Introduction
Sudden sensorineural hearing loss (SNHL) is defined as acute SNHL of 30 dB or greater over a minimum of three contiguous frequencies in the pure tone audiogram, which develops in under 72 hours.Reference Wilson and Alderson1 Vascular occlusion and viral infection are two feasible aetiological factors of sudden SNHL. An additional immune-mediated cause could be inflammation with abnormal sudden cellular stress in the cochlea.Reference Yamasoba, Kikuchi, Higo, O'Uchi and Tokumaru2–Reference Seo, Jeong, Choi and Moon5
Experimentally, immunocompetent cells from the blood stream that pass through the blood–labyrinth barrier to the cochlea can induce an immune response. This recruited immune response can produce sudden degenerative changes in the organ of Corti, stria vascularis and spiral ganglion.Reference Garcia-Berrocal, Trinidad, Ramirez-Camacho, Lobo, Verdaguer and Ibanez6, Reference Merchant, Adams and Nadol7 Autoimmune SNHL can rapidly progress between 30 and 60 years of age. However, sometimes it appears as sudden SNHL with bilateral involvement.Reference Xenellis, Nikolopoulos, Stavroulaki, Marangoudakis, Androulakis and Tsangaroulakis8 Clinically, unilateral sudden SNHL is more common than bilateral autoimmune SNHL. Merchant and colleagues reported that abnormal activation of cellular stress within the cochlea, especially activated nuclear factor kappa B that produces ancillary inflammatory cytokines, is a potential cause of sudden SNHL.Reference Merchant, Durand and Adams4, Reference Merchant, Adams and Nadol7
Cytokines are small proteins secreted by cells. Cytokines exert specific effects on the interactions between cells. Cytokines are autocrine in nature, which means that the producer cells possess corresponding surface receptors. These receptors exist in cochlear cells.Reference Demirhan, Eskut, Zorlu, Cukurova, Tuna and Kirkali9 Given the limited access to the cochlea and the difficulty in obtaining tissue samples, systemic inflammatory processes have been studied using peripheral blood to monitor possible pathogenetic mechanisms of sudden SNHL. Inflammatory cytokines and related proteins have been reported as potential markers in many cellular processes. However, to date, the role of inflammatory cytokines, specifically those in unilateral sudden SNHL, have not, to our knowledge, been extensively investigated. Few studies have discussed the involvement of inflammatory cytokines in sudden SNHL.Reference Haubner, Martin, Steffens, Strutz and Kleinjung10–Reference Singh, Dutta, Chattopadhyay, Chakravarty and Chaudhury12
This study investigated the levels of inflammatory cytokines, including tumour necrosis factor-α, interleukin (IL)-10 and IL-12, and interferon-γ, as well as peripheral blood mononuclear cells, and clusters of differentiation 11c and 86, in unilateral sudden SNHL.
Materials and methods
Participants
This study was given authoritative approval by the institutional review board of the tertiary university hospital (Chonnam National University Hospital; approval number CNUH-2013-042), and written informed consent was obtained from the participants.
Twenty-four patients with unilateral sudden SNHL were involved in the study between January 2013 and June 2014. Sudden SNHL was diagnosed based on clinical findings of SNHL exceeding 30 dB in three contiguous frequencies within 3 days. Patients with an objective cause of sudden SNHL, such as vestibular schwannoma or traumatic perilymph fistula, were excluded. Data on ototoxic mediation exposure, circulatory disease, and clinical and family history were obtained through self-reporting. Sudden SNHL patients with associated diseases, such as acute infection, systemic hypertension, hyperlipidaemia, diabetes mellitus, cardiovascular disease or liver disease, were also excluded.
Twenty-four normal-hearing patients, with no history of sudden SNHL, who were attending the clinic for other problems, were recruited to serve as the control group.
Blood samples
Blood samples were taken from the study group before corticosteroid treatment, to avoid the effects of corticosteroids on inflammatory cytokines or mononuclear cells. Peripheral blood mononuclear cells were separated using density gradient centrifugation of heparinised blood (Ficoll-Paque Plus; GE Healthcare Bio-Sciences, Piscataway, New Jersey, USA).
Flow cytometry
Two-colour cytometry analysis using fluorescence-activated cell sorting was performed using aliquots of 1 × 105 fixed cells and specific antibodies. The cells were incubated with staining buffer (phosphate-buffered saline containing 0.5 fetal bovine serum and 0.1 per cent sodium azide) containing anti-human cluster of differentiation 11c and anti-human cluster of differentiation 86 antibodies (both from BioLegend, San Diego, California, USA) for 30 minutes on ice. Cells stained using the applicable isotype-matched immunoglobulin G2b were considered as negative controls. After staining, the cells were processed using 1 per cent paraformaldehyde. Analysis was performed using a fluorescence-activated cell sorting Calibur instrument equipped with CellQuest software (BD Biosciences, San Diego, California, USA).
