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This study was designed to understand the contribution of the inflammasome and IL-1β activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1β and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc−/−-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1β maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc−/−mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.

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Serum levels of specific IgG and the sensitization of peripheral blood T-lymphocytes were measured in guinea pigs after single-dose antigenic sensitization by two routes-, intratympanic and intradermal injection. Keyhole limpet hemocyanin (KLH) served as the antigen. Intratympanic injection of antigen resulted in much lower levels of circulating anti-KLH IgC than intradermal injection. When KLH was conjugated with alum to produce nonspecific inflammation and serve as an adjuvant, the intratympanic route was considerably enhanced, but remained much less effective than the intradermal route. Development of an IgG response was also somewhat less rapid following intratympanic than following intradermal administration. Marked sensitization of circulating T-lymphocytes was seen after intradermal injection of alum-precipitated KLH. A much weaker, though still positive, response was seen after intradermal injection of KLH alone and with the intratympanic injection of alum-precipitated KLH. No T-lymphocyte sensitization could be detected after intratympanic injection of KLH alone. It was concluded that the afferent limb of both humoral (IgG) and cell-mediated immunity was operative in the middle ear. Therefore, the middle ear does not represent an immunologically “privileged” site. On the other hand, the afferent limb from the middle ear appears to operate less effectively and rapidly than that from the dermis. This observation is consistent with observations in other mucosal systems.

Guinea pig cytomegalovirus (GPCMV) has been used to establish a reproducible model of viral labyrinthitis and hearing loss. Cochlear function was assessed by electrophysiological recordings of cochlear microphonic (CM) and eighth nerve N1 compound action potential (AP) thresholds prior to and up to eight days following inoculation of the scala tympani. Inner ear inoculation of seronegative subjects with live GPCMV produced profound elevations in CM and AP thresholds: 70% of these subjects had their thresholds raised to the limits of the sound system throughout the tested frequency range of 0.10 to 32 kHz. Histopathologic effects associated with CM and AP threshold shifts were primarily limited to the perilabyrinthine compartment, and were greatest in the most basal cochlear turns. Systemic infection with GPCMV produced an immune response, but did not affect CM or AP thresholds. Subsequent inoculation of the inner ear of these seropositive animals with live GPCMV did not result in either CM or AP threshold shifts, or cochlear histopathology. Inoculations of inactivated virus into the inner ears of seronegative and seropositive animals produced only moderate CM and AP threshold effects. Primary GPCMV labyrinthitis thus results in significant cochlear dysfunction and histopathologic changes which are prevented by prior systemic infection with GPCMV.

Tympanosclerosis is a disease which affects the tympanic membrane (TM) and middle ear. The locus for the essential pathology is in the connective tissue component of the drum which is the lamina propria. In the middle ear, the pathology is in the basement membrane. In the early stages there is a minimal involvement of the mesenchymal component, usually seen as a small white macula, or scar, in the drum. In advanced disease, the hyalinization of the mesenchymal component together with the attraction of the calcium ion leads to a thick, dense, calcified scar. The theory of pathogenesis for this disease entity is that the connective tissue component of these structures is stimulated by infection, inflammation, or trauma involving some degree of local immunological hypersensitivity. With severe middle ear infection, the mucosa of the inner surface of the drum becomes permeable, and the ground substance of the lamina propria becomes edematous taking up water together with components of complement from the middle ear disease. The abnormal middle ear secretions contain the immunoglobulin components capable of participating in the process. If there is any damage to the connective tissue, the adsorption and repair permits the body to react immunologically against the destroyed tissue, thereby sensitizing this area. Experiments were performed with the guinea pig in which the tympanic membrane was removed and the lamina propria isolated and prepared as an immunological antigen. When this material was injected into the rabbits, a high, satisfactory antiguinea pig tympanic membrane antibody was formed (anti-GPTM). It was then possible to remove this anti-GPTM antibody from the serum. The tissue specificity was determined by immunofluorescent staining which showed it to be adsorbed onto the TM, middle ear mucosa, as well as the basement membrane of the respiratory tract. The guinea pigs which were passively sensitized with anti-GPTM antiserum demonstrated the binding of the antibodies on the tympanic membrane when subject to trauma, infection, or cautery. The manifestation was in the anatomical area related to the injury. Complement fixation was, likewise, demonstrated. Controlled animals which were not traumatized, or were not passively sensitized, included those which were injected with IgG from rabbits not sensitized against GPTM. They showed no fixation of antibody or complement in the TM. Following injury the antibody and complement bound to the TM provide the necessary elements for the subsequent immunopathologic disease entity known at tympanosclerosis.

Type II collagen autoimmunity has been reported to result in a number of middle and inner ear morphological and functional abnormalities. We investigated the immunological, electrophysiological, and anatomical effects of this autoimmune state in well-established Wistar-Furth rat models of type II collagen autoimmune arthritis. Seventeen animals immunized with bovine type II collagen were divided into short term (1–3 months postimmunization) and long term (9–11 months) groups. Thirty-three experimental ears were tested electrophysiologically and were compared to nine ears of unimmunized Wistar-Furth rats. No hearing loss was found in the immunized animals, except for four animals that showed middle ear abscess formation consistent with spontaneous, purulent otitis media. All immunized animals showed very significant serum and perilymph antibody titers to type II collagen. No morphological abnormalities of the external, middle, or inner ears could be identified in the noninfected experimental animals. These findings do not support previously reported observations that type II collagen autoimmunity results in ear disease.

It has been suggested that immune responses and resultant generation or release of inflammatory mediators play a role in otitis media with effusion (OME). To explore the potential of one such mediator to contribute to OME, the effects of histamine injected into the middle ear cavity were examined. Injection of histamine into the guinea pig middle ear was found to produce dilation and endothelial disjunction in capillaries, leading to striking mucosal edema and effusion which persisted for at least several hours. This response could be partially blocked by pretreatment with either H1 or H2 antihistamines. It is suggested that histamine released during immune responses in the middle ear may contribute to the formation of effusion and submucosal edema as observed in OME.

The effect of a middle ear immune response upon antibody levels in the perilymphatic compartment of the inner ear was investigated in the guinea pig. Animals were systemically sensitized with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) until high circulating levels were achieved. The middle ear cavity was then challenged with KLH, resulting in a vigorous immune response with effusion and mucosal inflammation. Antibody levels against KLH and BSA were then compared in serum, middle ear effusions, and perilymph. Anti-KLH levels in perilymph were found to increase substantially during middle ear response to challenge, while the anti-BSA levels did not, indicating a local origin for the anti-KLH antibody. The most likely explanations for these findings are 1) inner ear antibody originated in the middle ear and diffused across the round window membrane, or 2) antigen diffused across the round window membrane and evoked local production of antibody within the inner ear.