MW 48 KD) is a marker of hyperproliferatlon of keratinocytes. The use of immunoblot probes showed that CKs 13 and 16 were present in cholesteatoma. Immunofluorescenf staining showed the presence of CK 16 In the suprabasal layer of cholesteatoma, which was located near the external ear canal. CK 16 was also localized in the suprabasal layer of meatal skin and tympanic membrane. CK 13 was localized in the basal layer of the cholesteatoma, distal to the external ear canal, but not in the meatal skin and tympanic membrane. Taken together, the present data suggest that cholesteatoma is a hyperprollferative disease and that cholesteatoma expresses CK 16 near the external ear canal and transforms to express CK 13 during growth distally.

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.

Tumor necrosis factor‐alpha production in human head and neck squamous cell carcinoma

Recent studies suggest that tumor necrosis factor‐alpha (TNF‐α), a pleiotropic cytokine, is responsible for some of the systemic and local effects, including tumor‐associated cachexia and neoplastic bone destruction, seen in patients with cancer. This study was undertaken to determine if TNF‐α is produced by human squamous cell carcinoma of the head and neck, and, if so, to determine its source and cellular distribution.

Platelet-derived growth factor in middle ear cholesteatoma

Platelet-derived growth factor (PDGF) was localized in human middle ear cholesteatoma tissue by an immunoperoxidase technique using rabbit anti-human PDGF IgG. PDGF was found mainly in basal cells and in granulation tissue, and especially involved monocytes and fibroblast-like cells. The external ear canal epithelium was not significantly stained by anti-human PDGF. Findings demonstrate that the presence of PDGF in cholesteatoma is in response to inflammation and wound healing in the middle ear. PDGF in vitro was found to stimulate protein synthesis and cellular terminal differentiation of basal keratinocytes. PDGF also stimulated monocytes to form multinucleated osteoclast-like cells. These multinucleated cells, in turn, induced the resorption of devitalized bovine bone. This bone resorption was seen in co-cultures of osteoblasts and multinucleated osteoclast-like cells in the presence of PDGF, suggesting that cell-to-cell interaction plays a role in bone resorption. The present study suggests that PDGF takes part in the clinical development and the destructive effect of cholesteatoma.

An immunocytochemical study of cytokeratin expression in human middle ear cholesteatoma

SummaryAn indirect immunofluorescent method with monoclonal anti-cytokeratin antibodies was used to localize various cytokeratins in human middle ear cholesteatoma. The 50 K/58 K and 56.5 K/65–67 K paired cytokeratins are markers of skin type and were found in the specimens of human middle ear cholesteatomas studied. In contrast, the 40 K and 45 K cytokeratins (markers of simple epithelia), the 48 K cytokeratin (marker of hyperproliferative epidermal disease) and the 51K cytokeratin (marker of internal organ epithelia) were absent in human middle ear cholesteatoma. These findings indicate that the pattern of cytokeratins in human middle ear cholesteatoma is similar to that of skin but is different from those of simple epithelium, internal organ epithelia, and hyperproliferative epidermal disease. These findings also support the skin type epithelial origin of cholesteatoma and strongly favor the migration theory in the genesis of cholesteatomas.

Effect of endotoxin on keratin production of keratinocytes in vitro

The rate of keratin protein accumulation appears to be a crucial factor in the pathogenesis of middle ear cholesteatoma. The effect of endotoxin on keratin production of keratinocytes was studied. Endotoxins are lipopolysaccharides contained within the cell wall of gram-negative bacteria. Various concentrations of endotoxin were added to keratinocytes derived from newborn rats. Syntheses of keratin proteins were studied by incorporation of 3H-leucine into each protein fraction. Extraction of keratin proteins with various salt solutions showed that endotoxin stimulated keratinocytes to produce keratohyalin granules and its related proteins as well as proteins in the stratum corneum (keratin proteins). These findings suggest that accumulation of keratin debris in cholesteatoma may result from the effect of infection in the epithelial cells.