Many previous studies suggest that cochlear and vestibular functions, including hearing and balance, are influenced by immune responses in the inner ear (Ma et al. 2000
; Rahman et al. 2001
). Although immune function in the inner ear is very important in the protection from infectious diseases such as labyrinthitis, immune-related inflammatory responses also cause damage to the delicate tissues of the inner ear compartments and can often lead to cochlear degeneration and permanent hearing loss (Ryan et al. 2002
; Satoh et al. 2003
; Stone and Francis 2000
). However, the exact molecular mechanisms and roles of inflammatory mediators in hearing impairment still remain to be elucidated.
Cisplatin is one of the most widely used chemotherapeutic agents in treatment of human tumors. However, the risk of complications, including ototoxicity and nephrotoxicity, commonly hampers the use of higher doses to maximize its antineoplastic effects (Humes 1999
). Ototoxicity after cisplatin therapy is very common and occurs in the OHCs of the organ of Corti (Feghali et al. 2001
; Huang et al. 2000
; Kopke et al. 1997
; Watanabe et al. 2002
). In fact, cisplatin induces apoptosis of OHCs in the organ of Corti explants (Kopke et al. 1997
) and in vivo (Alam et al. 2000
; Watanabe et al. 2002
). It is also known that the cytotoxic effects of cisplatin occur via at least two major mechanisms, including the formation of DNA adducts (Huang et al. 2000
; Kartalou and Essigmann 2001
; Kharbanda et al. 2000
) and damage because of the production of ROS (Evans and Halliwell 1999
; Feghali et al. 2001
; Kopke et al. 1997
). These processes ultimately cause cell death by apoptosis or necrosis (Davis et al. 2001
; Jordan and Carmo-Fonseca 2000
). However, the roles of inflammatory cytokines in the pathogenesis of cisplatin ototoxicity have not been elucidated. In this study, we clearly demonstrate that the secretion and expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 were increased in HEI-OC1 cells after the addition of cisplatin in vitro, and in the rat cochleae after the peritoneal injection of cisplatin. In addition, our data show that proinflammatory cytokines are directly involved in the death of HEI-OC1 cells after cisplatin exposure. For the first time, we demonstrate that the early immediate secretion of proinflammatory cytokines occurs in auditory cells after cisplatin treatment independent of changes in mRNA expression. These results suggest that HEI-OC1 auditory cells may secrete proinflammatory cytokines from preexisting stores after cisplatin exposure. It has been reported that both TNF-α and IL-8 are stored in mast cells and are rapidly secreted after various stimuli, initiating late phase allergic reactions and other inflammatory responses (Gibbs et al. 2001
). Neutrophils are also known to contain stores of TNF-α, IL-12, IL-6, and MIP-2, which play a critical role in inflammation and antimicrobial activity (Bennouna et al. 2005
). Our results provide the first direct evidence that TNF-α, IL-1β and IL-6 are also secreted from preexisting stores in addition to their de novo synthesis in cisplatin-treated auditory cells.
NF-κB activation is pivotal in the expression of proinflammatory cytokines and other mediators involved in acute inflammatory responses and other conditions associated with increased ROS generation (Abraham 2003
). TNF-α and IL-1β can activate NF-κB through signal transduction involving the TNF-receptor-associated factor family of adaptor proteins (Dempsey et al. 2003
). TNF-α receptors phosphorylate IκB kinase, which in turn leads to IκB degradation in proteosomes and the intranuclear translocation of NF-κB subunits. NF-κB activation regulates a plethora of genes including proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 (Baldwin 1996
; Barnes and Karin 1997
). Association of NF-κB with IκB-α in the cytoplasm is well known to block the nuclear translocation of NF-κB, whereas exposure of cells to proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 induces NF-κB activation through IκB degradation and the subsequent nuclear translocation of NF-κB (Suzuki et al. 2000
; Wang et al. 2003
In this study, we also demonstrate that cisplatin induces the nuclear translocation of NF-κB through the degradation of cytoplasmic IκB and thereby the increase of transcriptional activity of NF-κB in vitro. In addition, cisplatin causes the increased NF-κB expression in the spiral ligament, stria vascularis, spiral limbus, and the three OHC layers of the organ of Corti in vivo. We speculate that the early immediate secretion of proinflammatory cytokines in auditory cells by cisplatin may be directly involved in NF-κB activation. This was further confirmed by our demonstration that the functional blocking of the early secretion of TNF-α with neutralizing antibody prevented the nuclear translocation of NF-κB in cisplatin-treated HEI-OC1 cells. Exogenous treatment with proinflammatory cytokines could also induce the nuclear translocation of NF-κB. Furthermore, NF-κB p65 protein expression largely overlapped with TNF-α, IL-1β, and IL-6 staining in the cochleae of cisplatin-injected rats.
