Neuroprotective Immunity: T Cell-Derived Glutamate Endows Astrocytes with a Neuroprotective Phenotype

Abstract

A well-controlled T cell response to CNS injury may result in increased neuronal survival. However, the precise mechanism of T cell-induced neuroprotection is unknown. In this study, we report the unexpected finding that during culture of T cells, high levels of glutamate accumulate, which are efficiently cleared if T cells are cocultured with astrocytes. The T cell-derived glutamate elicits in turn, the release of neuroprotective thiols (cysteine, glutathione, and cysteinyl-glycine) and lactate from astrocytes. Media obtained from astrocytes conditioned in the presence of T cells reduce neuronal apoptosis induced by oxidative stress in primary neuronal cultures from 48 ± 14 to 9 ± 4% (p < 0.001). Inhibition of glutamate-dependent signaling during astrocyte-T cell cocultivation by a glutamate uptake inhibitor, l-aspartic acid β-hydroxamate, abolishes this neuroprotective effect. The ability of astrocytes to clear extracellular glutamate is impaired under conditions of oxidative stress. We demonstrate that T cells, via secreted cytokines, restore glutamate clearance capacity of astrocytes under oxidative conditions. Furthermore, under normoxic conditions, glutamate-buffering capacity of astrocytes is increased upon cocultivation with T cells. It is known that, following CNS injury, astrocytes can respond with beneficial or destructive effects on neurons. However, the context and signaling mechanisms for this dual astrocytic response are unknown. Our results implicate T cells as potential determinants of the context that elicits a protective role for astrocytes in the damaged CNS.

Differential Dependence on Cysteine from TranssulfurationversusTransport During T Cell Activation

“…T cells were enriched by negative selection on T cell columns (R&D Systems, Minneapolis, MN) as per the vendor's protocol (6). For in vitro activation, as isolated naïve T cells were suspended in RPMI medium containing 2.5% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 1Âpenicillin=streptomycin (Invitrogen), 50 mM 2-mercaptoethanol (T cell media), and incubated with either anti-CD3 þ anti-CD28 antibodies (eBiosciences) or with anti-CD3 antibody þ dendritic cells or irradiated splenocytes (as APC, 1:4 ratio) for 48 to 72 h. After incubation, activated T cells were separated from APCs by gentle pipetting, and T cells were collected with low-speed centrifugation at 200 g for 10 min at 48C.…”

“…precipitated proteins were sedimented by centrifugation at 13,000 g for 10 min at 48C. Protein-free extracts were alkylated with monoiodoacetic acid, derivatized with 2,4-dinitrofluorobenzene solution, and analyzed with HPLC, as previously described (6,15). The concentration of metabolites in the control medium was subtracted from the final values.…”

Regulatory T Cells Interfere with Glutathione Metabolism in Dendritic Cells and T Cells

“…Measurement of Thiols and Disulfides by HPLC-Concentrations of extracellular cystine, cysteine, GSH, and intracellular GSH (GSH in ) were measured using an HPLC method as described (9,22). The GSH in values were normalized to protein concentration determined by the Bradford assay with bovine serum albumin as a standard.…”

Inflammation, Glutamate, and Glia: A Trio of Trouble in Mood Disorders

“…Unlike other cytokines, T lymphocytes are the primary producers of IFN-γ (Kipnis et al, 2012) and once released at high concentrations, IFN-γ can transform astrocytes into phagocytic, antigen-presenting cells (Fontana et al, 1984;Shrikant and Benveniste, 1996;Soos et al, 1998). In concert with the anti-inflammatory cytokine IL-4 (which is also produced by T cells), IFN-γ controls the transition of the astroglial phenotype from neuroprotective (A2) to neurotoxic (A1) with the drastic reduction in the ability of these cells to clear glutamate (Garg et al, 2008;Garg et al, 2009;Kipnis et al, 2012). However, at lower concentrations, IFN-γ increases glutamate buffering and clearance by stimulating EAATs with the opposite effects at higher concentrations (Hu et al, 2000;Ye and Sontheimer, 1996).…”