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.

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.

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.

Immunologic aspects of otologic disease: an overview.

The immunologic explosion has now reached the field of otology. By having better techniques to measure the changes at cellular and molecular levels, it is now possible to devise experiments to show morphologic anatomic changes as well as functional changes. The demonstration in 1980 (M.S.) that tympanosclerosis could be induced immunologically represents a concrete advancement in immunologic thinking in conceptualization of otologic disease. In 1974, one of the authors (M.S.) published work dealing with the treatment of vasculitis of immunologic origin for sudden hearing loss. This was aimed at inhibiting the complement cascade from starting its destructive action. Recently, the immunologic challenge in animals demonstrated by changes in the inner ear was shown by one of the authors (T.J.Y.). Such changes were compatible with labyrinthine hydrops, or Meniere's disease, otosclerosis, and sensorineural hearing loss and vestibular dysfunction.

Immunobiology of Langerhans' cells migrating into aural cholesteatomas.

Immunohistochemical and submicroscopic analyses of human cholesteatoma matrices reveal the presence of Langerhans' cells and T lymphocytes. Through cell-to-cell interaction, Langerhans' cells probably play a key role in skin-related disorders, including cholesteatomas. They originate from a mobile cell population of monocyte origin and migrate into and out of the body's lining. Their custodial function is always carried out in close relation with T lymphocytes. Various monoclonal antibodies directed against Langerhans' cell and T lymphocyte membrane receptors reveal the presence of these cell populations in cholesteatoma matrices but not in the tympanic membrane. Langerhans' cell and T cell traffic through cholesteatomas are discussed in relation to the pathogenesis, natural course, and recurrence rate of cholesteatomas. Through immunopathologic evaluation the clinical aggressiveness of a cholesteatoma will probably become predictable. Medical manipulation of Langerhans' cell and T cell functions- as an adjuvant to surgery - may have consequences for the future handling of cholesteatomas.

Human leukocyte antigen-A, -B, -C tissue types in patients with middle ear cholesteatoma.

Two series of consecutive, nonrelated patients with middle ear cholesteatoma verified by operation were tissue typed for human leukocyte antigens (HLAs) in order to demonstrate any associations. The first series (102 patients) showed only a possible increase of HLA-B8 and HLA-A2. Because HLA-B8 is one of the antigens most often involved in associations with organ-specific autoimmune diseases, and because of the close relationship between HLA-B8 and HLA-DR3, the second series (48 patients) was tissue typed for these two antigens. This second series showed a similar increase of HLA-B8/DR3, but the difference was not statistically significant.

Epidermal Langerhans cells in cholesteatoma.

The function of the epithelial component of a cholesteatoma has been the subject of intense research. The epithelium does not appear to have proteolytic enzyme activity, yet when it is present, increased amounts of bone resorption occur. Recent evidence suggests that epidermal Langerhans cells within epithelium have an immunologic alerting function. These cells were identified in normal tympanic membranes, canal skin, and cholesteatoma. The Langerhans cells in cholesteatoma displayed morphologic features similar to a contact hypersensitivity state. Langerhans cells within the epithelial matrix of the cholesteatoma may be responsible for generating and maintaining the chronic inflammatory reaction which induces bone resorption in this disease.

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.

Analysis of class II antigen expressing cells in cholesteatoma epithelium.

The present morphological study was designed to evaluate the significance of Langerhans cells in the pathogenesis of cholesteatoma. Biopsies of middle ear cholesteatomas were examined for Langerhans' cells expressing HLA-DR and anti-Leu 6 antigens by immunohistochemistry using monoclonal antibodies. No apparent difference in number of cells was observed when epithelium of cholesteatoma was compared with epithelium of healthy ear canals. Expression of HLA-DR antigens was detected on keratinocytes in Aspergillus flavus infected epidermis, used as a control tissue. This finding indicated an increased immunosurveillance of this tissue. However, no such expression of HLA-DR antigens was detected on epidermal cells of cholesteatomas. The results of the present study did not support the hypothesis of Langerhans' cells as having a primary role in the development of cholesteatoma.

Activation of peritoneal macrophages with human cholesteatoma debris and alpha-keratin.

The effect of human cholesteatoma debris on mouse peritoneal macrophages was studied in vivo. The number of macrophages and lymphocytes increased 5 days after injection of the debris into the peritoneal cavity. A similar increase in peritoneal cells was observed when an urea-extracted fraction of the cholesteatoma debris or alpha-keratin, a major component of the debris, was injected. Both cholesteatoma debris- and alpha-keratin-elicited macrophages exhibited a greater response of luminol-dependent chemiluminescence upon exposure to zymosan, suggesting that the elicited macrophages were activated. In contrast, other constituents of the debris, such as cholesterol or fatty acid--with the exception of lipopolysaccharide (LPS)--failed to elicit or activate peritoneal macrophages at the similar doses detected in the debris. The chemiluminescent response of macrophages obtained by injecting LPS was, however, much lower than that of alpha-keratin-induced macrophages. These results indicate that cholesteatoma debris is capable of eliciting and activating macrophages, and that alpha-keratin is responsible for the activation.

Immunohistochemical identification of inflammatory cells in secretory and chronic otitis media and cholesteatoma using monoclonal antibodies.

Monoclonal antibodies (OKT 3,4,6,8 and OKIa) were used in conjunction with the avidin-biotin-peroxidase complex method to classify inflammatory cells in 6 biopsies of the middle ear mucosa in patients with secretory otitis media (SOM) and in 19 middle ear or mastoidal biopsies in patients with chronic otitis media (COM). Mononuclear cell infiltrates under the mucosal epithelium were found to consist mainly of T4 positive (helper-inducer) T-lymphocytes (50-60%). T8 positive (suppressor-cytotoxic) T-lymphocytes accounted for 20-30% of the cells. T4 positive cells were confined to the round cell infiltrates, whereas T8 positive cells were also located under the mucosal epithelium and seemed to penetrate it. There were no differences between SOM or COM with regard to the distribution or localization of T4 and T8 cells. T6 and Ia positive Langerhans cells were found in the ingrowing tympanic membrane squamous epithelium and in the cavity skin. The number and distribution of these T6 positive cells were similar to those observed in the skin. In the thick cholesteatoma epithelium these cells were somewhat unevenly distributed but were more numerous than in other sites studied. The results are indicative of a normal cell-mediated mucosal response to infection.

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.

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.

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.

[Immunology of cholesteatoma]. / L'immunologie du cholestéatome.

The presence of Langerhans cells and T cells in human cholesteatoma is demonstrated by immunohistochemical and submicroscopic analyses. Various monoclonal antibodies directed against Langerhans cells and T lymphocytes reveal the presence of these cell populations in the cholesteatoma matrices but not in the normal tympanic membrane. Their quantitative and spatial distribution in and around the cholesteatoma matrix might be of importance in the pathophysiology of human cholesteatoma.

[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.

[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.

[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.

[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.

[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.