The elucidation of a cascade of proinflammatory cytokines, NF-κB activation, and ROS generation is very important for the understanding of the pathogenesis of cisplatin ototoxicity. TNF-α and IL-1β induce the generation of ROS produced mainly by NADPH oxidase in the membranes of various cell types, including fibroblasts, kidney mesangial cells, endothelial cells, and smooth muscle cells (Tolando et al. 2000
). However, a number of groups have also demonstrated that TNF-α induces apoptotic cell death via ROS generation (Garg and Aggarwal 2002
). In our study, we showed that antioxidants such as NAC and GSH significantly increased the viability of cisplatin- and proinflammatory cytokine-treated cells. However, neither antioxidant affected the early immediate secretion of proinflammatory cytokines in cisplatin-treated HEI-OC1 cells. These results suggest that the cisplatin-mediated secretion of proinflammatory cytokines is upstream signal of ROS production. This was further confirmed by the fact that exogenous treatment with proinflammatory cytokines significantly increased the generation of intracellular ROS. Thus, considering the time-dependent kinetics of NF-κB nuclear translocation (maximum at 1 or 2 h after cisplatin) and ROS generation (maximum at 24 h or 30 h) (So et al. 2005
), we speculate that the mechanism of cisplatin cytotoxicity comes from two different arms: (1) the immediate early activation of NF-κB because of the release of preexisting stores of proinflammatory cytokines, and (2) the de novo synthesis of proinflammatory cytokines and subsequent ROS production at later time points.
MAPKs regulate many cellular events, including differentiation, proliferation, and apoptosis (Lewis et al. 1998
). It has also been reported that p38 and ERK pathways play an important role in IL-1β and TNF-α production, respectively (Hsu and Wen 2002
; Shi et al. 2002
). Herein, our data demonstrate that all three types of MAPKs are activated in cisplatin-treated cells. Pharmacologic inhibition of ERK activity is much more effective in the suppression of proinflammatory cytokine secretion and the prevention of cell death in cisplatin-treated HEI-OC1 auditory cells compared to the inhibition of p38 and JNK. The biochemical/molecular mechanisms by which cisplatin induces early proinflammatory cytokine release are still not fully understood. However, considering that the early immediate secretion of proinflammatory cytokines was significantly blocked by the pharmacologic inhibition of ERK activity, we suggest that ERK activation is critical for the release of preexisting stores of inflammatory cytokines, as well as the delayed production of proinflammatory cytokines by de novo synthesis. In addition, intraperitoneal injection with cisplatin clearly resulted in an increase of proinflammatory cytokines in both serum and the cochleae. Interestingly, etanercept, a fusion protein derived from a soluble TNF-α receptor and the FC
chain of human IgG, significantly reduced the serum levels of proinflammatory cytokines. It also blunted the cisplatin-induced expression of mRNA and protein of proinflammatory cytokines in the cochleae. Immunohistochemical investigation of the location of proinflammatory cytokine production in the cochleae after cisplatin showed that these cytokines are predominantly expressed in the spiral ligament, spiral limbus, spiral modiolar veins, and lacunae. In addition, TNF-α expression was observed in the OHC layers of the organ of Corti. This implies that TNF-α may directly mediate damage to hair cell layers, causing hearing impairment.
