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

Purpose—The authors investigated the contributions of the transient receptor potential vanilloid-1 receptor (TRPV1) and Ca2+ to microglial IL-6 and nuclear factor kappa B (NFκB) translocation with elevated hydrostatic pressure. Methods—The authors first examined IL-6 colocalization with the microglia marker Iba-1 in the DBA/2 mouse model of glaucoma to establish relevance. They isolated microglia from rat retina and maintained them at ambient or elevated (+70 mm Hg) hydrostatic pressure in vitro and used ELISA and immunocytochemistry to measure changes in the IL-6 concentration and NFκB translocation induced by the Ca2+ chelator EGTA, the broad-spectrum Ca2+ channel inhibitor ruthenium red, and the TRPV1 antagonist iodo-resiniferatoxin (I-RTX). They applied the Ca2+ dye Fluo-4 AM to measure changes in intracellular Ca2+ at elevated pressure induced by I-RTX and confirmed TRPV1 expression in microglia using PCR and immunocytochemistry. Results—In DBA/2 retina, elevated intraocular pressure increased microglial IL-6 in the ganglion cell layer. Elevated hydrostatic pressure (24 hours) increased microglial IL-6 release, cytosolic NFκB, and NFκB translocation in vitro. These effects were reduced substantially by EGTA and ruthenium red. Antagonism of TRPV1 in microglia partially inhibited pressureinduced increases in IL-6 release and NFκB translocation. Brief elevated pressure (1 hour) induced a significant increase in microglial intracellular Ca2+ that was partially attenuated by TRPV1 antagonism. Conclusions—Elevated 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 and is at least partially dependent on the activation of TRPV1 and other ruthenium red-sensitive channels. 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 Copyright © Association for Research in Vision and Ophthalmology Corresponding author: David J. Calkins, Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Ophthalmology Research Laboratory, 1105 Medical Research Building IV, Nashville, TN 37232-0654; [email protected]. Disclosure: R.M. Sappington, None; D.J. Calkins, None NIH Public Access Author Manuscript Invest Ophthalmol Vis Sci. Author manuscript; available in PMC 2014 August 21. Published in final edited form as: Invest Ophthalmol Vis Sci. 2008 July ; 49(7): 3004–3017. doi:10.1167/iovs.07-1355. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript 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 pressureinduced 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. Sappington and Calkins Page 2 Invest Ophthalmol Vis Sci. Author manuscript; available in PMC 2014 August 21. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript MATERIALS AND METHODS Animals and Tissue Preparation This study was conducted in accordance with regulations set forth in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Vanderbilt University Medical Center. For DBA/2 studies, paraformaldehyde (4%)-fixed whole eyes from 6month-old DBA/2 mice with relatively low (average, 14.7 mm Hg) or higher (average, 21.2 mm Hg) IOP were obtained from Philip Horner (University of Washington, Seattle, WA) and were prepared for immunolabeling as whole mount specimens. As previously described, IOP was monitored monthly with a tonometer (Tono-Pen; Reichert, Depew, NY) before kill.46 For histology, adult Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA) were perfused with 4% paraformaldehyde (Sigma, St. Louis, MO), their eyes were enucleated, and their retinas were removed for whole mount preparations. For primary cultures of purified microglia, eyes from postnatal day (P) 4 to P10 Sprague–Dawley rats (Charles River Laboratories) were enucleated, and retinas were dissected, as previously described.13,14 Cell Separation and Primary Culture Primary cultures of purified retinal microglia were prepared as we previously described and published in this journal.13,14 Briefly, retinas from P4 to P10 Sprague–Dawley rats were dissociated by trituration and incubation at 37°C in 1 mg/mL papain + 0.005% DNase I (Worthington, Lakewood, NJ). Viability was assessed by trypan blue exclusion. To purify microglia, the cells were incubated in monoclonal anti-rat RT 1a/OX18 antibody (5 μg/mL; catalog number CBL1519; Chemicon, Temecula, CA) or monoclonal anti-rat RT 1a/OX18 antibody (8 μg/mL; catalog number MCA51R; AbD Serotec, Raleigh, NC) followed by incubation with a magnetic bead-conjugated secondary antibody (Miltenyi Biotec, Auburn, CA). The cells were then loaded into a preequilibrated column in the presence of a magnetic field (Miltenyi Biotec). Cells positively selected by the anti-OX18 antibody were eluted, plated at a density of 5 × 104 on two-chamber glass slides or 1 × 104 on eight-chamber glass slides (Nalge-Nunc, Rochester, NY), and maintained in a 50:50 mixture of Dulbecco modified Eagle medium and F12 medium (DMEM/F12; Invitrogen, Carlsbad, CA) plus supplements. Cultures were grown to approximately 80% confluence (10–14 days) in a standard incubator with 5% CO2 before our timed experiments. During this time, 50% of the culture media were replaced every 48 hours. As previously reported, the purity of our microglial cultures was determined by positive immunolabeling and PCR reactions for microglia-specific markers (OX18 and CD68) and by negative results for markers against astrocytes, Müller glia, and fibroblasts.13 Cultures not meeting a standard of 95% purity were discarded. Hydrostatic Pressure Experiments We exposed microglia cultures to either ambient or +70 mm Hg hydrostatic pressure for 24 hours. This magnitude of pressure was chosen to complement our previous work; the rationale for using 70 mm Hg and construction of the pressure chamber is described in detail.13,14 For elevated pressure, a humidified pressure chamber equipped with a regulator Sappington and Calkins Page 3 Invest Ophthalmol Vis Sci. Author manuscript; available in PMC 2014 August 21. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript and a gauge chamber was placed in a 37°C oven, and an air mixture of 95% air and 5% CO2 was pumped into the chamber to obtain +70 mm Hg pressure that was maintained by the regulator. For ambient pressure experiments, cells were kept in a standard incubator. As described previously, pH and dissolved O2 content were monitored experimentally to maintain values within the range for our ambient cultures.14 For all pressure experiments, at least three culture plates per condition were used.