In vitro study on immune response modifiers as novel medical treatment options for cholesteatoma.

OBJECTIVES: To investigate cytokine profile of cholesteatoma and to collect information about important intercellular signaling pathways by establishing two different cell culture models, to block important intercellular signaling pathways in cholesteatoma by applying immune system modifier drugs to develop alternative medical therapy options for cholesteatoma. METHODS: To observe the pathogenesis of cholesteatoma and to apply the immunomodulatory drugs, cholesteatoma tissue culture models were constituted with HEKa cells and cholesteatoma keratinocytes, which were obtained from 3 patients who underwent operations for cholesteatoma. Medicines including 5-fluorourasil, imiquimod, cyclosporine, and tacrolimus were applied on both cholesteatoma keratinocytes and HEKa cells. After 48 h of incubation, IL-1, IL-6, IL-8, IL-10, TNF-α, and Ki67 levels were measured to determine cell viability rates. RESULTS: In the cholesteatoma control group, IL-6 and TNF-α levels were found higher than in the HEKa control group. All repurposed drugs in the study demonstrated anti-inflammatory, anti-proliferative, and cytotoxic effects on cholesteatoma. Imiquimod and tacrolimus in particular are potential treatment prospects for cholesteatoma due to their strong anti-inflammatory and cytotoxic effects. CONCLUSION: Medical therapy options for cholesteatoma are still missing and surgery is not the ultimate solution. We have focused on intercellular inflammatory processes, which play significant roles in the pathogenesis of cholesteatoma in our paper. Inflammation and proliferation of cholesteatoma decreased after all repurposed drug applications in our study. Anti-inflammatory and anti-proliferative effects of tacrolimus and imiquimod was more significant than other drugs in the study. For this reason, tacrolimus and imiquimod should be examined in depth with in vivo studies in terms of efficacy and safety for medical treatment of cholesteatoma.

Cathelicidin antimicrobial peptide LL-37 in cholesteatoma enables keratinocyte reactivity with cytosolic DNA.

The purpose of this study was to determine whether self-DNA can trigger the inflammatory response in cholesteatoma. Specimens were collected from nine patients with invasive cholesteatoma, nine patients with attic-type cholesteatoma (pars flaccida was perforated in five patients and intact in four) and four healthy skins. Expression and localization of LL-37 and interferon-alpha were detected by immunofluorescence and immunoblot analysis. Cultures of human cholesteatomatous keratinocytes were exposed to CpG DNA, LL-37 or CpG DNA complexed to LL-37 for 24 h. Expression of interferon-alpha was detected by RT-PCR. We detected abundant cytosolic DNA, increased LL-37 and interferon-alpha in keratinocytes in invasive cholesteatoma and attic-type cholesteatoma with pars flaccida perforation, but not in attic-type cholesteatoma with pars flaccida intact and normal skin. In cultured keratinocytes, LL-37-DNA complexes induced IFN-α expression. These data suggest that cytosolic DNA is an important disease-associated molecular pattern that triggers the inflammation response in cholesteatoma. Furthermore, LL-37 played an important role in DNA-triggered inflammation. Thus, we have identified a link between cytosolic DNA, LL-37 and autoinflammation in cholesteatoma, providing new potential targets for the treatment of this disease.

Immune cell profile in invasive cholesteatomas: preliminary findings.

BACKGROUND: Cholesteatoma consists of keratinizing squamous epithelium, granulation tissue and keratin plugs. The pathogenesis of cholesteatoma may be related to alterations in the stromal immune cell infiltrate. OBJECTIVE: To examine the immunophenotypic characteristics of the immune cell infiltrate in invasive cholesteatomas. MATERIALS AND METHODS: This study included 12 patients with invasive cholesteatomas causing wide bone erosion of the mastoid, middle ear structures, and the bony plates of middle ear cleft. Diagnosis of invasiveness was based on the clinical, radiological and intraoperative findings. Canal wall-down surgical approach was done in all cases to control the disease process. We used the cholesteatomatous tissue specimens to perform immunohistochemical stains for B cells (CD20), T cells (CD3), histiocytes (CD68) and Langerhans' cells (CD1a). Mouse monoclonal antibodies and immunoperoxidase staining methods were used. The results of immunohistology were scored as mean values of positively stained immune cells. The data were compared with findings in 10 specimens of external ear skin (control group). RESULTS: Immunohistochemistry showed highly significant (p<0.00) counts of immune cells in invasive cholesteatomas (CD3: 4.7+/-0.4, CD68:4.6+/-0.5, CD20: 0.8+/-0.1 and CD1a: 0.8 +/-0.1) compared to those in external canal skin (control group: CD3:0.8+/-0.3, CD68: 1.0+/-0.4, CD20: 0.2+/-0.1 and CD1a: 0.1+/-0.1). In cholesteatomas, the predominant of CD3(+) T lymphocytes and CD68(+) cells (histiocytes). Rare CD20(+) cells and CD1a(+) cells (Langerhans' cells) were also observed. CONCLUSIONS: This preliminary study describes the profile of the immune cell infiltrate in invasive cholesteatomas. The numeric dominance of CD3(+) cells and CD68(+) cells suggests that cell-mediated immunity has important role in the development of cholesteatoma and in its autodestructive properties. Further studies are recommended to categorize the T cell subsets in different stages of cholesteatomas.