Enzyme-linked immunosorbent assay
The serum was separated from the blood samples of both groups and preserved at −20°C until analysis. Supernatant cytokine level was measured using human tumour necrosis factor-α, interferon-γ, interleukin (IL)-10 and IL-12 ELISA Max Deluxe enzyme-linked immunosorbent assay sets (BioLegend) according to the manufacturer's protocols. Absorbance was assessed by an enzyme-linked immunosorbent assay microplate reader at 405 nm and the results were expressed in pg/ml.
Statistical analyses
Statistical significance was analysed using the student's t-test for unpaired observations, and the differences were checked for statistical significance by one-way analysis of variance followed by Bonferroni's post-hoc test. A p-value of less than 0.05 was considered significant. Results are expressed as mean ± standard deviation or standard error of the total number of repeated measurements.
Results
The sudden SNHL group was composed of 15 males (62.5 per cent) and 9 females (37.5 per cent). The control group was composed of 12 males (50 per cent) and 12 females (50 per cent). The mean age was 46.91 years in the sudden SNHL group and 43.12 years in the control group. No significant difference was found with respect to mean age (Table 1). The hearing severity of the sudden SNHL group is shown in Table 2.
Table 1. Demographics of control and sudden SNHL groups
*n = 24; †n = 24. SNHL = sensorineural hearing loss.
Table 2. Initial hearing level in sudden SNHL patients
SNHL = sensorineural hearing loss.
The monocyte population percentage (mean ± standard error) was significantly higher in the sudden SNHL group (26.36 ± 4.3) than in the control group (14.32 ± 2.3) (Figure 1). The dendritic cell markers of cluster of differentiation 11c and cluster of differentiation 86 were identified in the sudden SNHL group (Figure 2). The cluster of differentiation 11c positive cell population was not significantly different between the groups. However, cluster of differentiation 86 expression was significantly higher in the sudden SNHL group than in the control group.
Fig. 1. Flow cytometry analysis of monocyte population in the (a) control group and (b) sudden sensorineural hearing loss (SNHL) group. (c) Monocyte population was significantly higher in the sudden SNHL group than the control group. SSC-H = side scatter height; FSC-H = forward scatter height; SS = side scatter; FS = forward scatter
Fig. 2. (a) Representative fluorescence-activated cell sorting analysis of cluster of differentiation 11c showed no difference between the two groups. (b) Cluster of differentiation 86 (CD86) expression was significantly higher in the sudden sensorineural hearing loss (SNHL) group than the control group. Histograms demonstrate the sudden SNHL group (red colour) and control (red colour) group. FL1/2-H = fluorescence pulse height; FSC-H = forward scatter height
The mean tumour necrosis factor-α level was significantly higher in the sudden SNHL group (15.8 ± 9.3 pg/ml) than in the control group (12.4 ± 8.7 pg/ml). Interferon-γ and interleukin (IL)-12 levels were significantly lower in the sudden SNHL group compared to the control group. There was no significant difference between the two groups in IL-10 level (Figure 3).
Fig. 3. Interleukin (IL)-10, IL-12, tumour necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in serum samples analysed by enzyme-linked immunosorbent assay. The mean TNF-α level in the sudden sensorineural hearing loss (SNHL) group (15.8±9.3 pg/ml) was significantly higher than that in the control group (12.4±8.7 pg/ml). Interferon-γ and IL-12 levels were significantly lower in the sudden SNHL group compared to the control group. There was no significant difference between the two groups in IL-10 level.
Discussion
Pro-inflammatory cytokines and anti-inflammatory cytokines have potential roles in immune responses. Among the cytokines, tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-2, IL-8 and IL-12, and interferon-γ are proinflammatory cytokines, while IL-4, IL-6 and IL-10 are anti-inflammatory cytokines.Reference Singh, Dutta, Chattopadhyay, Chakravarty and Chaudhury12 In the present study, we compared the cytokine levels and monocyte population between sudden SNHL and control groups. The TNF-α level, monocyte population and cluster of differentiation 86 expression were higher in the sudden SNHL group than in the control group.
Although the aetiology of sudden SNHL remains unclear, inflammation is reported to be one of the significant causative factors involved in sudden SNHL.Reference Hiramatsu, Teranishi, Uchida, Nishio, Suzuki and Kato13 Monocytes constitute a subset of circulating blood cells. Extravasation of circulating monocytes in the bloodstream is activated by inflammatory mediators, such as proinflammatory cytokines, local growth factors and microbial products.Reference Shi and Pamer14 The monocytes subsequently migrate into tissue, and can differentiate into a range of tissue dendritic cells or macrophages.
