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Autoantigen specific T cells inhibit glutamate uptake in astrocytes by decreasing expression of astrocytic glutamate transporter GLAST: a mechanism mediated by tumor necrosis factor-

Department of Neurology, Universität des Saarlandes, Homburg, Germany

¹ Correspondence: E-mail: tkorn{at}rics.bwh.harvard.edu

SPECIFIC AIMS

Glutamate excitotoxicity has been proposed to be of pathogenic relevance in autoimmune inflammatory disorders of the central nervous system (CNS) causing damage to both neurons and oligodendroglial cells. We studied the impact of autoantigen-activated myelin basic protein (MBP) specific T cells on the functional expression of GLAST that belongs to the predominant astrocytic transport proteins to clear extracellular glutamate.

PRINCIPAL FINDINGS

1. Interaction with activated MBP specific T cells decreases glutamate transport capacity in astrocytes
After coculture with MBP-specific encephalitogenic T cells, rat primary astrocytes exhibited reduced glutamate uptake capacities in vitro. v_max values were 7010 ± 447 pmol mg^–1 min^–1 and 5830 ± 447 pmol mg^–1 min^–1 in naive astrocytes and in astrocytes recovered from coculture with resting T cells; maximum glutamate uptake rates in astrocyte monolayers were suppressed to 4340 ± 178 pmol mg^–1 min^–1 after incubation with MBP specific T cells activated in the coculture dish through the presence of antigen and to 3860 ± 437 pmol mg^–1 min^–1 after incubation with preactivated T cell blasts, respectively. Thus, exposure of astrocyte monolayers to autoreactive T cells in the presence of their specific antigen or to purified preactivated T cell blasts led to a significant decrease in v_max of L-[³H]-glutamate uptake compared with naive astrocytes (P<0.002 and P<0.0005) or with astrocytes that had been cocultured with resting T cells (P<0.03 and P<0.005). The respective K_m values remained unchanged. These results indicated that, in vitro, the glutamate clearance capacity of astrocytes was significantly impaired by the presence of autoreactive T cells depending on their activation level.

2. MBP specific T cells decrease the expression of GLAST in astrocytes by secretion of TNF-
After coculture with autoreactive T cells, GLAST protein levels were reduced in astrocytes. Referred to naive astrocytes, the decrease of GLAST was negligible after coculture with resting T cells, but became significant when astrocytes had been exposed to MBP specific T cells either activated during coculture or preactivated and cocultured in the presence of IL-2. In the first setting reflecting local autoantigen activation, high TNF- levels were accompanied by marked concentrations of IL-10; in the second scenario reflecting encounter of systemically activated T cells, TNF- was lower and IL-10 absent in the coculture supernatant. To unravel the mechanism of this T cell-triggered down modulation of GLAST, astrocytes were incubated with variably diluted cell-free supernatants from freshly activated and purified T cell blasts. The supernatants suppressed the expression of GLAST in astrocytes concentration-dependently (Fig. 1 A). This argued in favor of a humoral factor inducing the decrease of astrocytic GLAST protein in the coculture settings. Since the predominant cytokines in the T cell blast supernatant were IFN- and TNF (Fig. 1B ), we focused on these first. Indeed, the down-regulation of GLAST could be blocked when the supernatant was preincubated with a neutralizing anti-TNF- antibody (Fig. 1C ). Recombinant TNF- recapitulated the GLAST-reducing effect. A dose-dependent decrease of GLAST protein was evident in astrocytes exposed to increasing concentrations of TNF- (Fig. 1D ). In contrast to TNF-, recombinant IFN- did not induce any significant change in the astrocytic expression level of GLAST as illustrated by immunoblot and flow cytometry (Fig. 1E ).