Regulation of apoptosis in external auditory canal cholesteatoma by hepatocyte growth factor/scatter factor.

OBJECTIVES: External auditory canal cholesteatomas (EACC) are characterized by focal invasion of squamous cell epithelium and accumulation of keratin debris in the apical part of the matrix. Apoptosis appears to be important in understanding the pathogenesis of EACC. Here the possible regulatory effect of the apoptosis mediated by hepatocyte growth factor (HGF)/scatter factor (SF)-c-Met-Fas in EACC is discussed. METHODS: We examined 17 EACC specimens for immunohistochemical expression of HGF/SF, c-Met, caspase 3 and Fas. The staining reaction was evaluated semiquantitatively. RESULTS: HGF/SF was detected in mesenchymal tissue below the EACC epithelium. c-Met was expressed throughout the epithelium. Fas and caspase 3 were detected at increasing levels towards the apical layers of the EACC matrix. CONCLUSIONS: High levels of HGF/SF result in binding of HGF/SF to c-Met, releasing Fas to aggregate and bind to its death-inducing signaling complex. The result is apoptosis, marked by formation of dead squamous cells and sequestered keratin debris on the apical side of the cholesteatoma.

Cytotoxicity of cytokeratin monoclonal antibody against keratinocytes: a possible therapeutic adjunct for cholesteatoma?

HYPOTHESIS: Monoclonal antibodies directed against cytokeratin subtypes in cholesteatoma produce growth inhibition of keratinocytes. BACKGROUND: Despite elegant surgical procedures for cholesteatoma, residual disease is an important clinical problem. Although gross cholesteatoma removal usually is feasible, microscopic foci of residual keratinocytes may develop into clinically significant disease. This study was designed to evaluate the keratinocyte cytotoxicity of monoclonal antibodies directed against a cytokeratin subtype relatively unique to cholesteatoma. METHODS: Keratinocytes and skin fibroblasts were trypsinized, counted, and seeded in multiwell plates. The cells were exposed to mouse monoclonal antibody to cytokeratin 10 at dilutions of 1:10, 1:25, 1:50, 1:100, and 1:200 with six replicates. After 24-, 48-, and 96-hour incubations, cells that had been pulsed with 3H-thymidine were harvested. Cellular DNA was processed for quantification of 3H-thymidine incorporation with a beta scintillation counter. Cells exposed to antibody are reported as percent inhibition relative to controls. RESULTS: Inhibition ranged from 88.9% for the 1:10 concentration to 26.9% for the 1:200 concentration after 24 hours of incubation. Similar effects were noted at the 48- and 96-hour intervals. Overall, the effect was significantly more pronounced on the keratinocytes than inhibition on skin fibroblasts. CONCLUSIONS: These results suggest that monoclonal antibodies have in vitro activity against keratinocytes. Additional investigation of a possible role for cytokeratin monoclonal antibodies should be pursued with a goal of developing a clinically useful biologic adjunct for cholesteatoma management.

Functional characterization of middle ear mucosa residues in cholesteatoma samples.

