Inflammatory mediators and immunocompetent cells in the middle ear with particular regards to otitis media and tympanosclerosis

Tympanosclerosis is a well known sequela after acute and chronic otitis media. The pathologically calcified plaques are most commonly found in the tympanic membrane and if located only at this site, it is termed myringosclerosis. Other localizations where it can be found are the epitympanon, the promontory and sometimes the middle ear ossicles are involved leading to hearing impairment. The only treatment for these patients is surgery but recurrencies are common and there is also a risk for iatrogenic sensorineural hearing loss associated with this kind of surgery. Hearing aids, thus, are then beneficial. It may be of great interest if predictable factors could be found to prevent pathological calcification. Earlier, tympanosclerosis has been investigated although usually not with immunohistochemical methods which gives a new possibility of a more exact identification of the cell types and the mediators involved in the early stages of the process.

A rat model was used in order to illuminate the inflammatory response after inoculation of a solution of Streptococcus pneumoniae into the middle ear. The rats developed acute otitis media and the tympanic membranes presented with sclerotic plaques in 30% of the inoculated animals. Different methods for fixation, decalcification and embedding were tested in order to find the best method for immunohistochemical staining of this fragile tissue and to identify macrophages, T-cells, B-cells, inducible nitric oxide synthase and interleukin-6 at different timepoints after inoculation of bacteria. A new immunohistochemical staining technique was developed, the free-floating technique. mRNA in situ hybridization was used to show the timepoint for expression of interleukin-6 mRNA. Results indicated that macrophages and T-cells were found mostly in the acute phase of the inflammatory process, three days postinoculation in the rat middle ear. B-cells were seen to a low extent but found to be most frequent at day six postinoculation. Inducible nitric oxide synthase was seen at the same time and in the same pattern as the macrophages, probably due to the fact that activated macrophages present inducible nitric oxide synthase. Interleukin-6 was shown with immunohistochemical technique from one hour postinoculation persisting throughout the study, lasting until day five postinoculation. Expression of interleukin-6 mRNA was seen at the earliest timepoint of sacrifice, one day postinoculation but not thereafter.

Biopsies from two groups of patients, children with secretory otitis media, a disease known to result in myringosclerosis and tympanosclerosis in a high percentage, and patients with already established tympanosclerosis associated with chronic otitis media, were analyzed with immunohistochemical technique. Antibodies against the same immunocompetent cells and parameters as in the rat studies were used. Macrophages were found in both patient groups. B-cells were most frequently seen in the biopsies from children with secretory otitis media as compared to the biopsies from patient with already established tympanosclerosis. There was no indication of T-cells. All biopsies from the tympanosclerotic middle ears but only one from the group of secretory otitis media patients showed labeling of inducible nitric oxide synthase. Interleukin-6 was seen most frequently in the secretory otitis media biopsies but not as often in the tympanosclerosis material. The above mentioned immunocompetent cells and the two parameters, interleukin-6 and inducible nitric oxide synthase, could be involved in an inflammatory sequence leading to the development of tympanosclerosis. Morphologically, tympanosclerosis has the appearance of bone and it is tempting to speculate that the pathological calcification can be seen as being ectopic production of bone. There are studies supporting involvement of macrophages differentiating into bone remodeling cells upon cytokine stimulation, among one is interleukin-6. Inducible nitric oxide synthases may also be involved, since it is known to be expressed in activated macrophages as well as in bone producing cells. Predictive factors for tympanosclerosis development could be, among others, macrophages, T-cells, B-cells, interleukin-6 and inducible nitric oxide synthase.

List of scientific papers

I. Forséni M, Eriksson A, Bagger-Sjöbäck D, Nilsson J, Hultcrantz M (1997). Development of tympanosclerosis: can predicting factors be identified? Am J Otol. 18(3):298-303.
https://pubmed.ncbi.nlm.nih.gov/9149821

II. Forséni M, Hansson GK, Bagger-Sjöbäck D, Hultcrantz M (1999). Infiltration of immunocompetent cells in the middle ear during acute otitis media: a temporal study. Am J Otol. 20(2):152-157.
https://pubmed.ncbi.nlm.nih.gov/10100514

III. Forséni M, Hansson GK, Bagger-Sjöbäck D, Hultcrantz M (1999). An immunohistochemical study of inducible nitric oxide synthase in the rat middle ear, with reference to tympanosclerosis. Acta Otolaryngol. 119(5):577-582.
https://pubmed.ncbi.nlm.nih.gov/10478599

IV. Forséni M, Melhus Å, Ryan AF, Bagger-Sjöbäck D, Hultcrantz M (2000). Detection and localization of interleukin-6 in the rat middle ear during experimental acute otitis media, using mRNA in situ hybridization and immunohistochemistry. [Submitted]

V. Forséni M, Bagger-Sjöbäck D, Hultcrantz M (2000). A study of inflammatory mediators in human tympanosclerotic middle ear. [Submitted]