View larger version (29K): [in this window] [in a new window]   Figure 1. Autoreactive T cells down modulate GLAST in astrocytes via TNF-. A) Astrocyte cultures were left untreated (medium) or incubated for 48 h with different dilutions of supernatant that had been collected from T cell blasts 12 h after isolation from bulk restimulation with MBP in the presence of syngeneic splenocytes as antigen presenting cells and subsequent expansion in IL-2-conditioned medium (TBL-sup). B) ELISA determination of IFN- and TNF- in the TBL supernatant to which astrocytes were exposed. C) Neutralizing anti-TNF- antibody reverses the GLAST suppressing effect of TBL supernatant. Astrocytes were left naive (medium, lane 1) and exposed to 1:10-diluted TBL supernatant plus irrelevant mouse IgG (lane 2) or to 1:10 diluted TBL supernatant preincubated with a neutralizing monoclonal anti-TNF- antibody (IgG1) at 200 µg/mL (lane 3). FACS analysis of GLAST and respective histogram plots of astrocytes that had been exposed to TBL supernatant (1:10) or TBL supernatant after TNF- neutralization (straight line) as compared with GLAST expression in naive astrocytes (dotted line). Note the rightward-shift of the GLAST-signal (straight line) after TNF- block. The shaded curves depict staining with isotype matched control antibody. D) Immunoblot analysis of lysates prepared from astrocytes that had been left untreated (medium) or incubated for 48 h with different concentrations of recombinant TNF-. The signals of monomeric GLAST were evaluated by densitometry. Astrocytic GLAST was significantly reduced only after incubation with 100 U/mL TNF- (*P<0.004) although there was a trend for lower TNF- concentrations. Mean +SD of n independent experiments. Reduction of GLAST by 100 U/mL TNF- was equally well detected by FACS-analysis of fixed astrocytes. E) Analogous experiments were performed with IFN- (n=3). Even with concentrations as high as 500 U/mL, no significant decrease of GLAST was detectable in astrocytes by immunoblot or FACS analysis.

3. Astroglial expression of GLAST is strongly reduced in experimental autoimmune encephalomyelits (EAE)
We wished to test whether the decrease of GLAST triggered by autoreactive T cells was paralleled in vivo. We used an adoptive transfer EAE model that simulates inflammatory features of multiple sclerosis (MS). Histolopathologic analyses of cerebella and spinal cords were performed on day 6 after cell transfer when animals were paraplegic. At this point, PBS-injected control-rats were healthy and showed intense GLAST expression in the cerebellar molecular layer and the spinal cord white matter (Fig. 2 A, G). In contrast, molecular layer and spinal cord white matter appeared virtually cleared of GLAST in paraplegic EAE-rats (Fig. 2B, H ). On day 6 after cell transfer, GFAP staining as marker for astrocytes did not seem to be altered due to autoimmune inflammation (Fig. 2C, D, I, K ). This argued against some unspecific response of astrocytic protein expression. The down-regulation of GLAST in astrocytes was widespread and not confined to inflammatory infiltrates that were mainly perivascular in the cerebellum (Fig. 2F ) and scattered in the spinal cord (Fig. 2M ) as illustrated by ED1 staining for macrophages. These findings indicated that in vivo also, a humoral factor (most likely TNF-) might be operative to decrease the expression of GLAST in astrocytes in the course of T cell-triggered autoimmunity.

View larger version (105K): [in this window] [in a new window]   Figure 2. In autoimmune encephalomyelitic rats, GLAST expression is reduced in widespread areas of the CNS. EAE was induced in Lewis rats by adoptive transfer of MBP-activated T cells. At the peak of clinical disease (paraplegia), animals were killed. Representative sections from the cerebellum (A–F) and the spinal cord (G–M) of a paraplegic rat (right columns) and a PBS-injected and clinically healthy control animal (left columns) are shown. Immunohistological staining was performed for GLAST (A, B and G, H), GFAP (C, D andI, K), and ED1 (E, F and L, M). Note that there are perivascular or scattered infiltrates of ED1-positive cells (macrophages) only in tissue sections from encephalomyelitic rats. Scale bars 50 µm (A–F) and 100 µm (G–M). The inserts in (A) and (B) display lower magnification fields of the cerebellum to give an overview how widespread the loss of GLAST was in the cerebellar molecular layer (arrow) of the diseased animal (B) as compared with the healthy control rat (A). Scale bar in the inserts 200 µm.