Cholesteatoma epithelium is characterized by a keratinocyte dysregulation with an aggressive growth that leads to the destruction of normal middle ear mucosa. The abnormal behavior of cholesteatoma epithelium seems to be induced by the presence of a heavy immune cell infiltrate releasing different cytokines and growth factors in high amounts. Middle ear mucosa rests are often observed within the cholesteatoma stroma or adjacent to the advancing front of cholesteatoma epithelium. This study investigated the presence of interleukin-1 (IL-1), transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and epidermal growth factor-receptor (EGF-R) in the mucosa rests as well as the expression of an activation marker, 4F2. The findings were correlated with the features of a surrounding stroma with an enhanced immune cell infiltrate. Cholesteatoma epithelium showed a high staining intensity of IL-1, TGF-alpha, and EGF-R. In contrast to this, middle ear mucosa did not show any positive reactions for the mentioned factors. Epidermal growth factor immunoreactivity was found in neither cholesteatoma epithelium nor in middle ear mucosa residues. The authors found a high concentration of lymphocytes and macrophages in the surrounding stroma. Most of these cells expressed TGF-alpha, IL-1, and 4F2, suggesting an activated form. Results indicate that keratinocytes present in the middle ear mucosa do not appear to react to the stimuli released by the inflamed stroma, reflecting important differences in the cell biological features of the keratinocytes that form parts of both types of epithelium.

[TNF alpha in serum of patients with cholesteatoma]. / TNF alfa w surowicy chorych z przewleklym zapaleniem ucha srodkowego z perlakiem.

TNF alfa levels in the sera of patients with cholesteatoma were investigated. Immunoradiometric method was used. TNF alfa levels in the sera of patients with cholesteatoma were higher than in the sera of a control group. The differences were statistically significant.

[New immunobiologic trends concerning etiopathogenicity of cholesteatoma]. / Nuevos aspectos inmunobiológicos sobre la etiopatogenia del colesteatoma.

After an epoch in which was pretended to explain the etiopathogenetic phenomena observed in Cholesteatoma through enzymatic studies, nowadays other investigations focus the topic in the possible presence of immunobiologic alterations at cellular level, so the research work is directed to the occurrence, distribution and activity of several growth factors and leukins. In this paper the AA. made a perusal of the new acquisitions and devote themselves to two important aspects of the cholesteatoma: the biologic behaviour of the squamous cell epithelia with an uncontrolled growth and to the immunobiologic mechanisms responsible for the bone resorption.

Possible autocrine growth stimulation of cholesteatoma epithelium by transforming growth factor alpha.

INTRODUCTION: Transforming growth factor alpha (TGF-alpha) is known to be produced by normal human keratinocytes and to stimulate their proliferation. The squamous epithelium of middle ear cholesteatoma is believed to exhibit hyperproliferative characteristics. This study was undertaken to determine if growth factors can be identified in cholesteatoma. MATERIALS AND METHODS: Cholesteatoma samples (n = 6) and retroauricular skin (n = 9) were obtained during surgery. Monoclonal antibody against epidermal growth factor (EGF) and TGF-alpha were evaluated in these specimens using immunohistochemical techniques. RESULTS: Epidermal growth factor receptor (EGF-R) was highly expressed in the basal layer of the epidermis, hair follicles, eccrine sweat glands, and the capillary system of normal skin. In the majority of cholesteatoma samples, expression of EGF-R was not confined to the basal layer but persisted in suprabasal cells of the stratum spinosum and stratum granulosum. In two cases, heterogenous standing was found in different parts of the same cryosection. Staining for TGF-alpha was consistently stronger in cholesteatoma than in normal skin, and encompassed all epithelial cell layers. Immune cells infiltrating the stroma of cholesteatoma stained positively for TGF-alpha. CONCLUSION: These data are consistent with autocrine stimulation of the squamous epithelium of cholesteatoma by TGF-alpha contributing to its unrestrained growth in the middle ear cavity.

Localization of proliferating cell nuclear antigen in aural cholesteatoma.

Middle ear cholesteatoma is not a genuine tumor but has a remarkable proliferative activity which causes serious destruction of the mastoid bone. In the present study, we used immunohistochemistry with antiproliferating cell nuclear antigen (PCNA) antibody on cholesteatomatous tissues to evaluate the localization of PCNA, as it has been said that PCNA is a very available protein for showing cell proliferative activity. Moderately concentrated PCNA was demonstrated within the germinal basal layer cells of the cholesteatomatous epithelium in three of eight surgical specimens. Furthermore, in one case of very active osteolytic cholesteatoma, PCNA activity was demonstrated in the mesenchymal cells, probably fibroblast-like cells, in direct contact to the destroying mastoid bone lesions. Although the etiology and histopathology of the invasive and proliferative activity of middle ear cholesteatomatous tissues are unclear, this observation suggests that immunohistopathological examination using PCNA antibody might be a useful tool for evaluating bone resorption activity and for establishing the prognosis of various types of cholesteatoma.

