Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

Abstract

The target-derived factors necessary for promoting initial outgrowth from the statoacoustic ganglion (SAG) to the inner ear have not been fully characterized. In the present study, conditioned medium from embryonic Immortomouse inner ear cell lines that maintain many characteristics of developing inner ear sensory epithelia were screened for neurite-promoting activity. Conditioned medium found to be positive for promoting SAG neurite outgrowth and neuronal survival was then tested for the presence of chemokines, molecules that have not previously been investigated for promoting SAG outgrowth. One candidate molecule, monocyte chemotactic protein 1 (MCP-1), was detected in the conditioned medium and subsequently localized to mouse hair cells by immunocytochemistry. In vitro studies demonstrated that function-blocking MCP-1 antibodies decreased the amount of SAG neurite outgrowth induced by the conditioned medium and that subsequent addition of MCP-1 protein was able to promote outgrowth when added to the antibodytreated conditioned medium. The use of the Immortomouse cell lines proved valuable in identifying this candidate cofactor that promotes outgrowth of earlystage SAG nerve fibers and is expressed in embryonic hair cells.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear

“…Subsequent in vitro experiments in the developing mouse and chick, as well as analysis of Mif knockout (KO) mice, demonstrated that MIF can account for the major functional activities of ODF, including promoting SAG neuron survival and directional neurite outgrowth, and is required for normal hearing and inner ear innervation patterning. We also demonstrate that residual bioactivity in chick or mouse otocyst-generated ODF that is not removed by function-blocking antibodies to MIF can be entirely eliminated by function-blocking antibodies to the cytokine MCP1, which we previously identified as a component of ODF (Bianchi et al, 2005). Together, these data and our studies of zebrafish otic development (Holmes et al, 2011;Shen et al, 2012) show that the cytokines MIF and MCP1 (Bianchi et al, 2005) play crucial roles in inner ear development, directly demonstrating another important connection between the immune and nervous systems.…”

“…Our laboratories have been investigating proteins that regulate innervation during the earliest stages of inner ear development (reviewed by Barald and Kelley, 2004;Gerlach et al, 2000), and our recent work highlights the importance of chemokines and cytokines in the early stage peripheral auditory system (Bianchi et al, 2005;Holmes et al, 2011;Shen et al, 2012).…”

“…We began investigating the neurotrophic role of immune system chemokines and cytokines during the earliest stages of neuronal innervation, using the ear as a model system (Bianchi and Cohan, 1991;Barald et al, 1997;Germiller et al, 2004;Bianchi et al, 2005;Holmes et al, 2011;Shen et al, 2012). In earlier reports, we used 4667 RESEARCH ARTICLE Cytokines as neurotrophins gain-and loss-of-function approaches to demonstrate that the cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophic factor in the developing zebrafish inner ear (Holmes et al, 2011;Shen et al, 2012).…”

“…Because so little ODF can be obtained from embryonic otocysts, we generated cell lines from the E9.5 Immortomouse otocyst (IMO) (Barald et al, 1997) that secrete a functionally bioactive ODF equivalent (Germiller et al, 2004;Bianchi et al, 2005). Using cytokine arrays on IMO-generated ODF from these cell lines we first identified the cytokine monocyte chemoattractant protein 1 (MCP1; also known as CCL2) as an active, but not the sole, component of ODF (Bianchi et al, 2005).…”

“…Using cytokine arrays on IMO-generated ODF from these cell lines we first identified the cytokine monocyte chemoattractant protein 1 (MCP1; also known as CCL2) as an active, but not the sole, component of ODF (Bianchi et al, 2005). Because MCP1 alone could not entirely replicate the activity of ODF, we investigated whether other cytokines or cytokine-interacting molecules are also present in ODF.…”