V. Schilling

Hyperproliferation-associated Keratin Expression in Human Middle Ear Cholesteatoma

Cholesteatoma is characterized by the presence of a squamous epithelium invading the middle ear altering its growth properties. This epithelium is believed to have hyperproliferative properties. Keratin 16 is accepted as a molecular marker for hyperproliferative epithelia. Two monoclonal antibodies K8.12 (directed against keratin 13) and KS.1A3 (directed against keratin 13 and 16) were used in an alkaline phosphatase anti-alkaline-phosphatase (APAAP)-technique to compare the expression of both keratin 13 and keratin 16 in normal human skin and aural cholesteatoma. Furthermore, the cytokeratin expression was compared to that of normal skin and palatine tonsil using one-dimensional gel electrophoresis. For both monoclonal antibodies, normal ear skin was stained only in the basal layer. In contrast, in the cholesteatoma samples the immunostaining of the antibody KS-1A3 was done not only in the basal cell layer but also in the suprabasal cells of the stratum spinosum and stratum granulosum. Using gel-electrophoresis, the presence of cytokeratin 16 was demonstrated in the cholesteatoma samples only. These results support the hyperproliferative character of cholesteatoma epithelium.

Aberrant expression of epidermal growth factor receptor in aural cholesteatoma.

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used in an alkaline phosphatase‐antialkaline phosphatase (APAAP) technique to compare the distribution of this protein in normal human skin and aural cholesteatoma. EGF receptors appear to be highly expressed on the basal layer of the epidermis, in hair follicle apocrine sweat glands, and in the capillary system of normal skin. Cholesteatoma epithelium showed increased positive reactions in the suprabasal layers. A heterogeneity in the expression was found in different parts of the cholesteatoma. These results suggest the presence of an aberrant regulation and persistence of EGF receptors in cholesteatoma and confirm the hyperproliferative character of the cholesteatoma epithelium.

Immunologically activated cells in aural cholesteatoma

In this immunohistochemical study, we characterized the cells infiltrating the stroma of acquired aural cholesteatomas in detail, using a panel of monoclonal antibodies directed against immune cell type-specific antigens, HLA class II antigens, and interleukin-2 receptor. For all antibodies used, normal ear skin was stained for comparison. The vast majority of the infiltrating cells was CD45-positive, ie, derived from bone marrow. Reactivity with anti-CD3 and anti-CD6 antibodies revealed an abundant infiltration of T lymphocytes beneath the squamous epithelium of cholesteatoma. The B lymphocyte-specific anti-CD19 and anti-CD22 antibodies detected only occasional positive cells. Hence, the cellular infiltrate in the stroma of aural cholesteatoma is made up primarily of T cells with macrophages scattered between them. Expression of HLA-DR was almost as high as that of CD45, whereas CD25-positive cells were detected in lower amounts. We infer that the majority of T cells and macrophages in the stroma of cholesteatoma are in an immunologically activated state. The characteristics of the infiltrating cell population suggest an antigen-driven process in cholesteatoma.

Localization of Transforming Growth Factor-β- Expressing Cells and Comparison with Major Extracellular Components in Aural Cholesteatoma

Transforming growth factor-β (TGF-β) plays an important role in the regulation of extracellular matrix (ECM) deposition by stimulating the synthesis of individual matrix proteins like tenascin and fibronectin. Cholesteatoma shows significant changes in the ECM, supporting the view of adisturbed cell-matrix interaction. The purpose of our present study was to evaluate the distribution of TGF-β in comparison to the deposition of tenascin, fibronectin, and collagen as major components of the ECM in cholesteatoma (n = 12) by means of histochemistry and immunohistochemistry. We found TGF-P in lymphocytes and fibrohistiocytes in the stroma of 7 cholesteatomas. In corresponding sections, a marked expression of tenascin and fibronectin was seen manifesting as a continuous band along the epidermal-stromal junction, extending to the deeper stroma. In addition, in those cases of TGF-β expression, beginning collagen fibril formation was seen in adjacent deeper stroma layers, indicating beginning stromal fibrosis. These results suggest that TGF-β may be involved in the stimulation of the synthesis of tenascin, fibronectin, and collagen. Furthermore, the enhanced expression of tenascin and fibronectin provides evidence for a deregulated cell-matrix interaction in cholesteatoma associated with the enhanced proliferative process of cholesteatoma formation.

Immunotype findings in macrophages in aural cholesteatomas.

SummarySince a heavy cellular infiltrate is seen in the stroma of most aural cholesteatomas, we attempted to characterize this cell population in more detail using monocyte/macrophage-specific monoclonal antibodies. KiM1+ (specific for CD 11c antigen, the 150kDa α-chain of a leukocyte integrin), and KiM6+ phagocytes were present in two- or fourfold higher numbers in the stroma of the six excised cholesteatomas than in the control tissues. Since the stroma of the cholesteatoma is devoid of microvessles, the typical perivascular localization of dermal macrophages was not seen in the cholesteatomas studied. The density of the macrophages in the normal ear skin was much higher in the upper dermis than in the lower dermis. In the cholesteatomatous specimens, the phagocytes were evenly scattered within the connective tissue and the cellular infiltrate. In contrast to diseased skin, no Mac 387+ macrophages were detected in the cholesteatomas. A great number of phagocytic cells closely resembling dermal macrophages was found in the stroma of the cholesteatomas and probably contributes to an active autoimmune process.

High levels of fibronectin in the stroma of aural cholesteatoma

PURPOSE Because abundant fibronectin deposition is a hallmark of healing cutaneous wounds and provides a matrix for hyperproliferative and migratory epidermal cells, the distribution of fibronectin in aural cholesteatoma was investigated immunohistochemically. MATERIALS AND METHODS A monoclonal antibody against the major cell binding domain of human fibronectin was used to stain 4-micron cryosections of cholesteatoma tissue by the alkaline phospatase-antialkaline phosphatase method. Section of normal retroauricular skin served as control. RESULTS When processed in parallel, fibronectin staining was much stronger in the stroma of cholesteatoma than in normal dermis. The squamous epithelium of both tissues did not show any staining for fibronectin. CONCLUSIONS These observations lend support to the view that the growth of cholesteatoma epithelium reflects an aberrant regenerative process.

Overexpression of Tenascin in Cholesteatoma and External Auditory Meatal Skin Compared to Retroauricular Epidermis

In the present study the distribution of tenascin in cholesteatoma was immunohistochemically investigated. The results were compared with those in external auditory meatal skin and in retroauricular skin of healthy controls. The staining pattern was additionally correlated to the degree of cell proliferation as detected by the monoclonal antibody MIB-1 (Ki-67 antigen). Retroauricular skin showed a limited distribution of tenascin in the papillary dermis and a sparse reactivity of MIB-1 in only a few epithelial cells. External auditory meatal skin revealed a more pronounced reaction for tenascin and MIB-1. In contrast, cholesteatoma tissue exhibited an abundant and continuous expression of tenascin covering the whole stroma compartment. This coincided with a significant increase of MIB-1-positive cells in the basal and suprabasal epithelial layers. Doublestaining experiments revealed most prominent stromal tenascin-expression in areas with marked signs for epithelial proliferation. This suggests that tenascin is selectively increased in response to epidermal hyperproliferation. This matrix protein thus shows a quantitatively and qualitatively enhanced expression under pathological conditions. Moreover, the abundant reactivity for tenascin in the cholesteatoma provides evidence of a deregulated cell-matrix interaction involved in the hyperproliferative process of cholesteatoma formation.