Interleukin-1 of cholesteatomatous keratinocytes.

Interleukin-1 (IL-1) has been thought to be one of the essential cytokines mainly produced by macrophages. It has recently been reported that epidermal keratinocytes produce IL-1, and attention is being paid to local immune reactions mediated with this cytokine. Interleukin-1 not only activates lymphocytes, but also acts as an osteoclast-activating factor. In this study, we used immunohistochemistry and immunoblotting on cholesteatomatous epithelium with anti-IL-1 alpha antibody and anti-IL-1 beta antibody. Next, the relationship of cholesteatomatous debris to the production of IL-1 by keratinocytes was evaluated. Highly concentrated IL-1 alpha was found in the cholesteatomatous epithelium, especially in the basal cell layer. The intensity of IL-1 beta staining was weaker than that of IL-1 alpha staining. In the immunoblotting study, the 31 kd band, an intracellular immature precursor molecule, was identified. The production of IL-1 alpha from keratinocytes was augmented to a greater degree by cholesteatomatous debris than by lipopolysaccharide or keratin. The keratinocytes did not produce IL-1 beta. These findings suggest that IL-1 alpha is derived from cholesteatomatous keratinocytes. Interleukin-1, mainly IL-1 alpha, from the stimulated cholesteatomatous keratinocytes may be an important factor in the markedly increased bone resorption observed in cholesteatoma.

[Immunohistochemical identification of cholesteatoma-associated macrophage populations]. / Immunhistochemische Identifizierung Cholesteatom-assoziierter Makrophagen-Populationen.

Extensive bone resorption occurring in aural cholesteatoma is responsible for the severe complications of this disease. In the area of active bone destruction, typical multinucleated osteoclasts are rarely seen, but a heavy cellular infiltrate is found. In the present study we tried to characterize the immunophenotype and the functional state of the cells infiltrating the stroma and the epithelial layer of aural cholesteatoma, using a panel of monoclonal antibodies directed against cell type specific antigens. The results were compared with normal retroauricular skin. The vast majority of cells infiltrating the stroma was bone marrow derived and consisted of T-cells and macrophages. By means of the activation markers HLA-DR and Interleukin-2 receptor an immunologically activated state of the majority of infiltrating cells in cholesteatomas was shown. The great number of activated macrophages in cholesteatomas seems to be very important in the cholesteatomatous immunological process. Because of their various immunological functions (antigen presentation to T-lymphocytes, participation in ingestion and killing of different invading microorganisms and synthesizing a great number of substances involved in host defence and inflammation) these cells play a central role in human immunological system. Langerhans cells, however, did not appear to be involved in the immune process of cholesteatoma. The characteristics of the infiltrating cell population with the great number of phagocytic cells suggest an active immune process resulting in autoaggressive bone resorption.

Immunotype findings in macrophages in aural cholesteatomas.

Since 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 CD11c antigen, the 150 kDa alpha-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 microvessels, 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.

[Human monocytes show chemotaxis in response to cholesteatoma debris].

Using a microchamber technique, we tested cholesteatoma debris and certain of its constituents for effects on the migration of human peripheral blood monocytes and polymorphonuclear leukocytes. Cholesteatoma debris induced significant migration of monocytes. When the individual constituents of cholesteatoma debris, i.e., alpha-keratin, cholesterol, lauric acid and lipopolysaccharides, were tested for monocyte chemotaxis, only alpha-keratin induced significant monocyte migration. alpha-keratin extracted from the cholesteatoma debris with 8 M urea also induced migration of monocytes with a bell-shaped dose-response curve, which is frequently encountered with chemoattractants. Therefore, cholesteatoma debris and one of its components, alpha-keratin, are potent chemoattractants for human monocytes. On the other hand, cholesteatoma debris showed no significant chemotactic effect on polymorphonuclear leukocytes. Based on the present and our previous results, cholesteatoma debris acts on monocytes/macrophages as a strong chemotactant, a potent activating (priming) factor, and an inducer of production of tumor necrosis factor, which is a bone-resorbing cytokine. Therefore, we concluded that macrophages induced by cholesteatoma debris may play an important role in the pathogenesis of bone resorption in cholesteatoma otitis.