It is very important to elucidate the cellular source of inflammatory cytokines in cisplatin ototoxicity. Many studies suggest that infiltration of inflammatory cells, including lymphocytes, polymorphonuclear leukocytes, monocytes, and macrophages, into the cochleae is an important cellular source of inflammatory cytokines. Inflammatory cells are assumed to migrate to the cochleae from the endolymphatic sac (Satoh et al. 2003
; Tomiyama and Harris 1986
), or from the systemic circulation through the spiral modiolar vein (Harris et al. 1990
). These inflammatory cells play a pivotal role in defending against invading pathogens and foreign proteins, but inflammatory reactions generated by such cells may cause cochlear damage and lead to hearing loss. However, interestingly, we could not observe the infiltrated leukocytes in the histological sections from cisplatin-injected rats stained with hematoxylin and eosin (data not shown). This result suggests a possibility that there are other cellular sources of inflammatory cytokines in cisplatin ototoxicity in addition to endolymphatic sac and spiral modiolar vein. Furthermore, it has been reported that spiral ligament fibrocytes can secrete chemokines and other inflammatory mediators after stimulation with TNF-α and IL-1β (Yoshida et al. 1999
), which might induce the infiltration of inflammatory cells and cause cochlear dysfunction because of cochlear inflammation. In this study, we demonstrate that TNF-α is ubiquitously expressed throughout the spiral ligament and stria vascularis after cisplatin injection. Immunoreactivity to IL-1β was also observed in the same region of the spiral ligament. These data suggest that spiral ligament fibrocytes may induce inflammatory cytokine production in cisplatin ototoxicity, and may also be cellular targets for inflammatory cytokines. Our study also suggests the possibility that auditory cells in the organ of Corti may be an important additional source of inflammatory cytokines.
It has been recently shown that the integrity of the spiral ligament is critical for the normal functioning of the stria vascularis and, consequently, of the organ of Corti. Many studies indicate that the potassium ions used in the transduction of auditory signals by the sensory cells of the organ of Corti are recycled (Kikuchi et al. 1995
), and that potassium uptake from the perilymphatic space is mediated primarily by Type II and IV fibrocytes in the ligament (Hibino et al. 2004
; Kikuchi et al. 1995
), which abundantly expresses Na+
-ATPase and Na+
cotransporter (Hibino et al. 2004
). Through these types of fibrocytes, potassium enters the stria vascularis via connective tissue gap junctions and potassium channels (Hibino et al. 2004
; Kikuchi et al. 1995
). The strial marginal cells then return potassium to the scala media, where it is once again available to sensory hair cells. Interestingly, it has been reported that the expression and function of gap junction proteins such as connexins in other systems (including lung and kidney) were controlled by inflammatory cytokines (Duffy et al. 2000
; Hu and Cotgreave 1997
; Temme et al. 1998
). Moreover, the function of ion channels is known to be altered by inflammatory cytokines (Carlson et al. 1999
; Soliven et al. 1991
). Therefore, it seems that both alteration of ion homeostasis and disruption of gap junctions within spiral ligaments by proinflammatory cytokines may be closely related to the destruction of cochlear structure and hearing loss caused by cisplatin.
Taken together, our data demonstrate for the first time that proinflammatory cytokines may play a critical role in cisplatin-induced cochlear injury. TNF-α plays a key role in this process because the inhibition of TNF-α action significantly attenuates the expression of other proinflammatory cytokines after cisplatin injection. These findings are particular interest because of inflammation being another mechanism in the pathogenesis of cisplatin ototoxicity. In addition, elucidation of the intracellular mechanisms that participate in cisplatin damage to the inner ear may provide new opportunities for pharmacotherapy with regard to cisplatin and immune-mediated disorders of hearing and balance.