A study of inflammatory mediators in the human tympanosclerotic middle ear.

OBJECTIVE: To analyze immunocompetent cells as well as 2 factors involved in inflammation and also thought to be involved in bone remodeling-interleukin 6 (IL-6) and inducible nitric oxide synthase in the human middle ear, including the tympanic membrane. DESIGN: Biopsy specimens were obtained from the human middle ear and tympanic membrane during surgery. Using an immunohistochemical technique, the expression of macrophages, T cells, B cells, IL-6, and inducible nitric oxide synthase were analyzed. MATERIALS: Nine biopsy specimens from tympanic membranes in children having a transtympanic ventilation tube inserted as a treatment for secretory otitis media and 11 biopsy specimens from tympanosclerotic plaques from patients with chronic otitis media and tympanosclerosis. RESULTS: More positively stained specimens showing macrophages, B cells, and IL-6 were seen in the biopsy specimens from children with secretory otitis media compared with the biopsy specimens from patients with chronic otitis media and tympanosclerosis. The biopsy specimens from patients with chronic otitis media and tympanosclerosis more often showed positive stainings for inducible nitric oxide synthase than the biopsy specimens from children with secretory otitis media. The presence of IL-6 and inducible nitric oxide synthase was shown by staining to be mostly in the surface cells, while macrophages and B cells were stained deeper in the tissues, in connective tissue, or around sclerotic lesions. CONCLUSIONS: The 2 patient groups differed in antigen presentation so that macrophages, B cells, and IL-6 were labeled more frequently in patients with secretory otitis media, that is, an early phase of the disease. Inducible nitric oxide synthase was seen more frequently in the patients with already established tympanosclerosis in a later phase of the disease.

Detection and localization of interleukin-6 in the rat middle ear during experimental acute otitis media, using mRNA in situ hybridization and immunohistochemistry.

OBJECTIVE: Otitis media is one of the most common diseases among children. A well-known sequela of acute, chronic, and secretory otitis media is tympanosclerosis. With the exception of surgery, there is no causal treatment available for this condition, which may cause hearing disabilities. This study aimed to describe the localization of interleukin (IL)-6 mRNA and its gene product in the rat middle ear during pneumococcal otitis media. IL-6 is known to be involved in inflammatory and bone remodeling processes. METHODS: Using an experimental model of pneumococcal acute otitis media, the expression of interleukin IL-6, was analyzed. Sprague-Dawley rats were sacrificed at different time points varying from 1 h to 6 days intervals after inoculation. The middle ears were analyzed by messenger RNA in situ hybridization, and by immunohistochemistry with cell-type specific antibodies directed against IL-6. RESULTS: Transcripts of IL-6 were observed only on day 1 post-inoculation, whereas the final gene product was observed at all intervals after inoculation. IL-6 was localized in the bony part of the bulla nearest to the mucosa, around mucosal vessels, and in the ciliae of the mucosal epithelium. The results demonstrated that IL-6 was synthesized locally as early as 1 h after bacterial middle ear challenge, and that although transcription could not be detected after 24 h, the cytokine product persisted for at least 5 days after the infection was introduced. CONCLUSIONS: IL-6 was shown to be produced early in the inflammatory process during induced pneumococcal otitis media in the rat. No production was seen after 24 h although the protein remained in the tissue for at least 5 days. IL-6 could initiate a differentiation of macrophages to osteoclasts and thereby participate in a bone remodeling process leading to tympanosclerosis development.

An immunohistochemical study of inducible nitric oxide synthase in the rat middle ear, with reference to tympanosclerosis.

Tympanosclerosis and myringosclerosis are well-known sequelae after acute and chronic otitis media and are also often seen after treatment of secretory otitis media with ventilation tubes. They sometimes cause serious hearing disability. There is no successful treatment for these conditions. There might be factors triggering an immunological or autoimmune chain reaction, which leads to tympanosclerosis. Intervention with the aim of abolishing this type of response might be possible if an interruption of the chain reaction can be found. Nitric oxide is a radical molecule with the ability to kill pathogens and is produced by the enzyme nitric oxide synthase. Expression of inducible nitric oxide synthase (iNOS) was analysed immunohistochemically in a rat model of acute otitis media. In rats sacrificed at days 3 and 6 after inoculation. iNOS was also strongly expressed in the middle ear mucosa and in the tympanic membrane as well as in the inner ear. In control specimens as well as in infected ones. iNOS was expressed in the tissue of the external ear canal. In rats sacrificed at day 10 and after 3 months, iNOS was expressed at the same locations, although less frequently. These data indicate that iNOS expression is induced during acute otitis media and suggest that nitric oxide may be important in the host defence against ear infections.