This event induces inflammation and activates the host-defence immune system.Reference Iwamoto, Iwai, Tsujiyama, Kurahashi, Takeshita and Naoe15 Proinflammatory cytokines, such as TNF-α, induce the differentiation of human monocytes into mature dendritic cells. The TNF-α-dependent dendritic cells elicit T helper 1 and 17 cell responses.Reference Iwamoto, Iwai, Tsujiyama, Kurahashi, Takeshita and Naoe15 Moreover, T helper 1 and 17 cell responses are essential for autoimmune SNHL.Reference Mijovic, Zeitouni and Colmegna16 To our knowledge, this is the first study to evaluate the role of dendritic cells in sudden SNHL. In the present study, we identified the presence of dendritic cell markers, namely, clusters of differentiation 11c and 86. Our results showed that the cluster of differentiation 86 level was significantly higher in the sudden SNHL group than in the control group. Although we did not clarify the correlation between dendritic cells and cytokines, we suggest that TNF-α can enhance the differentiation of monocytes into mature dendritic cells. Further studies are planned to clarify the mechanism of dendritic cells in sudden SNHL.
The role of TNF-α in sudden SNHL has rarely been reported.Reference Demirhan, Eskut, Zorlu, Cukurova, Tuna and Kirkali9, Reference Suslu, Yilmaz and Gursel17 Furthermore, different studies have reported different levels of TNF-α. One study showed that sudden SNHL patients had lower levels of TNF-α compared to the control group.Reference Suslu, Yilmaz and Gursel17 However, another study reported higher levels of TNF-α in sudden SNHL patients, with steroid-treated non-responders showing significantly higher levels of TNF-α post-treatment.Reference Demirhan, Eskut, Zorlu, Cukurova, Tuna and Kirkali9 In the present study, we did not compare TNF-α levels before and after treatment with steroids. The patients included in our study had unilateral sudden SNHL. None had bilateral symmetrical sudden SNHL with any history of autoimmune disease.
In this study, IL-12 and interferon-γ levels in the sudden SNHL group were lower than those in the control group. Interferon-γ is not a toxin designed to poison a key molecule in the cell. Normally, interferon and similar ILs mediate continuous interactions between cells related to growth and defence. Interferon-γ is produced by T-lymphocytes when stimulated with antigens or mitogens. Interleukin-12 is a central player in the generation of T helper 1 cells. Interleukin-12 is a regulatory cytokine that stimulates the production of interferon-γ, IL-2, IL-6 and granulocyte-macrophage colony-stimulating factor.Reference Trinchieri18
• Increses in tumor necrosis factor-α level and monocyte popluation might play critical roles in sudden sensorineural hearing loss
• Further studies of role of dendritic cells in sudden sensorineural hearing loss are necessary
Interleukin-10 is an important anti-inflammatory cytokine, which inhibits the production of proinflammatory cytokines (interferon-γ and TNF-α) by T helper 1 cells.Reference Demirhan, Eskut, Zorlu, Cukurova, Tuna and Kirkali9, Reference Joss, Akdis, Faith, Blaser and Akdis19 In the present study, we did not find any significant differences in IL-10 production between the two groups. The observed IL-10 levels might not be adequate to counteract the strong proinflammatory response that occurred in the patients prior to our study.
Recently, TNF-α inhibitors have been used for sudden SNHL treatment. Demirhan et al. suggested that increased TNF-α level can inhibit the response to steroid treatment, and insisted that TNF-α inhibitor plays an important role in sudden SNHL.Reference Demirhan, Eskut, Zorlu, Cukurova, Tuna and Kirkali9 Syrakic et al. also found that steroid-treated non-responders had a higher TNF-α level than control participants.Reference Svrakic, Pathak, Goldofsky, Hoffman, Chandrasekhar and Sperling20 In this study, we did not analyse the prognosis of sudden SNHL with regard to TNF-α levels because peripheral blood was collected only once, before steroid treatment. Although the underlying pathology of sudden SNHL is not fully understood, disturbed cochlear blood flow is a major cause.Reference Muzzi, Zennaro, Visentin, Soldano and Sacilotto21, Reference Narozny, Sicko, Przewozny, Stankiewicz, Kot and Kuczkowski22 Recent in vivo experiments showed that TNF-α inhibitor can reverse the decreased cochlear blood flow induced by TNF-α.Reference Ihler, Sharaf, Bertlich, Strieth, Reichel and Berghaus23
Thus, our results show that increased TNF-α level, monocyte population and cluster of differentiation 86 play important roles in sudden SNHL. Further studies are necessary to clarify the significance of TNF-α, monocyte population and cluster of differentiation 86 in the pathogenesis of sudden SNHL.
Competing interests
None declared