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.

[Chronic cholesteatomatous otitis media: the histopathological and clinical aspects]. / Otite media cronica colesteatomatosa: aspeti istopatologici e clinici.

In recent years the immunologic aspects of the normal and pathological ear have been studied by several authors, with particular attention given to the histopathologic aspects of the epidermis of the tympanic membranes of the outer ear canal and of the middle ear mucosa in normal physiologic as well as in inflammatory conditions. Such studies may help in giving a more precise definition to the pathogenesis and clinical behavior of middle ear cholesteatoma. In this paper we report the results of an immunohistopathologic study carried out using the immunohistochemical technique of monoclonal antibodies on cholesteatoma matrix samples taken during radical mastoidectomy or tympanoplasty. In particular, the presence of T-lymphocytes and Langerhans cells was evaluated using selective monoclonal antibodies and a relationship between the data collected and the clinical expression of the disease in each case was sought. In this study it was not possible to establish a close relationship between clinical behavior and immunohistopathological findings, which appeared rather similar in all the cases. The presence of Langerhans' cells may confirm the hypothesized role they play in phlogistic reactions and bone reabsorption due to the presence of the cholesteatoma in the middle ear. Yet, in order to evaluate their true role correctly, more detailed studies should be carried out on the spatial distribution of T-lymphocytes and Langerhans' cells in the cholesteatoma matrix as well as on their ultrastructural characteristics.

Immunohistologic analysis of the cholesteatoma matrix in children.

The immunohistological characteristics of retraction pockets, cholesteatoma matrix and granulomatous tissue were compared in 14 samples from pediatric cholesteatoma. The junction between epidermis and the middle ear mucosa appeared as the most inflammatory area, displaying the characteristics of delayed type hypersensitivity. CD1 + Langerhans cells were observed in all epidermic areas, but expressed class II molecules only in the vicinity of polymorphonuclear infiltrates. Numerous mast cells and IgA producing cells were also observed, suggesting that defenses from the mucosal immune system are summoned and contribute to the pathogenesis of cholesteatoma.

The nature of the epithelium in acquired cholesteatoma.

Monoclonal antibodies with defined specifications for individual cytokeratins were used to stain the epithelia of the external auditory meatus, the middle ear and cholesteatoma. The observed staining indicated that the epithelium of the external auditory meatus has a pattern of keratin expression typical of epidermis in general and the epithelium of the middle ear resembles simple columnar epithelia. The pattern of staining of cholesteatoma closely resembled that of the skin of the external auditory meatus.

[New concepts of the course of cholesteatoma from the immunohistological study of 96 samples]. / Nouvelles conceptions de l'évolution du cholestéatome à partir de l'étude immuno-histologique de 96 prélèvements.

The immunohistological characteristics of retraction pockets, cholesteatoma matrix and granulomatous tissue have been compared. Langerhans cells, epidemic folds and sub-epithelial tissue were labelled with specific markers (HLA II, adhesion receptors, KI 67). The junction between epidermis and sub-epithelial tissue of middle ear mucosa appeared as the most inflammatory area, displaying characteristics of delayed type hypersensitivity phenomenon. CD1+ Langerhans cells appear to express class II molecules only in the vicinity of polymorphonuclear infiltrates. Epidemic proliferation seems to able place mainly in the deepest recesses of the epidermis.

Cholesteatoma debris as an activator of human monocytes. Potentiation of the production of tumor necrosis factor.

Tumor necrosis factor (TNF) is a cytokine which stimulates osteoclastic bone resorption and inhibits collagen synthesis in vitro. In this study the effect of human cholesteatoma debris and its constituents on the production of TNF-alpha by human monocytes in vitro was studied. Cultured human peripheral monocytes secreted TNF into the culture medium when exposed to cholesteatoma debris in a dose-dependent manner. The TNF production, however, was partially inhibited by the treatment of the debris with polymyxin B which inhibits biological activities of lipopolysaccharide (LPS). When individual constituents of cholesteatoma debris, i.e. keratin, cholesterol, lauric acid and LPS, were added to the cultured monocytes at concentrations equivalent to those in the debris, significant production of TNF was observed only with the keratin and LPS. These data suggest that cholesteatoma debris is a potent activator of the TNF production of human monocytes in vitro, and that LPS and keratin are responsible for the production.