Infiltration of immunocompetent cells in the middle ear during acute otitis media: a temporal study.

HYPOTHESIS: The inflammatory response to acute otitis media (AOM) is a chain reaction involving, among others, macrophages, B lymphocytes, and T lymphocytes that vary in number on different days during the infection. The response is thought to eventually contribute to tympanosclerosis (TS). BACKGROUND: In humans, TS and myringosclerosis (MS) are obscure sequelae of chronic otitis media. MS is also commonly seen in children who have had acute purulent otitis media or secretory otitis media or after treatment with ventilation tubes in the tympanic membrane (TM). It causes hearing disability, especially if the ossicles or the inner ear are affected. No successful treatment is available. This study was performed to evaluate the inflammatory stages that may lead to TS or MS. METHODS: Sprague Dawley rats were exposed to a Pneumococcus type 3 solution into the middle ear. Groups of rats were killed at 3, 6, and 10 days after inoculation. Sections from the TM specimen were stained immunohistochemically according to the avidin-biotin method. Antibodies used were directed against macrophages, T cells, and B cells. Positive cells were counted and a mean value was estimated for each slide and section for each antibody in each rat. RESULTS: Results showed that macrophages, T cells, and B cells were presented time-dependently in the acute inflammatory response in AOM. At day 3, dendritic cells, macrophages, T cells, B cells, and other major histocompatibility complex (MHC)-restricted cells were richly expressed in the whole submucosal layer and especially in the annulus fibrosus. At day 6, the amount of all positive cells decreased except for B cells and other MHC-restricted cells, which slightly increased in number. At day 10, all of the cells were lower in number than at days 3 and 6. Macrophages and possible T cells could be detected in the TM, which has not been observed earlier. Large osteoclastlike cells were present close to the bone. CONCLUSIONS: Macrophages were the first cells to invade the tissue after AOM induction. Some cells were found in the TM. Large osteoclastlike cells could be seen adjacent to the bone in the submucosa. T cells and B cells were seen in the submucosa.

Development of tympanosclerosis: can predicting factors be identified?

HYPOTHESIS: The etiological hypothesis is that there might be factors triggering an immunological chain reaction that eventually leads to tympanosclerosis formation. BACKGROUND: Tympanosclerosis is a condition leading to a calcification process in the middle ear and, occasionally, also to the lining of the inner ear. This sometimes leads to hearing loss due to fixation of the middle ear ossicles. In severe cases. deafness may occur as a result of the inner ear impairment. Surgery is the treatment offered, often with poor long-term results, and, alternatively, prescription of hearing aids. Some patients develop tympanosclerosis after mild inflammatory otitis media processes whereas some heal without tympanosclerosis after more aggressive infections. This difference may be due to individual variations in the inflammatory response. The biological mechanism of calcification in tympanosclerosis is probably similar to that occurring in other calcifying tissues due to diseases. METHODS: The present investigation was performed to develop methods for immunohistochemical analyses of this delicate tissue consisting of both hard bone and the very thin tympanic membrane. Sprague-Dawley rats were inoculated with a suspension of Streptococcus pneumoniae, type 3, into the middle ear and sacrificed after 1 week up to 6 months. A new technique was elaborated where the whole specimen was prefixed briefly and then en bloc incubated with the primary antibodies and after that decalcified in edetic acid (EDTA). Primary antibodies against macrophages were used for the immunohistochemical staining. RESULTS: Acute otitis media was successfully induced in the rats and myringosclerosis was seen in 30% of the animals, often localized close to the bony frame where macrophages could also be detected. CONCLUSIONS: Acute otitis media and myringosclerosis were introduced in the animals. Conventional immunological techniques were tested on this delicate tissue. A new method for immunohistochemical staining was elaborated in which specimens were stained en bloc before decalcification and sectioning were performed. Expression of macrophages was demonstrated in the tympanic membrane.