Contribution of TRPV1 to Microglia-Derived IL-6 and NFκB Translocation with Elevated Hydrostatic Pressure

Glaucoma is a common optic neuropathy characterized by progressive loss of retinal ganglion cells (RGCs) and is often associated with increases in intraocular pressure.1–3 Although pathologic changes in the physiology of RGCs and their axons, which comprise the optic nerve, are primarily responsible for vision loss in glaucoma, other ocular cell types have also emerged as contributors to the disease process. In particular, astrocyte glia and microglia have been associated with various aspects of glaucoma. These include biochemical and structural changes in the optic nerve head, vascular pathology, and direct modulation of RGC survival.4–20 The contribution of glia to these events is often attributable to a change in the production or release of secreted factors. Increases in the level of inflammatory cytokines, such as tumor necrosis alpha (TNFα), interleukin (IL)-6, interferon gamma (IFNγ), IL-1α, IL-1β, IL-8, and IL-10, are evident in plasma and cerebral spinal fluid from a number of optic neuropathies, including neuromyelitis optica,21 optic neuritis,22 and AIDS-related optic neuropathy.23 Similarly, levels of IL-6 in the aqueous humor of patients with neovascular glaucoma are markedly increased.24 Growing evidence suggests that astrocyte glia and microglia produce these cytokines in glaucomatous optic neuropathy and AIDS-related optic neuropathy.4,7,23 Experimental models using elevated pressure or ischemic conditions reveal that many extracellular factors, including TNFα,4,5 nitric oxide,6 and IL-6,13,14,25,26 are released by astrocytes and microglia and can alter RGC survival. We recently identified IL-6 as a key component of pressure-induced signals from retinal microglia and described its protective properties for RGCs exposed to elevated pressure.13 We further identified the ubiquitin–proteasome pathway and activation of the transcription factor nuclear factor kappa B (NFκB), which are responsible for the production of IL-6 by astrocytes, microglia, and macrophages in other systems,27–37 as components of the pressure-induced release of IL-6.14 In other systems, the influx of extracellular Ca2+ can induce IL-6 production through the activation of NFκB,38–41 including that induced by cellular stretch.42 In microglia, Ca2+ mediates the response to a number of stimuli, including the activation of purinergic receptors, glutamate, and various proinflammatory cytokines.43 Here, to probe its relevance to glaucoma, we found that microglia-derived IL-6 increases with elevated intraocular pressure (IOP) in the DBA/2 mouse model of hereditary glaucoma. To probe the mechanisms of IL-6 release in vitro, we describe the influence of Ca2+ chelation and of blocking Ca2+ channels on pressure-induced activation of NFκB and subsequent IL-6 release by retinal microglia. We also describe the novel finding that retinal microglia, like microglia in the brain and spinal cord,44,45 express the capsaicin-sensitive, cation-selective transient receptor potential vanilloid-1 receptor (TRPV1). Using a hydrostatic pressure chamber, we determined that extracellular Ca2+ is required for pressure-induced IL-6 release and activation of NFκB in primary cultures of retinal microglia. Broad antagonism of ryanodine receptors and of TRPV1 with ruthenium red also inhibited IL-6 release and NFκB activation, though less efficiently. Specific antagonism of TRPV1 with iodo-resiniferatoxin (I-RTX; Alexis Biochemicals, Lausen, Switzerland) partially reduced the pressure-induced IL-6 release and activation of NFκB and the pressure-induced increases in intracellular Ca2+. Interestingly, the activation of TRPV1 alone, with its agonist capsaicin, was not sufficient to increase IL-6 concentration. Together these data suggest that elevated hydrostatic pressure induces an influx of extracellular Ca2+ in retinal microglia that precedes the activation of NFκB and the subsequent production and release of IL-6. This influx is mediated in part by the activation of TRPV1, though other pressure-induced events are necessary to promote increases in IL-6 release.