CONCLUSIONS AND SIGNIFICANCE

Glutamate excitotoxicity has long been recognized as pathogenic principle in ischemic brain lesions and degenerative disorders of the CNS. Only recently, it has been reported that glutamate receptor blocking agents were able to protect neurons and oligodendrocytes during EAE. Thus, excess extracellular glutamate might contribute to tissue damage apart from destructive processes that emanate directly from autoimmune inflammatory infiltrates. Astrocytes are the principal players in clearing glutamate from the extracellular space and have great spare capacities regarding secondary active glutamate reuptake. One of the predominant proteins to mediate glutamate transport in astrocytes is GLAST. Particularly in the cerebellar molecular layer it is very likely that synaptic spillover of glutamate and also ambient glutamate levels are controlled by astrocytes expressing GLAST.

In the present study, we show that interaction of astrocytes with autoantigen-activated encephalitogenic T cells leads to a partial loss of astrocytic glutamate uptake capacity on the basis of a substantial decrease of GLAST expression. It is evident that MBP specific T cells need to be activated but do not have to be in direct contact with astrocytes to mediate the down-regulation of astrocytic GLAST protein. T cell derived TNF- seems to be the driving agent. In accordance with the TNF- responsiveness of the EAAT1 promoter, the human homologue of rat GLAST, we assume a transcriptional mode of regulation of GLAST expression by TNF-. The delayed down-regulation of GLAST in the range of 48 h is in line with this concept. The decrease of GLAST in astrocytes upon TNF- is dose-dependent. Interestingly, IL-10 might partially antagonize the TNF- mediated down modulation of GLAST in astrocytes since higher TNF- levels do not further decrease GLAST in T cell/astrocyte cocultures when on site-stimulation of T cells allows for production of IL-10. TNF- antagonizing properties of IL-10 in terms of astrocyte effector functions have been described previously. TNF- mediated suppression of glutamate uptake into astrocytes has been reported in infectious diseases of the CNS. HTLV-1 infected T cells are likewise thought to impair astrocytic glutamate uptake by secretion of viral proteins or cytokines. Thus, excess extracellular glutamate accumulation might result and subsequent glutamate excitotoxicity could be a common pathogenic effector limb in chronic infectious and autoimmune inflammatory CNS disorders (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. MBP specific encephalitogenic T cells cause a decrease of GLAST expression in astrocytes by secretion of TNF-. As a consequence, glutamate reuptake into astrocytes is reduced and excess glutamate may accumulate in the extracellular space. Glutamate is able to trigger excitotoxicity in neurons via NMDA receptors and in oligodendrocytes via AMPA/kainate receptors. This might contribute to neuronal and oligodendroglial damage remote from actual autoimmune inflammatory infiltrates. IL-10 might partially antagonize the GLAST-suppressing effects of TNF- in astrocytes. The exact intracellular signaling pathway of TNF- that prompts the down-regulation of astrocytic GLAST is to be identified. This may provide us with new targets to combat glutamate excitotoxicity in T cell-triggered autoimmune inflammatory disorders of the CNS.

This may offer an explanation for the observation that in MS, markers of axonal and oligodendrocyte precursor integrity such as N-acetyl aspartate (as measured by magnetic resonance spectroscopy) are decreased early in the disease course and distant from truly demyelinating lesions. It is an open question whether, besides blockage of glutamate receptors, differential targeting of TNF- signaling pathways or strengthening of IL-10 effects might provide tools to combat glutamate excitotoxicity in chronic autoimmune inflammatory disorders of the CNS.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3748fje;

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